WO2012113022A1 - Therapeutic uses of slirp - Google Patents

Therapeutic uses of slirp Download PDF

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Publication number
WO2012113022A1
WO2012113022A1 PCT/AU2012/000166 AU2012000166W WO2012113022A1 WO 2012113022 A1 WO2012113022 A1 WO 2012113022A1 AU 2012000166 W AU2012000166 W AU 2012000166W WO 2012113022 A1 WO2012113022 A1 WO 2012113022A1
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Prior art keywords
seq
polypeptide
amino acids
slirp
cell
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PCT/AU2012/000166
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French (fr)
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Peter Leedman
Shane Colley
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The University Of Western Australia
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Priority claimed from AU2011900644A external-priority patent/AU2011900644A0/en
Application filed by The University Of Western Australia filed Critical The University Of Western Australia
Publication of WO2012113022A1 publication Critical patent/WO2012113022A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16Masculine contraceptives
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/36Gynecology or obstetrics
    • G01N2800/367Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the present invention relates to the modulation of SLIRP levels within a sperm cell to effect sperm function and motility.
  • the present invention further relates to methods for assessing the SLIRP gene and its products to predict fertility in males.
  • SRA Steroid receptor RNA activator
  • NRs nuclear receptors
  • ER estrogen receptor
  • SRA plays an important role in mediating 17p-estradiol (E 2 ) action. Its expression is both increased and aberrant in many human breast tumours, suggesting a potential role in pathogenesis.
  • E 2 17p-estradiol
  • SRA acts as an RNA-protein complex scaffold for other coregulators at transcription initiation sites, the precise mechanism by which SRA augments ER activity in general, and specifically in breast cancer, remains unclear.
  • protein interactors of SRA have been identified and provide insight into the putative mechanisms underlying SRA's transcriptional coactivation ability.
  • SLIRP SRA stem-loop-interacting RNA-binding protein
  • SLIRP is a widely expressed small SRA-binding protein and a repressor of ER and glucocorticoid receptor (GR) activity. While it has been shown to be predominantly localized to the mitochondria, it is actively recruited to the E 2 -responsive pS2 promoter where it modifies NR transactivation.
  • the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for increasing expression of a polypeptide in a sperm cell to improve the function of the cell, the polypeptide being any one or more selected from the group consisting of:
  • the present invention provides a method for increasing expression of a polypeptide in a sperm cell to improve the motility of the cell, the polypeptide being any one or more selected from the group consisting of:
  • the present invention provides a method for treating a disorder associated with an undesirable level of function and/or motility of sperm cells, the method comprising the step of administering an effective amount of an isolated polypeptide, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
  • polypeptide of the methods described herein may be encoded by a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • polypeptide may be encoded by a polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • the polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, may also comprise a nucleotide sequence 95% to 99% identical to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • one or more promoters may be used to increase the expression of a SLIRP polynucleotide encoding a SLIRP polypeptide as described herein.
  • antisense nucleic acids or siRNAs may be used to reduce or eliminate the expression of a polynucleotide encoding a SLIRP polypeptide as described herein.
  • the present invention also provides a method for sterilising or reducing the sterility of a male animal comprising a method as described herein, including the step of reducing levels or reducing activity of the SLIRP polypeptide within sperm cells of the male animal.
  • male animal as used herein includes both male humans and non-human male animals including, for example, but not limited to, mice, rats, sheep, cows/cattle, dogs, horses, pigs, orang-utan, amongst others. This sterilising of the male animal may be reversible by restoring the SLIRP levels in the sperm cells of the male animal.
  • the present invention provides a SLIRP polynucleotide as a biomarker for identification of dysmotile or dysfunctional sperm, the polynucleotide being any one or more selected from the group consisting of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • a microarray may be used to identify the presence of mutations in the SLIRP polynucleotide.
  • the present invention provides a use of a polypeptide as a biomarker for prediction of dysmotile or dysfunctional sperm, the polypeptide being any one or more selected from the group consisting of:
  • the present invention provides a use of a polypeptide as a biomarker for prediction of energy production in a sperm cell, the polypeptide being any one or more selected from the group consisting of:
  • polypeptide of the herein described methods may comprise a portion of a fusion protein.
  • a SLIRP polypeptide of the herein described methods may comprise a non-peptide mimetic of the polypeptide.
  • a selective binding agent may be used to detect the presence or levels of a SLIRP polypeptide as described herein.
  • a selective binding agent may be an antibody to the polypeptide, such as a polyclonal or monoclonal antibody, and said antibody may also be labelled.
  • the present invention provides a method for assessing the fertility of a male animal, comprising the steps:
  • These one or more cells may be isolated from any one of blood, sputum, or semen from the male animal. Moreover, the one or more cells may be quantified to assess the fertility, and wherein nucleotide sequencing may be used for identifying the nucleotide sequence.
  • the present invention further provides a kit for assessing the fertility of a male animal, the kit using any of the methods described herein, and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
  • the present invention also provides a kit for assessing the fertility of a male animal, the kit using the method of any of the methods described herein, and comprising at least one allele-specific oligonucleotide probe specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
  • the present invention provides a method for predicting the fertility of a male animal, comprising the steps:
  • the presence of the one or more mutations is predictive of a reduced fertility of the male animal when compared to a male animal without the one or more mutations.
  • the presence of the one or more mutations may be predictive that the male animal is infertile.
  • a mutant or wild-type SLIRP gene may be predictive of reduced fertility.
  • the mutations may be within any one or more of the coding region of the exons, non-coding exonic regions, intronic regions, or flanking regions of the SLIRP gene.
  • the one or more cells may be taken from blood, epithelial cells, semen, or other genetic- bearing material, from the male animal.
  • the present invention provides a method for predicting the fertility of a male animal, comprising the steps:
  • SLIRP gene product levels are predictive of a reduced fertility of the male animal when compared to SLIRP gene product levels in one or more cells from a normal male animal.
  • the SLIRP gene product levels may be measured using quantitative reverse transcriptase (QRT) PCR including real-time QRT-PCR, and the one or more cells may be isolated from semen or ejaculate from the male animal.
  • QRT quantitative reverse transcriptase
  • FIG. 1 SLIRP is expressed in murine testis and sperm.
  • FIG. 1 Northern blot of human RNA extracted from a range of tissues shows that SLIRP is highly expressed in the tissues that are mitochondria-rich and energy requiring: especially heart, liver, skeletal muscle and the testis. The blot is also probed with beta- actin as an RNA loading control.
  • SLIRP is a repressor of nuclear receptor activity and is present in nuclear receptor complexes but in some tissues is predominantly a mitochondrial protein.
  • Dex dexamethasone
  • DHT dihydrotestosterone
  • VitD vitamin D
  • GW501516 PPAR agonist
  • SRA steroid receptor RNA activator.
  • FIG. 4 SLIRP levels are frequently substantially lower in human males with teratozoospermia. Data presented compares the level of SLIRP expression between human males with normal fertility and those considered to have teratazoospermia (a condition in which less than 4 percent of sperm cells are morphologically normal). Data from Gene Expression Omnibus (GEO), National Center for Biotechnology Information, USA. Title: GDS2697 / 221434_s_at / C14orf156 / Homo sapiens. The image represents the abundance profile for an individual gene across each Sample in a DataSet.
  • GEO Gene Expression Omnibus
  • Ranks can also be a useful indicator for when a DataSet is not well normalized (you can verify that Sample values are well-distributed / normalized by viewing the 'value distribution' chart that is provided on each DataSet record under the 'analysis' button maintained by the GEO database).
  • FIG. 1 SLIRP is localised in the acrosome, midpiece and annulus of human sperm.
  • FIG. 7 SLIRP levels are depleted in HeLa cells 7 days post transfection with pSuperior SLIRP. Hela cells were transfected with either empty pSuperior. neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1.
  • FIG. 8 Schematic representation of the mouse SLIRP genomic locus.
  • the mouse SLIRP gene also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl/SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
  • FIG. 7 SLIRP levels are depleted in HeLa cells 7 days post transfection with pSuperior SLIRP. H.ela cells were transfected with either empty pSuperior.neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1.
  • FIG. 8 Schematic representation of the mouse SLIRP genomic locus.
  • the mouse SLIRP gene also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl/S IP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
  • Human SLIRP protein sequence aligned with non-human orthologues Human SLIRP protein (NP 1 12487), aligned with orthologues from other species including Orangutan (NP_001 126105), Pig (NP_001090941 ), Horse (XP_001494001 ), Sheep (NP_001 138656), Cow (NP_001032562), Dog (XP_547932), Mouse (NM_026958) and Rat (NM_001 109507). Numbers at top indicate residue number, numbers right refer to the length of individual proteins. Genebank (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA) accession numbers for individual sequences provided in brackets. A consensus sequence is also listed as is a graphical indicator of the conservation of individual residues between the species listed.
  • Figure 14 Human SLIRP mRNA sequence aligned with non-human orthologues.
  • Human SLIRP mRNA sequence (NM 031210) aligned with orthologues from other species including Orangutan (NM_001 132633), Pig (NM_001097472), Horse (XM_001493951 ), Sheep (NM_001 145184), Cow/cattle (NM_001037485), Dog (XM_547932), and Mouse (NM_026958). Numbers at top indicate nucleotide number, numbers right refer to the length of individual mRNAs.
  • Genebank National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA
  • accession numbers for individual sequences provided in brackets accession numbers for individual sequences provided in brackets. A consensus sequence is also listed along with a graphical indicator of the conservation of individual nucleotides between the species listed.
  • SLIRP polypeptides The high proportion of SLIRP which has been shown to localize within the mitochondria correlates with NRs being found in both mitochondrial and nuclear compartments of the cell. It is therefore unsurprising that the protein has been shown to have a role in modulating cellular metabolism and energy homeostasis in the mitochondria, a fundamental function for the organelle.
  • the modulation of SLIRP polypeptides to control motility and function of sperm cells by the present invention provides an unexpected role for SLIRP polypeptides.
  • the present invention provides a method for modulating the motility and/or function of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide or the activity of a polypeptide within the sperm cell, as compared to normal levels or normal activity of the polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
  • SEQ ID No: 2 and 4 are human SLIRP polypeptides, wherein SEQ ID No: 4 is SRA stem-loop-interacting RNA-binding protein, mitochondrial precursor [Homo sapiens], NCBI Reference Sequence NP_1 12487.
  • SEQ ID No: 6 is a cow SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial precursor [Bos taurus], NCBI Reference Sequence NP_001032562 XP_869675.
  • SEQ ID No: 8 is a dog SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Canis lupus familiaris], NCBI Reference Sequence XP_547932.
  • SEQ ID No: 10 is a sheep SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Ovis aries], NCBI Reference Sequence NP_001 138656.
  • SEQ ID No: 12 is a horse SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Equus caballus], NCBI Reference Sequence XM_001493951 .
  • SEQ ID No: 14 is a pig SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Sus scrofa], NCBI Reference Sequence NP 001090941 .
  • SEQ ID No: 16 is a rat SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Rattus norvegicus], NCBI Reference Sequence NP_001 102977 XP_001068036.
  • SEQ ID No: 18 is a Sumatran orangutan SLIRP polypeptide, specifically SRA stem-loop- interacting RNA-binding protein, mitochondrial [Pongo abelii], NCBI Reference Sequence NM_001 132633.
  • SEQ ID No: 20 is a mouse SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Mus musculus], NCBI Reference Sequence NP_081234 XP_354684.
  • SLIRP polypeptide(s) and SLIRP, as used herein, includes the above polypeptides [(i) to (vi)], unless the context specifically requires otherwise.
  • the species from which a polypeptide - 1 3 - originates will be selected to match the species of the sperm cell. For example, to modulate the motility and/or function of a human sperm cell, levels of a polypeptide or activity of a polypeptide of SEQ ID No: 2 or 4, or any one of (ii) to (vi) above can be increased or reduced as these polypeptides are of human origin.
  • levels of a polypeptide or activity of a polypeptide of SEQ ID No: 6 can be increased or reduced as the polypeptide is of Bos taurus origin.
  • the present invention also includes methods to modulate the motility and/or function of a sperm cell for species of animals not listed herein.
  • levels of SLIRP polypeptides or activities of SLIRP polypeptides may be increased or reduced within a sperm cell of an animal subject of a species not listed herein which can modulate the motility and/or function of the sperm cell.
  • the present invention also provides a method for increasing expression of a polypeptide in a sperm cell to improve the function and/or motility of the cell, the polypeptide being any one or more selected from the group consisting of:
  • the function and motility of sperm cells is central for their role in fertilisation. This is apparent with approximately 50% of infertile human males having low sperm motility and/or a low sperm count.
  • SLIRP is largely localised in the mitochondria, the site of cellular metabolism and energy homeostasis, and low sperm motility and/or a low sperm count are typical features of sperm mitochondrial defects.
  • the present invention also provides a method whereby increasing SLIRP levels in a sperm cell can improve the function and motility of the cell, particularly where the sperm cell is defective in that it has reduced motility and/or function.
  • sperm cell refers to a male animal gamete or reproductive cell which contains genetic information and participates in the act of fertilization of an ovum. - 14 -
  • sperm motility refers to the ability of a sperm cell to move actively forward. The mature sperm, after residence in the female reproductive tract, develops a forward progressive and then hyperactivated form of motility. Hyperactivated motility is thought to greatly facilitate progression and aid in penetration of the oocyte.
  • Sperm motility is one measure of fertility for a subject and can be assessed using a microscope (400x magnification) or by computerised imaging, amongst other methods.
  • improved sperm motility refers to the sperm cell(s) being able to move more actively forward than previously able.
  • the term “dysmotile sperm” refers to abnormal sperm which are unable to carry out the normal function and movement of a normal sperm cell.
  • sperm function refers to the ability of a sperm cell to carry out its normal functions including growth and development, and movement as is required for its ability to fertilise an ovum.
  • Full sperm functional competence is obtained by sperm as it goes along the epididymus.
  • Ejaculated sperm have the capacity for fertilization only after a period of residence in the female reproductive tract, during which they undergo a process known as capacitation.
  • capacitation sperm develop a forward progression and then hyperactivated motility (with exaggerated flagella bending).
  • Sperm also develop the capacity to undergo acrosome reaction and the ability to bind the oocyte, which are the end-points of sperm capacitation. Energy production in a sperm cell is carried out through the processes of cellular metabolism in the mitochondria.
  • the methods and uses of the present invention may involve SLIRP polypeptides which are recombinant, natural or synthetic.
  • the isolated SLIRP polypeptides may be mixed with carriers or diluents that will not interfere with the intended purpose of the polypeptide and still be regarded as isolated.
  • the SLIRP polypeptide may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which at least 90%, 95%, 98% or 99% of the protein in the preparation is a SLIRP polypeptide.
  • functional variants include isolated SLIRP polypeptides that have at least one important activity of the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, - 1 5 -
  • a variant SLIRP polypeptide may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the polypeptide is fused with another compound, such as a compound to decrease the half life of the polypeptide, or (iv) one in which the additional amino acids, such as a leader, signal or secretory sequence or a sequence which is employed for purification of the polypeptide sequence are fused to the mature polypeptide.
  • additional amino acids such as a leader, signal or secretory sequence or a sequence which is employed for purification of the polypeptide sequence are fused to the mature polypeptide.
  • the SLIRP variants may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.
  • the particular replacements may be determined by a skilled person as detailed more fully hereunder. Changes may be of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see for example the table hereunder).
  • Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • changes may be significant so as to disrupt binding with other molecules such as SRA.
  • Amino acids in the SLIRP polypeptides that are essential for function can be identified by methods known in the art, such as site directed mutagenesis or alanine-scanning mutagenesis. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as SRA binding. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization. Nuclear magnetic resonance or photoaffinity labelling may also be used when developing functional variants.
  • synthetic peptides corresponding to candidate functional or non-functional variants may be produced and their ability to display or show absence of one or more activities of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 assessed in vitro or in vivo.
  • SLIRP polypeptide variants can also be prepared as libraries using the sequence of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or other polypeptides herein. Phage display can also be effective in identifying useful SLIRP variants . Briefly, a phage library is prepared (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a biased degenerate array or may completely restrict the amino acids at one or more positions (e.g., for a library based on a protein from SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20).
  • phage-bearing inserts that have a relevant biological activity of the protein of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 such as SRA binding affinity or lack of, and/or regulation of nuclear receptor - 1 7 - signalling. This process can be repeated through several cycles of reselection of phage. Repeated rounds lead to enrichment of phage bearing particular sequences.
  • DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that confers the relevant activity can be determined.
  • One can repeat the procedure using a biased library containing inserts containing part or the entire minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof.
  • Polypeptides including variant polypeptides, can be tested for retention of any of the given activity.
  • the peptides can be tested for in vitro properties using transient transfection assays with a responsive reporter that assess the ability of the peptide to repress SRA-mediated coactivation to determine which of the variant peptides retain activity.
  • Preferred variant SLIRP polypeptides comprise an amino acid sequence that is at least 70-80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to a polypeptide sequence recited herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a polypeptide it is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular variant polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 herein can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 5371 1 ).
  • SLIRP polypeptides can be synthesized directly or obtained by chemical or mechanical disruption of larger molecules, fractioned and then tested for one or more activity of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • Functional or non-functional variants may also be produced by Northern blot analysis of total cellular RNA followed by cloning and sequencing of identified bands derived from different tissues/cells, or by PCR analysis of such RNA also followed by cloning and sequencing. Thus, synthesis or purification of an extremely large number of functional or non-functional variants is possible using the information contained herein.
  • SLIRP polypeptide variants also include fusion proteins, for example, where another peptide sequence is fused to the polypeptide of interest to aid in extraction and purification.
  • fusion protein partners include glutathione-S-transferase (GST), hexahistidine, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences.
  • the fusion protein will hinder SRA binding activity.
  • SLIRP polypeptides may also include conjugated proteins.
  • a protein may be modified by attachment of a moiety (e.g. a fluorescent, radioactive, or enzymatic label, or an unrelated sequence of amino acids to make a fusion protein) that does not correspond to a portion of the peptide in its native state.
  • SLIRP peptides may also comprise chimeric proteins comprising a fusion of an isolated peptide with another peptide.
  • a moiety fused to an isolated peptide or a fragment thereof also may provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labelling such as green fluorescent protein.
  • Purified isolated peptides include peptides isolated by methods including, but are not limited to, immunochromotography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
  • polypeptides herein can be conjugated by well-known methods, including bifunctional linkers, formation of a fusion polypeptide, and formation of biotin/streptavidin or biotin/avidin complexes by attaching either biotin or streptavid in/avid in to the peptide and the complementary molecule.
  • a conjugate can be formed by simultaneously or sequentially allowing the functional groups of the above-described components to react with one another.
  • Numerous art-recognized methods for forming a covalent linkage can be used. See, e.g., March, J., Advanced Organic Chemistry, 4th Ed., New York, N.Y., Wiley and Sons, 1985), pp.326-1 120.
  • conjugated SLIRP peptides may be prepared by using well-known methods for forming amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective conjugated peptide components.
  • reactive functional groups that are present in the amino acid side chains of the peptide preferably are protected, to minimize unwanted side reactions prior to coupling the peptide to the derivatizing agent and/or to the extracellular agent.
  • protecting group refers to a molecule which is bound to a functional group and which may be selectively removed therefrom to expose the functional group in a reactive form.
  • the protecting groups are reversibly attached to the functional groups and can be removed therefrom using, for example, chemical or other cleavage methods.
  • the peptides described herein can be synthesized using commercially available side-chain-blocked amino acids (e.g., FMOC-derivatized amino acids from Advanced Chemtech Inc., Louisville, Ky.).
  • the peptide side chains can be reacted with protecting groups after peptide synthesis, but prior to the covalent coupling reaction.
  • conjugated peptides can be prepared in which the amino acid side chains do not participate to any significant extent in the coupling reaction of the peptide to the other agent, such as a cell-type-specific targeting agent.
  • a peptide does not have a free amino- or carboxyl-terminal functional group that can participate in a coupling reaction, such a group can be introduced, e.g., by introducing a cysteine (containing a reactive thiol group) into the peptide by synthesis or site directed mutagenesis.
  • Disulfide linkages can be formed between thiol groups in, for example, the peptide and the targeting compound.
  • covalent linkages can be formed using bifunctional cross linking agents, such as bismaleimidohexane (which contains thiol-reactive maleimide groups and which forms covalent bonds with free thiols). See also the Pierce Co. Immunotechnology Catalogue and Handbook Vol. 1 for a list of exemplary homo- and hetero-bifunctional cross linking agents, thiol-containing amines and other molecules with reactive groups.
  • the covalent bond between the peptides and its conjugate is selected to be sufficiently labile (e.g., to enzymatic cleavage) so that it is cleaved following transport to its target, thereby - 20 - releasing the free peptides at the target.
  • Biologically labile covalent linkages e.g., imino bonds, and "active" esters can be used to form prodrugs where the covalently coupled peptides are found to exhibit reduced activity in comparison to the activity of the peptides alone.
  • amino acids in the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 that are required for activity may be incorporated into larger peptides and still maintain their function.
  • amino acids required for SRA binding include at least amino acids 21 to 26 or 60 to 67 or are other contiguous sequences of between about 5 and 20 amino acids and more preferably between about 6 and 15 amino acids.
  • the isolated peptides are non-hydrolyzable in that the bonds linking the amino acids of the peptide are less readily hydrolyzed than peptide bonds formed between L-amino acids.
  • isolated peptides from a library of non-hydrolyzable peptides, such as peptides containing one or more D- amino acids or peptides containing one or more non-hydrolyzable peptide bonds linking amino acids.
  • peptides that are optimal for a preferred function in suitable assay systems and then modify such peptides as necessary to reduce the potential for hydrolysis by proteases.
  • peptides may be labelled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing peptides and proteolytic fragments.
  • potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of an isolated peptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds that are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide.
  • Non-hydrolyzable bonds include - psi[CH 2 NH] ⁇ reduced amide peptide bonds, -psi[COCH 2 ] ⁇ ketomethylene peptide bonds, -psi[CH(CN)NH] ⁇ (cyanomethylene)amino peptide bonds, -psi[CH 2 CH(OH)] ⁇ hydroxyethylene peptide bonds, -psi[CH 2 0] ⁇ peptide bonds, and -psi[CH 2 S]- thiomethylene peptide bonds.
  • various changes may be made including the addition of various side groups that affect the manner in which the peptide functions, or which favourably affect the manner in which the peptide functions for the purposes of the present invention.
  • Such changes may involve adding or subtracting charge groups, - 21 - substituting amino acids, adding lipophilic moieties that may or may not affect binding but that affect the overall charge characteristics of the molecule facilitating a specific outcome with a physiological benefit.
  • no more than routine experimentation is required to test whether the molecule functions according to the invention.
  • the peptides herein may also be linked to a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG). Replacement of naturally occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify peptides.
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • bifunctional crosslinkers such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-(2 pyridyldithio) propionamido] hexanoate, and sulfosuccinimidyl 6-[3- (2 pyridyldithio) propionamido]hexanoate.
  • Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide.
  • Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.
  • the active conformation of the peptide may be stabilized by a covalent modification, such as cyclization or by incorporation of gamma-lactam or other types of bridges.
  • side chains can be cyclized to the backbone to create a L-g am ma- lactam moiety on each side of the interaction site.
  • Cyclization also can be achieved, for example, by formation of cystine bridges, coupling of amino and carboxy terminal groups of respective terminal amino acids, or coupling of the amino group of a lysine residue or a related homolog with a carboxy group of aspartic acid, glutamic acid or a related homolog.
  • Coupling of the alpha-amino group of a polypeptide with the epsilon- amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken.
  • Another approach is to include a metal-ion complexing backbone in the peptide structure.
  • the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the coordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a coordination number of four to six.
  • the nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulphur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, - 22 - or carboxyl functionalities.
  • the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino.
  • the peptide construct can be either linear or cyclic, however a linear construct is typically preferred.
  • One example of a small linear peptide is Gly-Gly-Gly-Gly that has four nitrogens (an N 4 complexation system) in the backbone that can complex to a metal ion with a coordination number of four.
  • Each motif describes a finite set of amino acid sequences in which the residues at each (relative) position may be (a) restricted to a single residue, (b) allowed to vary amongst a restricted set of residues, or (c) allowed to vary amongst all possible residues.
  • a motif might specify that the residue at a first position may be any one of valine, leucine, isoleucine, methionine, or phenylalanine; that the residue at the second position must be histidine; that the residue at the third position may be any amino acid residue; that the residue at the fourth position may be any one of the residues valine, leucine, isoleucine, methionine, phenylalanine, tyrosine or tryptophan; that the residue at the fifth position must be lysine, and so on.
  • the motifs in SEQ ID No:2 at amino acids 21 -26 and 60-67 provide further assistance to those skilled in the art as search, evaluation, or design criteria for functional variants of the polypeptides disclosed herein.
  • the present invention also provides methods for identifying functional variants of an isolated polypeptide.
  • the methods include selecting an isolated peptide, such as the isolated peptide identified herein as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Then a first amino acid residue of the isolated peptide is mutated to prepare a variant peptide.
  • the amino acid residue can be selected and mutated as indicated by a computer model of peptide conformation.
  • Peptides bearing mutated residues that maintain a similar conformation e.g. secondary structure
  • variant peptides Any method for preparing variant peptides can be employed, such as synthesis of the variant peptide, recombinantly producing the variant peptide using a mutated nucleic acid molecule, and the like.
  • the properties of the variant peptide in relation to the isolated peptides described previously are then determined according to standard procedures as described herein. - 23 -
  • Variants of the isolated peptides prepared by any of the foregoing methods can be sequenced, if necessary, to determine the amino acid sequence and thus deduce the nucleotide sequence which encodes such variants.
  • the present invention also provides fragments of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 comprising at least about 10, 20, 30, 50 or 100 amino acid residues.
  • “about” includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either extreme or at both extremes.
  • about 40-90 amino acids in this context means a polypeptide fragment of 40 plus or minus several, a few, 5, 4, 3, 2 or 1 amino acid residues to 90 plus or minus several a few, 5, 4, 3, 2 or 1 amino acid residues.
  • Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes.
  • the fragments include at least one biological activity of the polypeptide from which they are fragmented, such as SRA binding affinity and/or ability to bind an antibody to the full polypeptide.
  • the fragment may not have SRA binding affinity.
  • Fragments or portions of SLIRP polypeptides may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
  • SLIRP fragments may comprise an epitope-bearing portion of a polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the epitope is an immunogenic or antigenic epitope of the polypeptide.
  • An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • fragments bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence check Z-1 of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e. immunogenic epitopes) nor to the amino or carboxyl terminals.
  • Antigenic epitope- - 24 - bearing peptides and polypeptides may be contiguous or conformational epitopes and are useful to raise antibodies, including monoclonal antibodies that bind specifically to a SLIRP polypeptide.
  • the epitope-bearing fragments of the invention may be produced by any conventional means apparent to those skilled in the art.
  • the present invention also includes non-peptide mimetics.
  • a wide variety of techniques may be used to elucidate the precise structure of a peptide. These techniques include amino acid sequencing, x-ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, computer-assisted molecular modelling, peptide mapping, and combinations thereof. Structural analysis of a peptide provides a large body of data that comprise the amino acid sequence of the peptide as well as the three-dimensional positioning of its atomic components. From this information, non-peptide peptidomimetics may be designed that have the required chemical functionalities for therapeutic activity but are more stable, for example less susceptible to biological degradation.
  • variants of the present invention also include mimetics.
  • Nonpeptide analogs of peptides such as those that provide a stabilized structure or lessened biodegradation, may be employed in the methods and uses of the present invention.
  • Peptide mimetic analogs can be prepared based on a selected peptide by replacement of one or more residues by nonpeptide moieties.
  • the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, conformation such as a conformation that is not able to bind SRA.
  • the present invention also provides for the use of a polypeptide described herein for designing a mimetic thereof such as a non-peptide peptidomimetic.
  • selective binding agent refers to a molecule which has specificity for the polypeptides described herein.
  • Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable selective binding agents may be prepared using methods known in the art.
  • An exemplary selective binding agent of the present invention is capable of binding a portion of the polypeptides thereby inhibiting or enhancing the binding of the polypeptides to other molecules.
  • a selective binding agent is capable of binding a portion of the SLIRP polypeptides thereby inhibiting the binding of the SLIRP polypeptides to SRA.
  • Selective binding agents such as antibodies and antibody fragments that bind polypeptides herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, may be - 25 - used in the methods and uses of the present invention.
  • the antibodies may be polyclonal including monospecific polyclonal, monoclonal (MAbs), recombinant, chimeric, humanized such as CDR-grafted, human, single chain, and/or bispecific, as well as fragments, variants or derivatives thereof.
  • Antibody fragments include those portions of the antibody that bind to an epitope on the polypeptide. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full- length antibodies.
  • Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
  • Polyclonal antibodies generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of the polypeptide and an adjuvant. It may be useful to conjugate the polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for antibody titre.
  • a carrier protein that is immunogenic in the species to be immunized
  • aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for antibody titre.
  • Monoclonal antibodies are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture.
  • suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et at., Nature, 256:495-497 (1975) and the human B-cell hybridoma method, Kozbor, J. Immunol., 133:3001 (1984);(1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987).
  • hybridoma cell lines that produce monoclonal antibodies reactive with polypeptides herein.
  • Monoclonal antibodies of the invention may be modified for use as therapeutics.
  • One embodiment is a "chimeric" antibody in which a portion of the heavy and/or light chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies, so long as they exhibit the desired biological activity.
  • a monoclonal antibody of the invention is a "humanized" antibody.
  • Methods for humanizing non-human antibodies are well known in the art.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using - 26 - methods described in the art (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting at least a portion of a rodent complementarity-determining region (CDR) for the corresponding regions of a human antibody.
  • CDR rodent complementarity-determining region
  • transgenic animals e.g., mice
  • a polypeptide antigen i.e., having at least 6 contiguous amino acids
  • transgenic animals are produced by incapacitating the endogenous loci encoding the heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof.
  • Partially modified animals that is, those having less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications.
  • these transgenic animals When administered an immunogen, these transgenic animals produce antibodies with human (rather than e.g., murine) amino acid sequences, including variable regions that are immunospecific for these antigens. See PCT application nos. PCT/US96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT application nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP 546073A1 . Human antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
  • human antibodies can be produced from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991 );(1991 ) and Marks et al., J. Mol. Biol., 222:581 (1991 )). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • PCT Application No. PCT/US98/17364 which describes the isolation of high affinity and functional agonistic antibodies.
  • Chimeric, CDR grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
  • the antibodies of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and - 27 - immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)) for the detection and quantitation of polypeptides.
  • the antibodies will bind polypeptides with an affinity that is appropriate for the assay method being employed.
  • antibodies may be labelled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 4 C, 32 P, 35 S, or 25 l; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase.
  • a labelled standard e.g., a polypeptide described herein or an immunologically reactive portion thereof
  • the amount of the candidate polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibody.
  • the antibodies typically are insolubilized before or after the competition, so that the standard and candidate polypeptide that are bound to the antibodies may conveniently be separated from the standard and candidate polypeptide which remain unbound.
  • Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and/or quantitated.
  • the test sample analyte
  • a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labelled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labelled with a detectable moiety (indirect sandwich assays).
  • sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.
  • the selective binding agents are also useful for in vivo imaging.
  • An antibody labelled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labelled antibody in the host is assayed.
  • the antibody may be labelled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • Selective binding agents which may be used in the methods and uses of the invention, including antibodies, may be used as therapeutics. These therapeutic agents are generally agonists or antagonists, in that they either enhance or reduce, respectively, at least one of the biological activities of a polypeptide herein, including SRA binding.
  • antagonist antibodies of the invention are antibodies or binding fragments thereof which are capable of specifically binding to a polypeptide herein and which are capable of inhibiting or eliminating the functional activity of the polypeptide in vivo or in vitro.
  • the selective binding agent e.g., an antagonist antibody will inhibit the functional activity of the polypeptide by at least about 50%, and preferably by at least about 80%.
  • the selective binding agent may be an antibody that is capable of interacting with SRA or some other binding partner (a ligand or receptor) of the polypeptides described herein thereby inhibiting or eliminating SRA binding activity in vitro or in vivo.
  • Selective binding agents including agonist and antagonist like antibodies, are identified by screening assays that are well known in the art.
  • the invention also relates to a kit comprising selective binding agents (such as antibodies) and other reagents useful for detecting the levels of the polypeptides described herein in biological samples.
  • selective binding agents such as antibodies
  • Such reagents may include, a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • the invention further provides a method for assessing the fertility of a male animal, comprising the steps:
  • the one or more cells may be isolated from any one of blood, sputum, or semen from the male animal. Moreover, the one or more cells may be quantified to assess the fertility. Sequencing using techniques known in the art may be employed to identify said nucleotide sequence.
  • the invention also provides a kit for assessing the fertility of a male animal, the kit using the preceding method and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
  • kit for assessing the fertility of a male animal the kit using the preceding method and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
  • kits for assessing the fertility of a male animal, the kit also using the preceding method and comprising at least one allele-specific oligonucleotide probe specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
  • the polypeptides of the present invention can be used to clone its binding partners, using an expression cloning strategy.
  • Radiolabeled ( 25 -lodine) polypeptide or affinity/activity-tagged polypeptide can be used in binding assays to identify a cell type or cell line or tissue that expresses the receptor(s).
  • RNA isolated from such cells or tissues can be converted to cDNA, cloned into a mammalian expression vector, and transfected into mammalian cells (such as COS or 293 cells) to create an expression library.
  • a radiolabeled SLIRP polypeptide can then be used as an affinity ligand to identify and isolate from this library the subset of cells which express the receptor(s).
  • DNA can then be isolated from these cells and transfected into mammalian cells to create a secondary expression library in which the fraction of cells expressing receptor(s) is many-fold higher than in the original library. This enrichment process can be repeated iteratively until a single recombinant clone containing the receptor is isolated. Isolation of the receptor(s) is useful for identifying or developing novel agonists and antagonists of the polypeptide signalling pathway.
  • Such agonists and antagonists include soluble polypeptide receptor(s), receptor antibodies, small molecules, proteins, peptides, carbohydrates, lipids, or antisense oligonucleotides, and they may be used for treating, preventing, or diagnosing one or more disease or disorder, such as sperm dysfunction or dysmotility.
  • antibodies may be used to detect polypeptides of the invention present in biological samples by a method that comprises:
  • Suitable samples include extracts of tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, prostate and bone tissues or from neoplastic growths derived from such tissues.
  • Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. - 30 -
  • One type of assay for identifying substances that bind to the polypeptides of the present invention involves contacting a SRA binding protein comprising a SLIRP polypeptide described herein, which is immobilised on a solid support, with a non-immobilised candidate substance determining whether and/or to what extent the SLIRP polypeptide and candidate substance bind to each other.
  • the candidate substance may be immobilised and the SLIRP polypeptide non-immobilised.
  • the SLIRP polypeptide is immobilised on beads such as agarose beads. Typically this is achieved by expressing the component as a GST- fusion protein in bacteria, yeast or higher eukaryotic cell lines and purifying the GST- fusion protein from crude cell extracts using glutathione-agarose beads.
  • binding of the candidate substance, which is not a GST-fusion protein, to the immobilised SLIRP polypeptide is determined in the absence of the SLIRP polypeptide. The binding of the candidate substance to the immobilised SLIRP polypeptide is then determined.
  • This type of assay is known in the art as a GST pulldown assay. Again, the candidate substance may be immobilised and the SLIRP polypeptide non-immobilised.
  • Binding of the SLIRP polypeptide to the candidate substance may be determined by a variety of methods well-known in the art.
  • the non-immobilised component may be labelled (with for example, a radioactive label, an epitope tag or an enzyme- antibody conjugate).
  • binding may be determined by immunological detection techniques.
  • the reaction mixture can be Western blotted and the blot probed with an antibody that detects the non-immobilised component. ELISA techniques may also be used.
  • Candidate substances are typically added to a final concentration of from 1 to 1000 nmol/ml, more preferably from 1 to 100 nmol/ml.
  • the final concentration used is typically from 100 to 500 ⁇ g ml, more preferably from 200 to 300 ng/ml.
  • Another type of in vitro assay involves determining whether a candidate substance modulates binding of a protein/agent known to interact with SLIRP, such as SRA.
  • SLIRP protein/agent known to interact with SLIRP
  • Such an assay typically comprises contacting SLIRP protein with the known interacting protein in the presence or absence of the candidate substance and determining if the candidate substance has an effect on SLIRP binding to the known interacting protein.
  • This - 31 - candidate may be used according to the present invention to competitively bind SLIRP thereby inhibiting SLIRP binding to SRA, or may be a modified form of the candidate where the candidate is modified according to any of the methods described herein.
  • Candidate substances may also be tested on whole cells for their effect on sperm cell function and/or motility.
  • the candidate substances Preferably have been identified by the above-described in vitro methods.
  • rapid throughput screens for substances capable of increasing sperm cell motility and/or function may be used as a preliminary screen and then used in the in vitro assay described above to confirm that the affect is on SLIRP.
  • the candidate substance i.e. the test compound
  • the cell may be transfected with a nucleic acid construct which directs expression of the polypeptide in the cell.
  • the expression of the polypeptide is under the control of a regulatable promoter.
  • an assay to determine the effect of a candidate substance identified by the method of the invention on sperm cell motility and/or function comprises administering the candidate substance to a cell and determining whether the substance affects either motility and/or function within the cell.
  • the concentration of candidate substances used will typically be such that the final concentration in the cells is similar to that described above for the in vitro assays.
  • the candidate substance is administered to the cell together with functional SLIRP.
  • a substance that enhances SLIRP may serve to increase sperm cell function and/or motility.
  • this invention is also particularly useful for screening compounds by using the SLIRP polypeptide or fragment thereof in any of a variety of drug screening techniques to identify candidate substances that enhance SLIRP function.
  • the SLIRP polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with a SLIRP polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the SLIRP polypeptide or fragment, or (ii) for the presence of a complex between the SLIRP polypeptide or fragment and a ligand, by methods well known in the art.
  • the SLIRP polypeptide or fragment is typically labelled.
  • Free SLIRP polypeptide or fragment is separated from that present in a protein protein, protein:RNA or protein:DNA complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to SLIRP or its interference with SLIRP:ligand binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the SLIRP polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with SLIRP polypeptide and washed. Bound SLIRP polypeptide is then detected by methods well known in the art.
  • Purified SLIRP can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • antibodies to the polypeptide can be used to immobilize the SLIRP polypeptide on the solid phase.
  • the present invention further provides a method for modulating the motility and/or function of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
  • the present invention further provides the preceding method wherein the polypeptide is encoded by a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • polynucleotide sequences are species specific and encode the polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, respectively.
  • SLIRP polynucleotides may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti- sense strand.
  • the human SLIRP gene also known as DC50; PD04872; C14orf156, is present on chromosome 14 (position 14q24.3). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl /SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
  • isolated SLIRP polynucleotide(s) means a polynucleotide, DNA or RNA, which has been removed from its native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules.
  • Isolated SLIRP polynucleotides further include such molecules produced synthetically.
  • SLIRP polynucleotides include those that comprise a nucleotide sequence different to those specifically described herein but which, due to the degeneracy of the genetic code, still encode the same polypeptide.
  • the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants of SLIRP polynucleotides such as SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
  • the present invention also provides fragments of SLIRP polynucleotides.
  • Preferred fragments comprise at least 10, 20, 30, 40, 50, 60 or 70 contiguous nucleotides.
  • Other preferred fragments encode SLIRP polynucleotides with at least one important property of the full length polypeptide or epitope bearing portions of the larger polypeptide. Methods for determining fragments would be readily apparent to one skilled in the art and are exemplified in more detail below.
  • SLIRP polynucleotides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the present invention may also use isolated polynucleotides that selectively hybridize with at least a portion of a SLIRP polynucleotide.
  • the term "selectively hybridize” excludes the occasional randomly hybridizing nucleic acids under at least moderate stringency conditions.
  • selectively hybridizing polynucleotides preferably hybridize under at least moderate stringency conditions and more preferably under high stringency conditions.
  • the hybridising polynucleotides may be used, for example, as probes or primers for detecting the presence of SLIRP polynucleotides encoding polypeptides in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 such as cDNA or mRNA.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55 'C, can be used, e.g., 5x SSC, 0.1 % SDS, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SSC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x (or less) SCC.
  • SSC Buffer Concentrate (20X) contains 3M sodium chloride and 0.3M sodium citrate (pH 7.0).
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating T m have been derived and are known to those skilled in the art.
  • a minimum length for a hybridizable nucleic acid is at - 35 - least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20, 30 or 40-70 nucleotides.
  • a polynucleotide which hybridizes only to a poly A sequence such as a 3' terminal poly(A) tail of a SLIRP polynucleotide), or to a complementary stretch of T (or U) residues, would not be included as a selectively hybridizable SLIRP polynucleotide, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • compositions including at least two nucleic acids that selectively hybridize with different regions of the target nucleic acid so as to amplify a desired region.
  • the target region can range between 70% complementary bases and full complementarity.
  • the selectively hybridisable SLIRP polynucleotides described herein or more particularly portions thereof can be used to detect SLIRP nucleic acid in samples by methods such as the polymerase chain reaction, ligase chain reaction, hybridization, and the like. Alternatively, these sequences can be utilized to produce an antigenic protein or protein portion, or an active protein or protein portion.
  • portions of the selectively hybridisable SLIRP polynucleotides described herein can be selected to selectively hybridize with homologous SLIRP polynucleotides in other organisms.
  • These selectively hybridisable polynucleotides can be used, for example, to simultaneously detect related sequences for cloning of homologues of SLIRP polynucleotides.
  • SLIRP polynucleotides that encode a SLIRP polypeptide include, but are not limited to, those encoding the amino acid sequences of the polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • SLIRP polynucleotides may comprise the coding sequence for the polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA - 36 - processing, including splicing and polyadenylation signals, for example ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • SLIRP polynucleotides also include those encoding a polypeptide, such as the entire protein, lacking the N terminal methionine.
  • SLIRP polynucleotides include those with a sequence encoding a SLIRP polypeptide fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein.
  • the "GFP” tag is another peptide useful for purification and a reporter of expression which corresponds to an epitope for a derived from the jellyfish Aequorea victoria.
  • the methods and uses of the present invention may use variants of SLIRP nucleic acid molecules which encode variants of SLIRP polypeptides. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Non-naturally occurring variants may be produced using mutagenesis techniques known to those in the art.
  • variants include those produced by nucleotide substitutions, deletions or additions that may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the encoded polypeptide. Also especially preferred in this regard are conservative substitutions.
  • the present invention may also use isolated SLIRP polynucleotides comprising a nucleotide sequence at least 60, 70, 80 or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence encoding the polypeptide having the complete amino acid sequence in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • a nucleotide sequence that is 95% identical to a reference sequence is identical to the reference sequence except that it may include - 37 - up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleotide sequence encoding a polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 5371 1 ). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981 ), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • nucleic acid molecules having a sequence at least 60, 70, 80, 90, 95, 96, 97, 98 or 99 percent identical to the nucleic acid sequence of the polypeptides in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 will encode a SLIRP polypeptide.
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having one or more properties of the full polypeptide such as SRA binding. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
  • the present invention also extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of amino acid sequences of SLIRP polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 at the translational level.
  • This approach utilises antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • the present invention further provides a method for modulating the motility and/or function of a sperm cell, wherein antisense nucleic acids or siRNAs are used to reduce or eliminate the expression of a SLIRP polynucleotide.
  • the present invention further provides a method for sterilising a male animal including the step of reducing levels or reducing activity of SLIRP polypeptide within the cell, for example, amongst others, using antisense nucleic acids or siRNAs to reduce or eliminate the expression of a SLIRP polynucleotide in the sperm cells of the male animal.
  • a method for sterilising a male animal including the step of reducing levels or reducing activity of SLIRP polypeptide within the cell, for example, amongst others, using antisense nucleic acids or siRNAs to reduce or eliminate the expression of a SLIRP polynucleotide in the sperm cells of the male animal.
  • expression of the SLIRP polynucleotide may be restored upon removal of, for example, the antisense nucleic acids, the sterilising of the male animal can be reversed.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule [See: Weintraub, (1990) Sci. Am., 262:40-46; Marcus-Sekura, (1988) Anal. Biochem., 172:289-295]. In the cell, they hybridise to that mRNA, forming a double-stranded molecule. The cell does not translate an mRNA complexed in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein.
  • Oligomers of about fifteen nucleotides and molecules that hybridise to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into target cells.
  • Antisense methods have been used to inhibit the expression of many genes in vitro [Hambor et at., (1988) J. Exp. Med., 168:1237-1245].
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns.
  • RNA molecules that recognise specific nucleotide sequences in an RNA molecule and cleave it [Cech, (1988) J. Am. Med. Assoc., 260:3030-3034]. Because they are sequence-specific, only mRNAs with particular sequences are inactivated. - 39 -
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead”-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • SLIRP polynucleotide sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave, mRNAs for amino acid sequences of SLIRP polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, thus inhibiting expression of the SLIRP polynucleotide sequences.
  • the SLIRP polynucleotides herein may be used to screen for mutations in a gene encoding a SLIRP polypeptide and/or to secure expression of SLIRP polypeptides described herein with SRA binding activity or another biological activity of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • SRA binding activity or another biological activity of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • mutations that result in the expression of SLIRP polypeptides described herein which prevent or alter SRA binding activity may also be screened for.
  • Such mutations may affect expression of a SLIRP polynucleotide and can have an effect on SLIRP polypeptide expression in sperm cells causing dysmotile or dysfunctional sperm.
  • use of SLIRP polynucleotides and identification of mutations in SLIRP polynucleotides may be used as a biomarker of dysmotile or dysfunctional sperm
  • a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • cDNA or genomic libraries of various types may be screened as natural sources of SLIRP polynucleotides, or such polynucleotides may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR.
  • the choice of cDNA libraries normally corresponds to a tissue source that is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences. - 40 -
  • nucleic acid sequences used in the methods and uses of the invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a polynucleotide sequence of interest.
  • Reagents useful in applying such techniques are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear, and a number of other sources.
  • the recombinant nucleic acid sequences used to produce SLIRP polypeptides may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
  • Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes.
  • the preparation of such probes and suitable hybridisation conditions are well known in the art.
  • nucleic acid molecules hybridisable to a DNA molecule of the invention include nucleic acid molecules hybridisable to a non-coding region of a nucleic acid encoding a polypeptide of the present invention, which non-coding region is selected from the group consisting of an intron, a 5 ' non-coding region, and a 3 ' non- coding region.
  • Polynucleotide polymorphisms associated with alleles of the SLIRP polynucleotides of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19 which predispose to certain disorders such as dysmotile or dysfunctional sperm can be detected by hybridisation with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under stringent to moderately stringent hybridisation and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, stringent conditions will be used. Hybridisation stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary.
  • Conditions which rule out - 41 - nonspecific/adventitious bindings, that is, which minimize noise. Since such indications identify neutral DNA polymorphisms as well as mutations, these indications need further analysis to demonstrate detection of a disorder susceptible allele.
  • Probes for the alleles may be derived from the sequences of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19 or its corresponding gene.
  • the probes may be of any suitable length, which span all or a portion of the gene or SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and which allow specific hybridisation to a region of interest. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridises to the target sequence with the requisite specificity.
  • the probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods.
  • techniques for preparing and labeling probes see, e.g. Sambrook et al., 1989 : "Molecular Cloning: a laboratory manual. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Coldspring Harbour Laboratory Press, Coldspring Harbour, NY or Ausubel et al., 1992 Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., guitarist, R.E., Moore, D.D., Seidman, J.G., Smith, J.G. and Struhl, K.
  • polypeptide degradation or turnover rate may be selected by using homologous polynucleotides.
  • polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code.
  • Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change ligand-binding affinities, interchain affinities, or the polypeptide degradation or turnover rate.
  • Probes comprising synthetic oligonucleotides or other SLIRP polynucleotides may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labelled by nick translation, Klenow fill-in reaction, or other methods known in the art.
  • Portions of the SLIRP polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1 .0 kb, from a polynucleotide sequence encoding a SLIRP polypeptide are preferred as probes.
  • the probes may also be used to determine whether mRNA - 42 - encoding the polypeptide is present in a cell or tissue and whether the genomic organisation of the genes locus is deleted or otherwise damaged.
  • the present invention provides one or more SLIRP polynucleotides or fragments thereof as described herein comprising mutations with respect to the wild type sequence.
  • the present invention provides a plurality of SLIRP polynucleotides or fragments thereof as described herein for use in screening the DNA of an individual for the presence of one or more mutations/polymorphisms. The plurality of sequences is conveniently provided immobilized to a solid substrate as is described below.
  • SLIRP polynucleotides of the invention including probes that may be used to detect both normal (wild type) and abnormal SLIRP sequences in nucleic acid samples, may be immobilized to a solid phase support.
  • the probes will typically form part of a library of DNA molecules that may be used to detect simultaneously a number of different genes in a given genome.
  • single-stranded molecules may be synthesised off the solid substrate and each pre-formed sequence applied to a discrete position on the solid substrate.
  • nucleic acids may be printed directly onto the substrate using robotic devices equipped with either pins or pizo electric devices. - 43 -
  • the library sequences are typically immobilised onto or in discrete regions of a solid substrate.
  • the substrate may be porous to allow immobilisation within the substrate or substantially non-porous, in which case the library sequences are typically immobilised on the surface of the substrate.
  • the solid substrate may be made of any material to which polypeptides can bind, either directly or indirectly.
  • suitable solid substrates include flat glass, silicon wafers, mica, ceramics and organic polymers such as plastics, including polystyrene and polymethacrylate. It may also be possible to use semi-permeable membranes such as nitrocellulose or nylon membranes, which are widely available.
  • the semi-permeable membranes may be mounted on a more robust solid surface such as glass.
  • the surfaces may optionally be coated with a layer of metal, such as gold, platinum or other transition metal.
  • a particular example of a suitable solid substrate is the commercially available BiaCoreTM chip (Pharmacia BiosensorsTM).
  • the solid substrate is generally a material having a rigid or semi-rigid surface.
  • at least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different polymers with, for example, raised regions or etched trenches.
  • the solid substrate is suitable for the high density application of DNA sequences in discrete areas of typically from 50 to 100 ⁇ , giving a density of 10000 to 40000 cm 2 .
  • the solid substrate is conveniently divided up into sections. This may be achieved by techniques such as photoetching, or by the application of hydrophobic inks, for example teflon-based inks (Cel-lineTM, USA).
  • Discrete positions, in which each different member of the library is located may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
  • Attachment of the nucleic acid sequences to the substrate may be by covalent or non- covalent means.
  • the nucleic acid sequences may be attached to the substrate via a layer of molecules to which the library sequences bind.
  • the nucleic acid sequences may be labelled with biotin and the substrate coated with avidin and/or streptavidin.
  • biotinylated nucleic acid sequences A convenient feature of using biotinylated nucleic acid sequences is that the efficiency of coupling to the solid substrate can be determined easily. Since the nucleic acid sequences may bind only poorly to some solid substrates, it is often necessary to provide a chemical interface between the solid substrate (such as in the case of glass) and the nucleic acid sequences. Examples of suitable chemical interfaces include hexaethylene glycol.
  • polylysine coated glass Another example is the use of polylysine coated glass, the polylysine then being chemically modified using standard procedures to - 44 - introduce an affinity ligand.
  • Other methods for attaching molecules to the surfaces of solid substrate by the use of coupling agents are known in the art, see for example W098/49557.
  • Binding of complementary nucleic acid sequence to the immobilised nucleic acid library may be determined by a variety of means such as changes in the optical characteristics of the bound nucleic acid (i.e. by the use of ethidium bromide) or by the use of labelled nucleic acids, such as polypeptides labelled with fluorophores.
  • Other detection techniques that do not require the use of labels include optical techniques such as optoacoustics, reflectometry, ellipsometry and surface plasmon resonance (SPR) - see W097/49989, incorporated herein by reference.
  • the present invention provides a solid substrate having immobilized thereon at least one SLIRP polynucleotide, preferably two or more different SLIRP polynucleotides, for example two or more different polynucleotides corresponding to different alleles.
  • the solid substrate further comprises polynucleotides derived from genes other than the SLIRP gene.
  • High throughput expression profiling has a broad range of applications with respect to SLIRP polypeptides, including, but not limited to: the identification and validation of disease-related genes as targets for therapeutics; molecular toxicology of polypeptides of the invention and inhibitors thereof; stratification of populations and generation of surrogate markers for clinical trials; and enhancing polypeptide related small molecule drug discovery by aiding in the identification of selective compounds in high throughput screens (HTS), particularly with respect to disorders affecting the function and activity of sperm affecting fertility.
  • HTS high throughput screens
  • the step of increasing SLIRP polypeptide levels within the sperm or other cell may be accomplished by plasmid mediated transfection of the cell with vectors including SLIRP polynucleotides that are able to express SLIRP polypeptides within the cell.
  • Plasmid mediated transfection of target cells has been used to increase wild type and amino and carboxy terminal tagged SLIRP over expression. This has been achieved using CMV promoter driven vectors eg pFLAG-CMVTM-5a (carboxy tag), p3xFLAG- CMVTM-7.1 (amino tag), both from Sigma-AldrichTM. Also pcDNA4/TO derivative vectors from InvitrogenTM to express carboxy terminal tagged SLIRP.
  • the step of decreasing SLIRP polypeptide levels within the cell may be accomplished using siRNA technologies to deplete expression of SLIRP polypeptides of the invention from the cell. - 45 -
  • SLIRP levels have been depleted using siRNA technologies as described in Hatchell et al., 2006 using reagents supplied by Dharmacon, Inc.TM, specific for the SLIRP gene.
  • a nucleic acid molecule encoding the amino acid sequence of a SLIRP polypeptide may be inserted into an appropriate expression vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • a nucleic acid molecule encoding the amino acid sequence of polypeptide herein may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems), and/or eukaryotic host cells. Selection of the host cell will depend in part on whether the polypeptide is to be post-translationally modified (e.g., glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable.
  • expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences collectively referred to as "flanking sequences" in certain embodiments, will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secret
  • the vector may contain a "tag"-encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another "tag” such as FLAG, HA (hemaglutinin influenza virus) or myc for which commercially available antibodies exist.
  • This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix.
  • the tag can subsequently be removed from the purified polypeptide by various means such as using certain peptidases for cleavage.
  • Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source) or synthetic, or the flanking sequences may be native sequences that normally function to - 46 - regulate polypeptide expression.
  • the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.
  • flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein other than the gene flanking sequences will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
  • flanking sequence may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or flanking sequence fragments from the same or another species.
  • flanking sequence may be not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen ® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for the optimal expression of the polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (Product No.
  • 303-3s New England Biolabs, Beverly, MA
  • various origins e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such as HPV or BPV
  • VSV vesicular stomatitus virus
  • HPV vesicular stomatitus virus
  • BPV papillomaviruses
  • the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).
  • a transcription termination sequence is typically located 3' of the end of a polypeptide coding region and serves to terminate transcription.
  • a transcription termination - 47 - sequence in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.
  • selection genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells.
  • suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase.
  • Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to the amplification of both the selection gene and the DNA that encodes a polypeptide described herein. As a result, increased quantities of the polypeptide are synthesized from the amplified DNA.
  • a ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth herein and used in a prokaryotic vector.
  • a leader, or signal, sequence may be used to direct the polypeptide out of the host cell.
  • a nucleotide sequence encoding the signal sequence is positioned in the coding region of the nucleic acid molecule encoding the polypeptide, or directly at the 5' end of the polypeptide coding region.
  • Many signal sequences have been identified, and any of those that are functional in the selected host cell may be used in conjunction with - 48 - the nucleic acid molecule. Therefore, a signal sequence may be homologous (naturally occurring) or heterologous to the gene or cDNA encoding the polypeptide. Additionally, a signal sequence may be chemically synthesized using methods described herein.
  • the signal sequence may be a component of the vector, or it may be a part of the nucleic acid molecule that is inserted into the vector.
  • nucleotide sequence encoding a native signal sequence joined to a polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence joined to a polypeptide coding region.
  • the heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • the native polypeptide signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders.
  • the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incidental to expression, which may not have been totally removed.
  • the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the N- terminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.
  • transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is particularly true where a polypeptide is produced in eukaryotic host cells, especially mammalian host cells.
  • the introns used may be naturally occurring within the gene, especially where the gene used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the gene (as for most cDNAs), the intron(s) may be obtained from - 49 - another source.
  • the position of the intron with respect to flanking sequences and the gene is generally important, as the intron must be transcribed to be effective.
  • the preferred position for the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence.
  • the intron or introns will be located on one side or the other (i.e., 5' or 3') of the cDNA such that it does not interrupt the coding sequence.
  • Any intron from any source including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell(s) into which it is inserted.
  • synthetic introns may be used to practice this invention, provided that it is compatible with the host cell(s) into which it is inserted.
  • synthetic introns may be used in the vector.
  • the expression and cloning vectors that may be used in the methods and uses of the present invention will each typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the polypeptide. Promoters are untranscribed sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes, inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
  • Constitutive promoters initiate continual gene product production; that is, there is little or no control over gene expression.
  • a large number of promoters, recognized by a variety of potential host cells, are well known.
  • a suitable promoter is operably linked to the DNA encoding the polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.
  • the native gene promoter sequence may be used to direct amplification and/or expression of a nucleic acid molecule.
  • a heterologous promoter is preferred, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter, and if it is compatible with the host cell system that has been selected for use.
  • Promoters suitable for use with prokaryotic hosts include the beta- lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence(s), using linkers or adapters as needed to supply any useful restriction sites. - 50 -
  • Suitable promoters for use with yeast hosts are also well known in the art.
  • Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • Additional promoters which may be of interest in controlling gene transcription include, but are not limited to: the SV40 early promoter region; the CMV promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus; the herpes thymidine kinase promoter, the regulatory sequences of the metallothionine gene, prokaryotic expression vectors such as the beta-lactamase promoter; or the tac promoter.
  • the elastase I gene control region which is active in pancreatic acinar cells; the insulin gene control region which is active in pancreatic beta cells; the immunoglobulin gene control region which is active in lymphoid cells; the mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells; the albumin gene control region which is active in liver; the alphafetoprotein gene control region which is active in liver; the alpha 1 -antitrypsin gene control region which is active in the liver; the beta-globin gene control region which is active in myeloid cells; the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain; the myosin light chain-2 gene control region which is active in skeletal muscle; and the gonadotropic releasing hormone gene control region which is active in the hypothalamus.
  • Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto- protein and insulin). Typically, however, an enhancer from a virus will be used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically located at a site 5' from the promoter. - 51 -
  • Expression vectors may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the desired flanking sequences are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
  • Preferred vectors for practicing this invention are those that are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, Carlsbad, CA), pBSII (Stratagene Company, La Jolla, CA), pET15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacll; Invitrogen), pDSR-alpha (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco/BRL, Grand Island, NY).
  • vectors include, but are not limited to, cosmids, plasmids or modified viruses, but it will be appreciated that the vector system must be compatible with the selected host cell.
  • vectors include, but are not limited to, plasmids such as
  • Bluescript plasmid derivatives (a high copy number ColE1 -based phagemid, Stratagene Cloning Systems Inc., La Jolla CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products ⁇ e.g., TOPOTM TA Cloning ® Kit, pCR2.1 ® plasmid derivatives, Invitrogen, Carlsbad, CA), and mammalian, yeast, or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA).
  • Further suitable vectors include inducible vectors, for example, amongst others, heavy metal, tetracycline or estrogen inducible vectors. [SC: please include specific examples you have used or could be used inc company info]
  • the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector for a polypeptide into a selected host cell may be accomplished by well known methods including transfection, infection, calcium chloride, calcium phosphate, electroporation, microinjection, lipofection or the DEAE- dextran method or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et at., supra. - 52 -
  • Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell).
  • the host cell when cultured under appropriate conditions, synthesizes the polypeptide that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity, such activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.
  • a number of suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10-2209. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61 ); CHO DHFR-cells (Uriaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)); human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573); or 3T3 cells (ATCC No. CCL92).
  • CHO Chinese hamster ovary cells
  • CHO DHFR-cells Uriaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)
  • human embryonic kidney (HEK) 293 or 293T cells ATCC No. CRL1573)
  • suitable mammalian host cells are the monkey COS-1 (ATCC No. CRL1650) and COS-7cell lines (ATCC No. CRL1651 ) cell lines, and the CV-1 cell line (ATCC No. CCL70).
  • suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7cell lines (ATCC No. CRL1651 ) cell lines, and the CV-1 cell line (ATCC No. CCL70).
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which are available from the ATCC. Each of these cell lines is known by and available to those skilled in the art of protein expression.
  • E. coli e.g., HB101 , (ATCC No. 33694) DH5a, DH10, and MC1061 (ATCC No. 53338)
  • HB101 ATCC No. 33694
  • DH5a DH5a
  • DH10 DH10
  • MC1061 ATCC No. 533378
  • B. subtilis Pseudomonas spp.
  • B. subtilis Pseudomonas spp.
  • Streptomyces spp. and the like may also be employed in this method.
  • yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris. - 53 -
  • insect cell systems may be utilized in the methods of the present invention. Such systems are described for example in Kitts et at., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. ⁇ J. Virol., 67:4566-4579 (1993).
  • Preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).
  • transgenic animals to express glycosylated SLIRP polypeptides or variants thereof.
  • a transgenic milk-producing animal a cow or goat, for example
  • plants to produce polypeptides.
  • the glycosylation occurring in plants is different from that produced in mammalian cells, and may result in a glycosylated product which is not suitable for human therapeutic use.
  • compositions are within the scope of the methods and uses of the present invention. Such compositions may comprise a therapeutically effective amount of a SLIRP polypeptide or SLIRP polynucleotide including those described herein in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Pharmaceutical compositions may also comprise a therapeutically effective amount of one or more selective binding agents described herein in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Benefits of methods using such pharmaceutical compositions described herein may include increasing SLIRP levels within a sperm cell in able to restore function and motility to the sperm cell.
  • the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colouring, - 54 - flavoring and diluting agents; e
  • the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the active agent.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution, artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
  • compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution.
  • the polypeptide product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions can be capable of parenteral delivery.
  • compositions may be capable of inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. - 55 -
  • the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired polypeptide or nucleotide in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which the active agent is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid, acid or polyglycolic acid), or beads or liposomes, that provides for the controlled or sustained release of the product which may then be delivered as a depot injection.
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition may be formulated for inhalation.
  • a polypeptide or nucleotide may be formulated as a dry powder for inhalation.
  • the polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. It is also contemplated that certain formulations may be administered orally.
  • SLIRP polypeptides or SLIRP nucleotides that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the active agent. Diluents, flavourings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • Another pharmaceutical composition may involve an effective quantity of the polypeptide or nucleotide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate
  • binding agents such as starch, gelatin, or acacia
  • lubricating agents such as magnesium stearate, stearic acid, or talc.
  • sustained-sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using these methods may be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration may be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
  • the effective amount of the active agent in the pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the active agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titre the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • a typical dosage may range from about 0.1 ⁇ g kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 ⁇ g kg up to about 100 mg/kg; or 1 ⁇ g kg up to about 100 mg/kg; or 5 ⁇ g kg up to about 100 mg/kg. - 57 -
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the active agent and the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implants.
  • the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition may be administered locally via implantation of a membrane, sponge or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge or another appropriate material on to which the desired molecule has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, such as the scrotum, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • compositions herein in an ex vivo manner.
  • cells, tissues, or organs such as the testis that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.
  • An active agent herein such as a polypeptide or selective binding agent can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide or selective binding agent.
  • Such cells may be animal or human cells, and may be autologous, heterologous, or xenogenic.
  • the cells may be immortalized.
  • the cells may be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semipermeable polymeric enclosures or membranes that allow the release of the protein - 58 - product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • Additional embodiments of the methods and uses of the present invention relate to cells and methods (e.g., homologous recombination and/or other recombinant production methods) for both the in vitro production of therapeutic polypeptides and for the production and delivery of therapeutic polypeptides by gene therapy or cell therapy.
  • Homologous and other recombination methods may be used to modify a cell that contains a normally transcriptionally silent gene encoding a polypeptide described herein, or an under expressed gene, and thereby produce a cell which expresses therapeutically efficacious amounts of the polypeptides.
  • This may assist to restore function and/or motility to sperm cell which are, for example, amongst others, affected by low levels or mutated SLIRP polypeptides.
  • Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes.
  • the basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome or to correct specific mutations within defective genes.
  • a given DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to targeting DNA.
  • the targeting DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process.
  • a promoter/enhancer element, a suppresser or an exogenous transcription modulatory element is inserted in the genome of the intended host cell in proximity and orientation sufficient to influence the transcription of DNA encoding the desired polypeptide.
  • the control element controls a portion of the DNA present in the host cell genome.
  • DNA that encodes the polypeptide itself but rather by the use of targeting DNA (containing regions of homology with the endogenous gene of interest), coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals for transcription of the gene encoding the polypeptide.
  • the expression of a desired targeted gene in a cell is altered via homologous recombination into the cellular genome at a preselected site, by the introduction of DNA that includes at least a regulatory sequence, an exon and a splice donor site.
  • DNA that includes at least a regulatory sequence, an exon and a splice donor site.
  • These components are introduced into the chromosomal (genomic) DNA in such a manner that this, in effect, results in the production of a new transcription unit (in which the regulatory sequence, the exon and the splice donor site present in the DNA construct are operatively linked to the endogenous gene).
  • the expression of the desired endogenous gene is altered.
  • Altered gene expression encompasses activating (or causing to be expressed) a gene which is normally silent (unexpressed) in the cell as obtained, as well as increasing the expression of a gene which is not expressed at physiologically significant levels in the cell as obtained.
  • the embodiments further encompass changing the pattern of regulation or induction such that it is different from the pattern of regulation or induction that occurs in the cell as obtained, and reducing (including eliminating) the expression of a gene which is expressed in the cell as obtained.
  • homologous recombination can be used to increase, or cause production of a SLIRP polypeptide described herein from a cell's endogenous gene involves first using homologous recombination to place a recombination sequence from a site-specific recombination system (e.g., Cre/loxP, FLP/FRT) (see, Sauer, Current Opinion In Biotechnology, 5:521 -527, 1994; and Sauer, Methods In Enzymology, 225:890-900, 1993) upstream (that is, 5' to) of the cell's endogenous genomic polypeptide coding region.
  • a site-specific recombination system e.g., Cre/loxP, FLP/FRT
  • a plasmid containing a recombination site homologous to the site that was placed just upstream of the genomic polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme.
  • This recombinase enzyme causes the plasmid to integrate, via the plasmid's recombination site, into the recombination site located just upstream of the genomic polypeptide coding region in the cell line (Baubonis and Sauer, Nucleic Acids Res., 21 :2025-2029, 1993; and O'Gorman et al., Science, 251 : 1351 -1355, 1991 ).
  • flanking sequences known to increase transcription e.g., enhancer/promoter, intron or translational enhancer
  • if properly positioned in this plasmid would integrate in such a - 60 - manner as to create a new or modified transcriptional unit resulting in de novo or increased polypeptide production from the cell's endogenous gene.
  • a further method to use the cell line in which the site-specific recombination sequence has been placed just upstream of the cell's endogenous genomic polypeptide coding region is to use homologous recombination to introduce a second recombination site elsewhere in the cell line's genome.
  • the appropriate recombinase enzyme is then introduced into the two-recombination-site cell line, causing a recombination event (deletion, inversion or translocation) (Sauer, Current Opinion In Biotechnology, supra, 1994 and Sauer, Methods In Enzymology, supra, 1993) that would create a new or modified transcriptional unit resulting in de novo or increased polypeptide production from the cell's endogenous gene.
  • Another approach for increasing, or causing, the expression of the polypeptide from a cell's endogenous gene involves increasing, or causing, the expression of a gene or genes (e.g., transcription factors) and/or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner which results in de novo or increased polypeptide production from the cell's endogenous gene.
  • This method includes the introduction of a non-naturally occurring polypeptide (e.g., a polypeptide comprising a site-specific DNA binding domain fused to a transcriptional factor domain) into the cell such that de novo or increased polypeptide production from the cell's endogenous gene results.
  • the present invention may further employ DNA constructs useful in the method of altering expression of a target gene.
  • the exemplary DNA constructs comprise: (a) one or more targeting sequences; (b) a regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site.
  • the targeting sequence in the DNA construct directs the integration of elements (a)-(d) into a target gene in a cell such that the elements (b)-(d) are operatively linked to sequences of the endogenous target gene.
  • the DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)-(f) such that the elements of (b)-(f) are operatively linked to the endogenous gene.
  • the targeting sequence is homologous to the preselected site in the cellular chromosomal DNA with which homologous recombination is to occur.
  • the exon is generally 3' of the regulatory sequence and the splice-donor site is 3' of the exon.
  • sequence of a particular gene is known, such as the nucleic acid sequence of the polypeptides presented herein
  • a piece of DNA that is complementary to a selected - 61 - region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest.
  • This piece serves as a targeting sequence(s) upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be incorporated into the newly synthesized daughter strand of DNA.
  • the methods and use of the present invention therefore, includes nucleotides encoding a polypeptide, which nucleotides may be used as targeting sequences.
  • Polypeptide cell therapy e.g., the implantation of cells producing polypeptides described herein, is also contemplated.
  • This embodiment involves implanting cells capable of synthesizing and secreting a biologically active form of the polypeptide.
  • Such polypeptide-producing cells can be cells that are natural producers of the polypeptides or may be recombinant cells whose ability to produce the polypeptides has been augmented by transformation with a gene encoding the desired polypeptide or with a gene augmenting the expression of the polypeptide.
  • Such a modification may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion.
  • the natural cells producing polypeptide be of human origin and produce human polypeptide.
  • the recombinant cells producing polypeptide be transformed with an expression vector containing a gene encoding a human polypeptide.
  • Implanted cells may be encapsulated to avoid the infiltration of surrounding tissue.
  • Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or in membranes that allow the release of polypeptide, but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue.
  • the patient's own cells, transformed to produce polypeptides ex vivo may be implanted directly into the patient without such encapsulation.
  • PCT Application no. PCT/US91/00157 of Aebischer et al. See also, PCT Application no. PCT/US91/00155 of Aebischer et al..; Winn et al., Exper. Neurol., 1 13:322-329 (1991 ), Aebischer et al., Exper. Neurol., rM :269-275 (1991 ); and Tresco et al., ASAIO, 38:17-23 (1992).
  • SLIRP polypeptides are also part of the methods and use of the present invention.
  • One example of a gene therapy technique is to use the gene (either genomic DNA, cDNA, and/or synthetic DNA) encoding a SLIRP polypeptide described herein that may be operably linked to a constitutive or inducible promoter to form a "gene therapy DNA construct".
  • the promoter may be homologous or heterologous to the endogenous gene, provided that it is active in the cell or tissue type into which the construct will be inserted.
  • DNA molecules designed for site-specific integration e.g., endogenous sequences useful for homologous recombination
  • tissue-specific promoter, enhancer(s) or silencer(s) DNA molecules capable of providing a selective advantage over the parent cell
  • DNA molecules useful as labels to identify transformed cells negative selection systems, cell specific systems
  • cell-specific binding agents as, for example, for cell targeting
  • cell-specific internalization factors cell-specific internalization factors
  • transcription factors to enhance expression by a vector, as well as factors to enable vector manufacture e.g., endogenous sequences useful for homologous recombination
  • tissue-specific promoter, enhancer(s) or silencer(s) DNA molecules capable of providing a selective advantage over the parent cell
  • DNA molecules useful as labels to identify transformed cells negative selection systems, cell specific systems
  • cell-specific binding agents as, for example, for cell targeting
  • cell-specific internalization factors e.g., cell-specific internalization factors
  • transcription factors to enhance expression by a vector, as well as factors to enable vector manufacture
  • a gene therapy DNA construct can then be introduced into cells (either ex vivo or in vivo) using viral or non-viral vectors.
  • Certain vectors such as retroviral vectors, will deliver the DNA construct to the chromosomal DNA of the cells, and the gene can integrate into the chromosomal DNA.
  • Other vectors will function as episomes, and the gene therapy DNA construct will remain in the cytoplasm.
  • regulatory elements can be included for the controlled expression of the gene in the target cell. Such elements are turned on in response to an appropriate effector. In this way, a therapeutic polypeptide can be expressed when desired.
  • One conventional control means involves the use of small molecule dimerizers or rapalogs (as described in WO 9641865 (PCT/US96/099486); WO 9731898 (PCT/US97/03137) and W09731899 (PCT/US95/03157) used to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating biological process, such as a DNA-binding protein or a transcriptional activation - 63 - protein. The dimerization of the proteins can be used to initiate transcription of the transgene.
  • An alternative regulation technology uses a method of storing proteins expressed from the gene of interest inside the cell as an aggregate or cluster.
  • the gene of interest is expressed as a fusion protein that includes a conditional aggregation domain that results in the retention of the aggregated protein in the endoplasmic reticulum.
  • the stored proteins are stable and inactive inside the cell.
  • the proteins can be released, however, by administering a drug (e.g., small molecule ligand) that removes the conditional aggregation domain and thereby specifically breaks apart the aggregates or clusters so that the proteins may be secreted from the cell.
  • a drug e.g., small molecule ligand
  • Another control means uses a positive tetracycline-controllable transactivator.
  • This system involves a mutated tet repressor protein DNA-binding domain (mutated tet R- 4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline) linked to a polypeptide that activates transcription.
  • In vivo gene therapy may be accomplished by introducing the gene encoding a polypeptide into cells via local injection of a nucleic acid molecule or by other appropriate viral or non-viral delivery vectors.
  • a nucleic acid molecule encoding a SLIRP polypeptide may be contained in an adeno-associated virus (AAV) vector for delivery to the targeted cells (e.g., Johnson, International Publication No. WO95/34670; and International Application No. PCT/US95/07178).
  • AAV adeno-associated virus
  • the recombinant AAV genome typically contains AAV inverted terminal repeats flanking a DNA sequence encoding a polypeptide operably linked to functional promoter and polyadenylation sequences.
  • Alternative suitable viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma virus vectors.
  • U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • U.S. Patent No. 5,399,346 provides examples of a process for providing a patient with a therapeutic protein by the delivery of human cells that have been treated in vitro to insert a DNA segment encoding a therapeutic protein.
  • Nonviral delivery methods include, but are not limited to, liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene gun).
  • Gene therapy materials and methods may also include the use of inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site- specific integration, DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture.
  • inducible promoters tissue-specific enhancer-promoters
  • DNA sequences designed for site- specific integration DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and
  • Patent No. 4,970,154 involving electroporation techniques; WO96/40958 involving nuclear ligands; U.S. Patent No. 5,679,559 describing a lipoprotein-containing system for gene delivery; U.S. Patent No. 5,676,954 involving liposome carriers; U.S. Patent No. 5,593,875 concerning methods for calcium phosphate transfection; and U.S. Patent No. 4,945,050 wherein biologically active particles are propelled at cells at a speed whereby the particles penetrate the surface of the cells and become incorporated into the interior of the cells.
  • gene therapy or cell therapy can further include the delivery of one or more additional SLIRP polypeptide(s) in the same or a different cell(s), particularly sperm cells.
  • additional SLIRP polypeptide(s) in the same or a different cell(s), particularly sperm cells.
  • Such cells may be separately introduced into the patient, or the cells may be contained in a single implantable device, such as the encapsulating membrane described above, or the cells may be separately modified by means of viral vectors.
  • a means to increase endogenous polypeptide expression in a cell via gene therapy is to insert one or more enhancer element into the polypeptide promoter, where the enhancer element(s) can serve to increase transcriptional activity of the gene.
  • the enhancer element(s) used will be selected based on the tissue in which one desires to activate the gene(s); enhancer elements known to confer promoter activation in that tissue will be selected.
  • the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing the polypeptide promoter (and optionally, inserted into a vector and/or 5' and/or 3' flanking sequence(s), etc.) using standard cloning techniques.
  • This construct known as a "homologous recombination construct" can then be introduced into the desired cells either ex vivo or in vivo.
  • Gene therapy also can be used to decrease polypeptide expression by modifying the nucleotide sequence of the endogenous promoter(s). Such modification is typically - 65 - accomplished via homologous recombination methods.
  • a DNA molecule containing all or a portion of the promoter of the gene selected for inactivation can be engineered to remove and/or replace pieces of the promoter that regulate transcription.
  • the TATA box and/or the binding site of a transcriptional activator of the promoter may be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity thereby repressing the transcription of the corresponding gene.
  • the deletion of the TATA box or the transcription activator binding site in the promoter may be accomplished by generating a DNA construct comprising all or the relevant portion of the polypeptide promoter(s) (from the same or a related species as the polypeptide gene to be regulated) in which one or more of the TATA box and/or transcriptional activator binding site nucleotides are mutated via substitution, deletion and/or insertion of one or more nucleotides.
  • the TATA box and/or activator binding site has decreased activity or is rendered completely inactive.
  • the construct will typically contain at least about 500 bases of DNA that correspond to the native (endogenous) 5' and 3' DNA sequences adjacent to the promoter segment that has been modified.
  • the construct may be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described herein.
  • the integration of the construct into the genomic DNA of the cells will be via homologous recombination, where the 5' and 3' DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.
  • the identification and characterisation of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 as a corepressor of SRA mediated nuclear receptor coactivation and the recognition of the role of SLIRP in sperm motility and function means that there are a range of diseases and disorders, including those associated with fertility that may be treated using therapies based on the polypeptides described herein. This includes either reducing SLIRP levels or using a SLIRP antagonist to reduce fertility and function as a contraceptive, as well as increasing SLIRP levels or using an agonist to enhance fertility and the chances of successful conception.
  • the present invention provides a method for treating a disorder associated with an undesirable level of function and/or motility of sperm cells, the method comprising the step of administering an effective amount of an isolated polypeptide comprising:
  • Therapeutic methods in accordance with the present invention will vary depending on whether the intention is to increase, decrease or remove the amount and/or physiological effects of the SLIRP polypeptides described herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • SLIRP polypeptides described herein such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • Methods that may be used to increase, decrease or remove the effects of the polypeptide as deemed appropriate by a clinician with a view to achieving a therapeutic outcome.
  • the present invention also provides a method of treating a subject suffering from a disorder associated with undesirable physiological levels of a SLIRP polypeptide including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 comprising the step of manipulating the physiological levels of the polypeptide.
  • This manipulation may be intended to affect the activity of downstream signalling pathways and thereby the function and/or motility within sperm cells of the subject being treated.
  • the physiological levels of the polypeptide can be increased or decreased as required to treat particular disorders. These increases or decreases can be achieved using the polypeptides, polynucleotides and/or selective binding agents described herein. These agents are capable of increasing or decreasing the endogenous production of the polypeptide or can be administered directly to increase or decrease the physiological levels of the polypeptide using the methods described herein.
  • selective binding agents such as antibodies could be administered to decrease the physiological levels of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 by binding the polypeptide to prevent binding with SRA.
  • SLIRP polypeptides, polynucleotides or binding agents of the present invention may be preferable to administer the SLIRP polypeptides, polynucleotides or binding agents of the present invention in combination with other therapeutic agents that are useful for treating a given disease or disorder.
  • Such combinations could use conjugates comprising the polypeptide or the therapy could be concomitant or involve the sequential administration of the agents.
  • chemotherapeutic agents and/or radiotherapy may be administered in combination with polypeptides, polynucleotides or binding agents of the present invention.
  • the polypeptides and/or polynucleotides herein may be used in combination with one or more agents that are adapted to interfere with the natural action of androgens.
  • agents that are adapted to interfere with the natural action of androgens.
  • These - 67 - agents include the androgen receptor antagonists such as flutamide and bicalutamide.
  • Other combination partners include agents that block other receptors on cancer cells that may be responsible for cell proliferation e.g. agents that block erbB-2 or EGF- receptor.
  • Estrogen antagonists are known to those skilled in the art and may be selected from the group consisting of: SERMs and ICI 182,780 (also Faslodex, AstraZeneca).
  • Other inhibitors of estrogen production include aromatase inhibitors (e.g. Letrozole). These may also represent good combination partners for therapy according to the present invention.
  • SLIRP based therapies include inhibitors of the tyrosine kinase signalling pathway (EGF-receptor monoclonal antibodies) or small molecule inhibitors such as Iressa ® .
  • Inhibitors of the erbB-2 pathway may also be used (e.g. Herceptin ® )
  • the effect of the administered therapeutic composition can be monitored by standard diagnostic procedures.
  • the patient outcomes from the administration of a therapeutic composition can be monitored by assessing any one or more of the recognised markers for disease progression.
  • polypeptides may be administered as a therapeutic or a prophylactic depending on the particular circumstances and as deemed appropriate by a medical practitioner.
  • fertility treatment for a male subject may comprise enhancing the expression of SLIRP polypeptides in sperm cells of the subject ex vivo. This could be achieved by transiently expressing SLIRP in sperm obtained from infertile men prior to its use in a range of in vitro fertilization techniques (eg ICSI, intracytoplasmic sperm injection).
  • in vitro fertilization techniques eg ICSI, intracytoplasmic sperm injection.
  • the present invention also provides the use of a polypeptide as a biomarker for identification of dysmotile and/or dysfunctional sperm, the polypeptide being any one or more selected from the group consisting of:
  • the present invention provides the use of a polypeptide as a biomarker for identification of energy production in a sperm cell, the polypeptide being any one or more selected from the group consisting of:
  • Microsatellite marker information (against which primers can be developed and comparative analysis can occur) can be obtained from the Genethon Genetic Linkage Map (http://www.genethon.fr) and the Johns Hopkins University Biolnformatics Genome Database (http://www.bis.med.jhmi.edu/). Their relative order can be determined via The Center of Medical Genetics Database
  • the present invention also provides a method for performing a diagnosis on a patient for a disorder associated with undesirable levels of motility and/or function of the sperm of the patient, comprising:
  • step (ii) comparing the level determined in step (i) to the concentration range of the polypeptide known to be present in normal subjects;
  • step (iii) diagnosing whether the patient has the disorder based on the comparison in step (ii).
  • Diagnostic information may be provided for a male subject wherein, for example, decreased expression of SLIRP polypeptides including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 in the subject's sperm cells, as compared to expression levels in normally functioning sperm cells, may indicate that the subject's sperm have reduced motility and/or function.
  • SLIRP polypeptides including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 in the subject's sperm cells, as compared to expression levels in normally functioning sperm cells, may indicate that the subject's sperm have reduced motility and/or function.
  • the diagnostic method of the present invention can be used for any disorder associated with undesirable levels of motility and/or function of the sperm of a patient.
  • step (a) above is performed using a binding agent described herein.
  • the diagnostic method may be applied to patients known to be suffering from a disorder associated with undesirable levels of motility and/or function of the patient's sperm with a view to assessing their response to treatment for the disorder by modulation of the levels of the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • One method for monitoring levels involves using antibodies including monoclonal antibodies to the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 as described herein.
  • the present invention may be applied to assess the prognosis of a patient with undesirable levels of motility and/or function of the patient's sperm.
  • the present invention also provides a method for prognostic evaluation of a patient comprising:
  • step (ii) comparing the level determined in step (i) to the concentration range of the polypeptide known to be present in normal subjects;
  • step (iii) evaluating the prognosis of said patient based on the comparison in step (ii).
  • expression levels of SLIRP polypeptides including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 may be used to provide prognostic information such as future fertility.
  • quantitative histology may be used to score SLIRP polypeptide levels to provide prognostic information.
  • microarray data may be used to observe expression levels of nucleic acids of the invention in sperm cells of a subject to provide prognostic information as described above.
  • the methods and use of the present invention may also employ non-human animals such as mice, rats, or other rodents, rabbits, dogs, goats, sheep, cows, horses, pigs, or other farm animals, in which the gene encoding the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or a variant thereof has been disrupted ("knocked out") such that the level of expression of this gene or genes is(are) significantly decreased or completely abolished.
  • Such animals may be prepared using techniques and methods - 70 - such as those described in U.S. Patent No. 5,557,032. Increased apoptotic activity may be observed in such animals.
  • the methods and use of the present invention may also employ non-human animals such as mice, rats, or other rodents, rabbits, dogs, goats, sheep, cows, horses, pigs, or other farm animals, in which either the native form of the gene encoding the polypeptide of SEQ ID No: 2, 4, 6, 8, 1 0, 1 2, 14, 16, 1 8, or 20 or variant thereof for that animal or a heterologous gene is over-expressed by the animal, thereby creating a "transgenic" animal.
  • Such transgenic animals may be prepared using well known methods such as those described in U.S. Patent No. 5,489,743 and PCT Application No. W094/28122. Decreased apoptotic activity may be observed in such animals.
  • the methods and use of the present invention may also employ non-human animals in which the promoter for one or more of the polypeptides of the present invention is either activated or inactivated (e.g., by using homologous recombination methods) to alter the level of expression of one or more of the native polypeptides that can result in the modulation of apoptotic activity.
  • non-human animals may be used for drug candidate screening.
  • the impact of a drug candidate on the animal may be measured.
  • drug candidates may decrease or increase the expression of the gene encoding the polypeptide.
  • the amount of the polypeptide, which is produced may be measured after the exposure of the animal to the drug candidate.
  • one may detect the actual impact of the drug candidate on the animal.
  • the overexpression of a particular gene may result in, or be associated with, a disease or pathological condition.
  • one may test a drug candidate's ability to decrease expression of the gene or its ability to prevent or inhibit a pathological condition such as a condition in a subject resulting in decreased and/or dysmotile sperm.
  • the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition.
  • a drug candidate may test a drug candidate's ability to decrease the production of such a metabolic product or its ability to prevent or inhibit a pathological condition.
  • the transgene of interest encoding a SLIRP polypeptide or variant thereof is selected.
  • the simultaneous use of more than one transgene for insertion into a single embryo is within the scope of this invention.
  • the structural gene may be obtained from any source, if obtained from vertebrate mammals, the structural gene may be from a homologous source (i.e., a gene from one mouse implanted into another mouse), or from a non-homologous source (i.e., a structural gene from rabbit implanted into a mouse).
  • the transgene may have additional effects on the phenotype of the transgenic mammal, and these effects may be related or unrelated to the physiological action of the polypeptides herein.
  • Preferred transgenes for use in the present invention include myc, myb, E2F (Nevins, J. R., Science 258:424-429 [1992]), abl, ras, pim.1 , src, E1 A (Nevins, supra), HPVE7 (human papilloma virus E7, Nevins, supra), and SV40 early region, SV40 large T antigen, SV40 large T antigen tsA58 mutant, and mutants and fragments thereof. More preferred genes include myc, E2F, SV40 early region, and the SV40 early region tsA58 mutant. The most preferred gene is SV40 early region tsA58 mutant.
  • Promoters useful in practicing this invention are those that are highly regulated with respect to activity, both temporally and spatially.
  • the promoters of choice are those that are active only in particular tissues or cell types.
  • the source of the promoter may be from any prokaryotic or eukaryotic organism, any vertebrate or invertebrate, or any plant. Where the promoter is obtained from a mammal, the mammal may be homologous (the same species as the mammal to be transfected) or non-homologous (a different species).
  • the vectors of this invention preferably contain other elements useful for optimal functioning of the vector in the mammal into which the vector is inserted. These elements are well known to those of ordinary skill in the art, and are described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, 1989.
  • Vectors used for transforming mammalian embryos are constructed using methods well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, plasmid and DNA and RNA purification, DNA sequencing, and the like as described, for example in Sambrook, Fritsch, and Maniatis, eds., Molecular Cloning: A Laboratory Manual., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]). - 72 -
  • the specific lines of any mammalian species used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryos, and good reproductive fitness.
  • lines such as C57/BL6 x DBA2 F1 cross, or FVB lines are used (obtained commercially from Charles River Labs).
  • the age of the mammals that are used to obtain embryos and to serve as surrogate hosts is a function of the species used, but is readily determined by one of ordinary skill in the art. For example, when mice are used, pre-puberal females are preferred, as they yield more embryos and respond better to hormone injections.
  • the male mammal to be used as a stud will normally be selected by age of sexual maturity, among other criteria.
  • hormones or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos.
  • the type of hormones/cofactors and the quantity used, as well as the timing of administration of the hormones will vary for each species of mammal. Such considerations will be readily apparent to one of ordinary skill in the art.
  • a primed female i.e., one that is producing eggs that can be fertilized
  • a stud male i.e., one that is producing eggs that can be fertilized
  • the resulting fertilized embryos are then removed for introduction of the transgene(s).
  • eggs and sperm may be obtained from suitable females and males and used for in vitro fertilization to produce an embryo suitable for introduction of the transgene.
  • fertilized embryos are incubated in suitable media until the pronuclei appear.
  • exogenous nucleic acid comprising the transgene of interest is introduced into the female or male pronucleus.
  • the male pronucleus is preferred.
  • nucleic acid may be accomplished by any means known in the art such as, for example, microinjection. Following introduction of the nucleic acid into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method is to incubate the embryos in vitro for 1 -7 days and then reimplant them into the surrogate host.
  • Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos - 73 - implanted into a particular host will vary, but will usually be comparable to the number of offspring the species naturally produces.
  • Transgenic offspring of the surrogate host may be screened for the presence of the transgene by any suitable method. Screening is often accomplished by Southern or Northern analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening. Typically, the tissues or cells believed to express the transgene at the highest levels are tested, although any tissues or cell types may be used for this analysis.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular markers or enzyme activities, and the like. Blood cell count data is useful for evaluation of thrombocytopenia.
  • Progeny of the transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic mammal. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Where mating is used to produce transgenic progeny, the transgenic mammal may be backcrossed to a parental line. SLIRP transgenic mice may also be crossed with other transgenic or knock out lines to further highlight SLIRP's effects. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the transformed mammals, their progeny, and cell lines of the present invention provide several important uses that will be readily apparent to one of ordinary skill in the art.
  • the mammals and cell lines are particularly useful in screening compounds that have potential as prophylactic or therapeutic treatments for diseases associated with undesirable levels of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or a variant thereof.
  • screening of candidate compounds is conducted by administering the compound(s) to be tested to the mammal, over a range of doses, and evaluating the mammal's physiological response to the compound(s) over time.
  • Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound. - 74 -
  • Example 1 - SLIRP is present within the testis and sperm cells
  • SLIRP is abundantly expressed in the testis ( Figure 2). In a Northern blot of human RNA from various tissues, SLIRP is abundantly expressed in testis tissue further supporting a role for its function within this tissue. - 75 -
  • SLIRP is predominantly mitochondrial.
  • Figure 3 shows that although SLIRP is a nuclear receptor corepressor of nuclear receptor action (Figure 3A), and is closely associated with promoter DNA (as evidenced by ChIP studies, Hatchell et al, 2006, Figure 3B this document), the majority of SLIRP resides in the mitochondria ( Figure 3C). HeLa cells were stained with antibodies against the mitochondrial protein Hsp 60 (red) and SLIRP (green) and the resultant merge (yellow) demonstrates colocalization of each protein.
  • SLIRP has been evidenced in the head and annulus of human sperm indicating that morphology and motility could be influenced by a loss of SLIRP ( Figure 5). This suggests that SLIRP is important for maintaining sperm function.
  • siRNAs are double stranded, complementary RNA molecules generally composed of a 19 nucleotide sequence having exact homology with a specific sequence within a target mRNA with a limited number of nucleotides, such as a diuridine sequence, at its 3' end.
  • siRNAs When introduced into cells, siRNAs will reduce the expression of a given target gene by a combination of enhancing the rate at which a target mRNA is degraded and inhibition of its translation.
  • siRNAs may be used individually, in combination with one another or with siRNAs directed against other targets (ie two or more genes may be knocked down simultaneously).
  • SiRNAs directed against human SLIRP mRNA target sequences we have used are listed in table 1 and have been used to deplete SLIRP in a range of cell lines including HeLa (cervical), SW620 (colon), MCF-7 (breast) LnCAP (prostate) cell lines.
  • Typical knockdowns produced by the first 4 siRNAs listed in Table 1 are presented in Figure 6.
  • SiRNAs targeted to the sequences as in Table 1 or others regions within the SLIRP mRNA may be introduced into tissues by different methods. These include, chemically synthesised molecules transfected into cells via cationic lipid transfer e.g. using Lipofectamine 2000 (Invitrogen ® ) or electroporation of similar molecules. Cells or tissue may also be transfected with viruses expressing the siRNA of interest or a plasmid directing their expression.
  • Hela cells were transfected with either empty pSuperior.neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1 .
  • Figure 7 shows SLIRP levels are decreased in pSupSLIRP transfected cells following FACS sorting of GFP positive cells and 7 days post transfection compared with the empty vector, ⁇ -actin expression assessed as loading control.
  • the normal expression of the SLIRP gene in the mouse may be disrupted using knockout technologies. Multiple strategies may be utilised to achieve altered SLIRP expression in cell lines and/or whole animals. The following strategy provides one such approach.
  • the mouse SLIRP gene also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl /SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA. In one nucleotide listing, the SLIRP exon is present on mouse Chromosome 12 in the region 88,784,866-88,790,828 as defined by Genbank Mus musculus Build 37.1 ( Figure 8).
  • This gene is composed of 4 exons, the initiating methionine being in exon 1 .
  • its genome may be genetically reengineered ( Figure 9) to place DNA recombinase recognition sites such as the loxp domain recognised by ere recombinase or Flippase Recognition Target (FRT) sites as recognised by Flippase either side of the region of the gene to be removed.
  • the region to be removed would include the initiating methionine of the gene or a region critical to the normal function of the gene to be targeted.
  • mice may either have one (heterozygous) or both (homozygous) copies of the wild type gene replaced with the floxed allele.
  • a floxed gene will express the same protein as a wild type animal, in a manner highly similar to or identical with the wild type animal. This may be achieved by having floxed gene transcription driven by the wild type promoter region.
  • the genome of a mouse may be reengineered such that all or part of the SLIRP gene is floxed and when exposed to ere a region of the DNA - 78 - removed such that transcripts initiated from the endogenous promoter are targeted for rapid destruction as a result of splicing to an RNA destabilisation sequence.
  • transcripts initiated within exon 1 may be spiced to include a c-fos 3' untranslated region containing signals resulting in the rapid destruction of the mRNA resulting from the recombination event.
  • an exon encoding a protein degradation signal e.g. a ubiquitination site that when spliced in frame to a transcript from an endogenous promoter the protein resulting post recombination will be rapidly destroyed thereby minimising its expression.
  • heterozygous and homozygous floxed SLIRP mice respectively may be bred with mice that express a suitable recombinase e.g. ere.
  • a suitable recombinase e.g. ere.
  • Cre expression will depend upon the promoter used to direct its expression. For example, in the case of actin-cre, the recombinase is present in all tissues where actin is present and considered ubiquitously expressed.
  • actin- cre and floxed SLIRP mouse are crossed, wild type expression of SLIRP will be lost from all tissues.
  • floxed SLIRP mice were crossed with villin-cre mice, the SLIRP gene would only be deleted from gastrointestinal epithelium.
  • Isolated tissues may also be exposed to a suitable recombinase to assess the effects of SLIRP removal via for example transfection with a virus or plasmid expressing the required recombinase.
  • Example 4 - SLIRP KO influences fertility - localised in areas of mitochondrial energy, and morphology.
  • SLIRP k0/k0 A global SLIRP knockout mouse (SLIRP k0/k0 ) was generated to provide an in vivo model to investigate SLIRP's role in male fertility and spermatogenesis. Although SLIRP wt/k0 male mice are fertile, SLIRP k0/k0 males were shown to have reduced fertility ( Figure 10). The SLIRP k0/k0 animal is viable, however when homozygous knockout males were crossed with wild type (wt) females the resultant litter size was reduced by -30% (ko/ko x wt/wt, 4.8 pups/litter) compared with those produced by wt males with comparable females (wt/wt x wt/wt, 6.6).

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Abstract

The present invention provides methods for modulating the motility and/or function of a sperm cell, the method including the step of increasing or reducing levels of SLIRP polypeptide or the activity of SLIRP polypeptide within the sperm cell, as compared to normal levels or normal activity of SLIRP polypeptide within the sperm cell. The present invention further provides methods for assessing the SLIRP gene and its products to predict fertility in males.

Description

THERAPEUTIC USES OF SLIRP
FIELD OF THE INVENTION
The present invention relates to the modulation of SLIRP levels within a sperm cell to effect sperm function and motility. The present invention further relates to methods for assessing the SLIRP gene and its products to predict fertility in males.
BACKGROUND
Steroid receptor RNA activator (SRA) is an RNA coactivator which augments transactivation by nuclear receptors (NRs) including the estrogen receptor (ER), an NR that plays a key role in the proliferation of breast cancer cells. SRA plays an important role in mediating 17p-estradiol (E2) action. Its expression is both increased and aberrant in many human breast tumours, suggesting a potential role in pathogenesis. Although it is hypothesised that SRA acts as an RNA-protein complex scaffold for other coregulators at transcription initiation sites, the precise mechanism by which SRA augments ER activity in general, and specifically in breast cancer, remains unclear. More recently, protein interactors of SRA have been identified and provide insight into the putative mechanisms underlying SRA's transcriptional coactivation ability. One of these interactors, SRA stem-loop-interacting RNA-binding protein (SLIRP) was identified and characterised in WO2007/009194 and shown to bind to STR7, a functional substructure of SRA.
SLIRP is a widely expressed small SRA-binding protein and a repressor of ER and glucocorticoid receptor (GR) activity. While it has been shown to be predominantly localized to the mitochondria, it is actively recruited to the E2-responsive pS2 promoter where it modifies NR transactivation.
Increased expression of SLIRP in energy- and mitochondria-rich tissues, such as skeletal muscle, heart, liver and testis was identified by Hatchell, et al. ((2006) Molecular Cell 22, 657-68).
However, cellular signalling mechanisms are complex and cellular proteins are commonly found to have multiple roles within a cell or alternative roles in cells of different tissues or between species. Thus, it is unclear whether SLIRP is involved in cellular pathways other than modulating the activity of several NR pathways and its role in cell metabolism and homeostasis.
It is against this background that the present invention has been developed. SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In a second aspect, the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In a third aspect, the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and (vi) a functional variant of any one of (i) to (v).
In a fourth aspect, the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In a fifth aspect, the present invention provides a method for increasing expression of a polypeptide in a sperm cell to improve the function of the cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In a sixth aspect, the present invention provides a method for increasing expression of a polypeptide in a sperm cell to improve the motility of the cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v). In a seventh aspect, the present invention provides a method for treating a disorder associated with an undesirable level of function and/or motility of sperm cells, the method comprising the step of administering an effective amount of an isolated polypeptide, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In an eighth aspect, the present invention provides a method for modulating the motility of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or
(vi) a functional variant of any one of (i) to (v).
In a ninth aspect, the present invention provides a method for modulating the function of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or
(vi) a functional variant of any one of (i) to (v). The polypeptide of the methods described herein may be encoded by a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19. In another form of the invention, the polypeptide may be encoded by a polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19. The polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, may also comprise a nucleotide sequence 95% to 99% identical to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
In one embodiment of the invention, one or more promoters may be used to increase the expression of a SLIRP polynucleotide encoding a SLIRP polypeptide as described herein.
In another embodiment of the invention, antisense nucleic acids or siRNAs may be used to reduce or eliminate the expression of a polynucleotide encoding a SLIRP polypeptide as described herein. Thus, the present invention also provides a method for sterilising or reducing the sterility of a male animal comprising a method as described herein, including the step of reducing levels or reducing activity of the SLIRP polypeptide within sperm cells of the male animal. The term "male animal" as used herein includes both male humans and non-human male animals including, for example, but not limited to, mice, rats, sheep, cows/cattle, dogs, horses, pigs, orang-utan, amongst others. This sterilising of the male animal may be reversible by restoring the SLIRP levels in the sperm cells of the male animal.
In a tenth aspect, the present invention provides a SLIRP polynucleotide as a biomarker for identification of dysmotile or dysfunctional sperm, the polynucleotide being any one or more selected from the group consisting of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19. In this respect, a microarray may be used to identify the presence of mutations in the SLIRP polynucleotide.
In a eleventh aspect, the present invention provides a use of a polypeptide as a biomarker for prediction of dysmotile or dysfunctional sperm, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2; (v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
In a twelfth aspect, the present invention provides a use of a polypeptide as a biomarker for prediction of energy production in a sperm cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
The polypeptide of the herein described methods may comprise a portion of a fusion protein. Alternatively, a SLIRP polypeptide of the herein described methods may comprise a non-peptide mimetic of the polypeptide.
In one form of the invention a selective binding agent may be used to detect the presence or levels of a SLIRP polypeptide as described herein. Such a selective binding agent may be an antibody to the polypeptide, such as a polyclonal or monoclonal antibody, and said antibody may also be labelled.
In a thirteenth aspect, the present invention provides a method for assessing the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal;
(ii) identifying the nucleotide sequence encoding a SLIRP polypeptide described herein;
(iii) analysing the nucleotide sequence for one or more mutations which will reduce the fertility of the male animal.
These one or more cells may be isolated from any one of blood, sputum, or semen from the male animal. Moreover, the one or more cells may be quantified to assess the fertility, and wherein nucleotide sequencing may be used for identifying the nucleotide sequence.
The present invention further provides a kit for assessing the fertility of a male animal, the kit using any of the methods described herein, and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
The present invention also provides a kit for assessing the fertility of a male animal, the kit using the method of any of the methods described herein, and comprising at least one allele-specific oligonucleotide probe specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
In a fourteenth aspect, the present invention provides a method for predicting the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal;
(ii) sequencing the SLIRP gene from nucleotides present in the one or more cells; and
(iii) analysing the nucleotide sequence of the SLIRP gene for one or more mutations which will reduce the fertility of the male animal;
wherein the presence of the one or more mutations is predictive of a reduced fertility of the male animal when compared to a male animal without the one or more mutations. The presence of the one or more mutations may be predictive that the male animal is infertile. In addition, a mutant or wild-type SLIRP gene may be predictive of reduced fertility. The mutations may be within any one or more of the coding region of the exons, non-coding exonic regions, intronic regions, or flanking regions of the SLIRP gene. The one or more cells may be taken from blood, epithelial cells, semen, or other genetic- bearing material, from the male animal.
In a fifteenth aspect, the present invention provides a method for predicting the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal; and
(ii) measurement of SLIRP gene product levels from nucleotides present in the one or more cells;
wherein the presence of reduced SLIRP gene product levels is predictive of a reduced fertility of the male animal when compared to SLIRP gene product levels in one or more cells from a normal male animal. The SLIRP gene product levels may be measured using quantitative reverse transcriptase (QRT) PCR including real-time QRT-PCR, and the one or more cells may be isolated from semen or ejaculate from the male animal. BRIEF DESCRIPTION OF THE FIGURES
Figure 1. SLIRP is expressed in murine testis and sperm. A) SLIRP staining detected by immunohistochemistry in mouse seminiferous tubules and sperm visualized using DAB and counter stained with Haematoxylin. Dark, round nuclei of the spermatogonia are highlighted with short white arrows. Smaller, SLIRP positive spots, darker than the nuclear stain, present in spermatocytes and spermatids highlighted with longer arrows. SLIRP staining in the tails of sperm within the lumen also highlighted with longer arrows. B) Immunofluorescent detection of PCNA in round nuclei of immature, proliferating spermatogonia (short arrows) contrasts with the smaller granules of SLIRP (longer arrows) in spermatids and the tails of sperm. L, lumen. .
Figure 2. Northern blot of human RNA extracted from a range of tissues shows that SLIRP is highly expressed in the tissues that are mitochondria-rich and energy requiring: especially heart, liver, skeletal muscle and the testis. The blot is also probed with beta- actin as an RNA loading control.
Figure 3. SLIRP is a repressor of nuclear receptor activity and is present in nuclear receptor complexes but in some tissues is predominantly a mitochondrial protein. A) Over expression of SLIRP represses nuclear receptor activity in Hela cells transfected with appropriate nuclear receptor expression plasmids and reporter constructs. Dex, dexamethasone", DHT, dihydrotestosterone, T3, triiodothyronine; VitD, vitamin D; GW501516, PPAR agonist; SRA, steroid receptor RNA activator. B) Chromatin immunoprecipitation assays demonstrate that SLIRP is more abundant at the metallothionein promoter in HeLa cervical cancer cells in the presence of Dexamethasone (+ Dex) than in its absence (- Dex; lane 5). C) Confocal microscopy of HeLa cells simultaneously stained with a nuclear dye (top left quadrant) and probed for Hsp 60 (top right quadrant) and SLIRP (bottom left quadrant) by immunofluorescence shows co-localization ('Overlay': bottom right quadrant) of these proteins within the mitochondria.
Figure 4. SLIRP levels are frequently substantially lower in human males with teratozoospermia. Data presented compares the level of SLIRP expression between human males with normal fertility and those considered to have teratazoospermia (a condition in which less than 4 percent of sperm cells are morphologically normal). Data from Gene Expression Omnibus (GEO), National Center for Biotechnology Information, USA. Title: GDS2697 / 221434_s_at / C14orf156 / Homo sapiens. The image represents the abundance profile for an individual gene across each Sample in a DataSet. Vertical bar: Represents value measurements as extracted from original submitter-supplied GEO Sample records (GSMxxx) and reflect the measured level of abundance of an individual transcript across the Samples that make up a DataSet. Values are presented as arbitrary units. Single channel experiments: Values are assumed to be submitted as normalized (scaled) signal count data. Dual channel experiments: Values are assumed to be submitted as normalized log ratios (typically test/reference). Square: Represents rank order; all values within a Sample are rank ordered, and then placed into percentile 'bins'. In other words, all the values of one hybridization are sorted, then split into 100 groups. So, the rank bars on charts give an indication of where the expression of that, gene falls with respect to all other genes on that array. Ranks can also be a useful indicator for when a DataSet is not well normalized (you can verify that Sample values are well-distributed / normalized by viewing the 'value distribution' chart that is provided on each DataSet record under the 'analysis' button maintained by the GEO database).
Figure 5. SLIRP is localised in the acrosome, midpiece and annulus of human sperm. A) Immunohistochemical detection of SLIRP in human sperm. Acrosomal (small vertical arrow), midpiece (bar) and annulus (arrow head) staining with DAB highlighted. Whole sperm outlined by haematoxylin. B) Human sperm stained with haematoxylin only. C) Immunofluorescent detection of SLIRP in human sperm. Arrows highlight SLIRP expression in acrosome, midpiece and annulus. Nuclear staining with DAPI indicated by bar. Figure 6. siRNA mediated depletion of human SLIRP expression in Hela cells. The ability of individual and pooled mixtures of siRNAs to deplete cells of SLIRP protein were compared by western analysis. Hela cells were transfected with either Lipofectmine alone (LF), individual siRNAs (si#1-4), a combination of the 4 SLIRP directed siRNAs (SP) or a non-specific (non-SLIRP (NS) targeting) siRNA to a final concentration of 20u . Three days post transfection, lysates from duplicate knockdown treatments were prepared and the expression of SLIRP and β-actin assessed by western analysis. Blots demonstrate that each of the individual siRNAs and a pool of all four were able to deplete SLIRP protein levels in Hela cells without affecting β-actin expression when compared to LF and NS treated cultures. Individual siRNAs as listed in table 1 corresponding to catalogue numbers D-014696-01 to 04 (Thermo Scientific®).
Figure 7. SLIRP levels are depleted in HeLa cells 7 days post transfection with pSuperior SLIRP. Hela cells were transfected with either empty pSuperior. neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1. Figure shows SLIRP levels detected by western analysis are decreased in lysates prepared from pSupSLIRP compared with - 9B - empty vector transfected cells following FACS sorting, of GFP pbsitive cells 7 days post transfection, β-actin expression assessed as loading control.
Figure 8. Schematic representation of the mouse SLIRP genomic locus. The mouse SLIRP gene, also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl/SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
- 10 -
Figure 7. SLIRP levels are depleted in HeLa cells 7 days post transfection with pSuperior SLIRP. H.ela cells were transfected with either empty pSuperior.neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1. Figure shows SLIRP levels detected by western analysis are decreased in lysates prepared from pSupSLIRP compared with empty vector transfected cells following FACS sorting of GFP positive cells 7 days post transfection. β-actin expression assessed as loading control.
Figure 8. Schematic representation of the mouse SLIRP genomic locus. The mouse SLIRP gene, also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl/S IP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
- 1 1 -
Figure 13. Human SLIRP protein sequence aligned with non-human orthologues. Human SLIRP protein (NP 1 12487), aligned with orthologues from other species including Orangutan (NP_001 126105), Pig (NP_001090941 ), Horse (XP_001494001 ), Sheep (NP_001 138656), Cow (NP_001032562), Dog (XP_547932), Mouse (NM_026958) and Rat (NM_001 109507). Numbers at top indicate residue number, numbers right refer to the length of individual proteins. Genebank (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA) accession numbers for individual sequences provided in brackets. A consensus sequence is also listed as is a graphical indicator of the conservation of individual residues between the species listed.
Figure 14. Human SLIRP mRNA sequence aligned with non-human orthologues. Human SLIRP mRNA sequence (NM 031210) aligned with orthologues from other species including Orangutan (NM_001 132633), Pig (NM_001097472), Horse (XM_001493951 ), Sheep (NM_001 145184), Cow/cattle (NM_001037485), Dog (XM_547932), and Mouse (NM_026958). Numbers at top indicate nucleotide number, numbers right refer to the length of individual mRNAs. Genebank (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA) accession numbers for individual sequences provided in brackets. A consensus sequence is also listed along with a graphical indicator of the conservation of individual nucleotides between the species listed.
DETAILED DESCRIPTION OF THE INVENTION
The high proportion of SLIRP which has been shown to localize within the mitochondria correlates with NRs being found in both mitochondrial and nuclear compartments of the cell. It is therefore unsurprising that the protein has been shown to have a role in modulating cellular metabolism and energy homeostasis in the mitochondria, a fundamental function for the organelle. However, the modulation of SLIRP polypeptides to control motility and function of sperm cells by the present invention provides an unexpected role for SLIRP polypeptides.
The present invention provides a method for modulating the motility and/or function of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide or the activity of a polypeptide within the sperm cell, as compared to normal levels or normal activity of the polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2; - 1 2 -
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
SEQ ID No: 2 and 4 are human SLIRP polypeptides, wherein SEQ ID No: 4 is SRA stem-loop-interacting RNA-binding protein, mitochondrial precursor [Homo sapiens], NCBI Reference Sequence NP_1 12487.
SEQ ID No: 6 is a cow SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial precursor [Bos taurus], NCBI Reference Sequence NP_001032562 XP_869675.
SEQ ID No: 8 is a dog SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Canis lupus familiaris], NCBI Reference Sequence XP_547932.
SEQ ID No: 10 is a sheep SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Ovis aries], NCBI Reference Sequence NP_001 138656.
SEQ ID No: 12 is a horse SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Equus caballus], NCBI Reference Sequence XM_001493951 .
SEQ ID No: 14 is a pig SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Sus scrofa], NCBI Reference Sequence NP 001090941 .
SEQ ID No: 16 is a rat SLIRP polypeptide, specifically SRA stem-loop-interacting RNA- binding protein, mitochondrial [Rattus norvegicus], NCBI Reference Sequence NP_001 102977 XP_001068036.
SEQ ID No: 18 is a Sumatran orangutan SLIRP polypeptide, specifically SRA stem-loop- interacting RNA-binding protein, mitochondrial [Pongo abelii], NCBI Reference Sequence NM_001 132633.
SEQ ID No: 20 is a mouse SLIRP polypeptide, specifically SRA stem-loop-interacting RNA-binding protein, mitochondrial [Mus musculus], NCBI Reference Sequence NP_081234 XP_354684.
For the purposes of describing the present invention the term "SLIRP polypeptide(s)" and SLIRP, as used herein, includes the above polypeptides [(i) to (vi)], unless the context specifically requires otherwise. Moreover, the species from which a polypeptide - 1 3 - originates will be selected to match the species of the sperm cell. For example, to modulate the motility and/or function of a human sperm cell, levels of a polypeptide or activity of a polypeptide of SEQ ID No: 2 or 4, or any one of (ii) to (vi) above can be increased or reduced as these polypeptides are of human origin. In another example, to modulate the motility and/or function of a cow (Bos taurus) sperm cell, levels of a polypeptide or activity of a polypeptide of SEQ ID No: 6 can be increased or reduced as the polypeptide is of Bos taurus origin.
In addition, the present invention also includes methods to modulate the motility and/or function of a sperm cell for species of animals not listed herein. In this respect, levels of SLIRP polypeptides or activities of SLIRP polypeptides may be increased or reduced within a sperm cell of an animal subject of a species not listed herein which can modulate the motility and/or function of the sperm cell.
The present invention also provides a method for increasing expression of a polypeptide in a sperm cell to improve the function and/or motility of the cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
The function and motility of sperm cells is central for their role in fertilisation. This is apparent with approximately 50% of infertile human males having low sperm motility and/or a low sperm count. SLIRP is largely localised in the mitochondria, the site of cellular metabolism and energy homeostasis, and low sperm motility and/or a low sperm count are typical features of sperm mitochondrial defects. Thus, the present invention also provides a method whereby increasing SLIRP levels in a sperm cell can improve the function and motility of the cell, particularly where the sperm cell is defective in that it has reduced motility and/or function.
For the purposes of describing the present invention, a "sperm cell" refers to a male animal gamete or reproductive cell which contains genetic information and participates in the act of fertilization of an ovum. - 14 -
For the purposes of describing the present invention, "sperm motility" refers to the ability of a sperm cell to move actively forward. The mature sperm, after residence in the female reproductive tract, develops a forward progressive and then hyperactivated form of motility. Hyperactivated motility is thought to greatly facilitate progression and aid in penetration of the oocyte. Sperm motility is one measure of fertility for a subject and can be assessed using a microscope (400x magnification) or by computerised imaging, amongst other methods. In this regard, "improved sperm motility" refers to the sperm cell(s) being able to move more actively forward than previously able. Furthermore, the term "dysmotile sperm", for the purposes of describing the invention, refers to abnormal sperm which are unable to carry out the normal function and movement of a normal sperm cell.
Similarly, "sperm function" refers to the ability of a sperm cell to carry out its normal functions including growth and development, and movement as is required for its ability to fertilise an ovum. Full sperm functional competence is obtained by sperm as it goes along the epididymus. Ejaculated sperm have the capacity for fertilization only after a period of residence in the female reproductive tract, during which they undergo a process known as capacitation. During capacitation, sperm develop a forward progression and then hyperactivated motility (with exaggerated flagella bending). Sperm also develop the capacity to undergo acrosome reaction and the ability to bind the oocyte, which are the end-points of sperm capacitation. Energy production in a sperm cell is carried out through the processes of cellular metabolism in the mitochondria.
Polypeptides
The methods and uses of the present invention may involve SLIRP polypeptides which are recombinant, natural or synthetic. The isolated SLIRP polypeptides may be mixed with carriers or diluents that will not interfere with the intended purpose of the polypeptide and still be regarded as isolated. The SLIRP polypeptide may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which at least 90%, 95%, 98% or 99% of the protein in the preparation is a SLIRP polypeptide.
It will be recognized that some amino acid sequences of the SLIRP polypeptides can be varied without significantly affecting the structure or function of the polypeptide. When such differences in sequence are contemplated, it should be remembered that there would be critical areas on the protein that determine activity.
Thus, functional variants include isolated SLIRP polypeptides that have at least one important activity of the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, - 1 5 -
18, or 20. For example, the ability to inhibit binding to SRA. Such variants include the recited sequences with deletions, insertions, inversions, repeats, and type substitutions. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J.U., et al, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990).
Thus, a variant SLIRP polypeptide may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which one or more of the amino acid residues includes a substituent group, (iii) one in which the polypeptide is fused with another compound, such as a compound to decrease the half life of the polypeptide, or (iv) one in which the additional amino acids, such as a leader, signal or secretory sequence or a sequence which is employed for purification of the polypeptide sequence are fused to the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
As indicated above, the SLIRP variants may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. The particular replacements may be determined by a skilled person as detailed more fully hereunder. Changes may be of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see for example the table hereunder). Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
- 1 6 -
Figure imgf000018_0001
Alternatively, changes may be significant so as to disrupt binding with other molecules such as SRA.
Amino acids in the SLIRP polypeptides that are essential for function can be identified by methods known in the art, such as site directed mutagenesis or alanine-scanning mutagenesis. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as SRA binding. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization. Nuclear magnetic resonance or photoaffinity labelling may also be used when developing functional variants. Alternatively, synthetic peptides corresponding to candidate functional or non-functional variants may be produced and their ability to display or show absence of one or more activities of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 assessed in vitro or in vivo.
SLIRP polypeptide variants can also be prepared as libraries using the sequence of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or other polypeptides herein. Phage display can also be effective in identifying useful SLIRP variants . Briefly, a phage library is prepared (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a biased degenerate array or may completely restrict the amino acids at one or more positions (e.g., for a library based on a protein from SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20). One then can select phage-bearing inserts that have a relevant biological activity of the protein of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 such as SRA binding affinity or lack of, and/or regulation of nuclear receptor - 1 7 - signalling. This process can be repeated through several cycles of reselection of phage. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences of the expressed polypeptides. The minimal linear portion of the sequence that confers the relevant activity can be determined. One can repeat the procedure using a biased library containing inserts containing part or the entire minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof.
Polypeptides, including variant polypeptides, can be tested for retention of any of the given activity. For example, the peptides can be tested for in vitro properties using transient transfection assays with a responsive reporter that assess the ability of the peptide to repress SRA-mediated coactivation to determine which of the variant peptides retain activity.
Preferred variant SLIRP polypeptides comprise an amino acid sequence that is at least 70-80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to a polypeptide sequence recited herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a polypeptide it is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular variant polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 herein can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 5371 1 ). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, - 1 8 - the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
In general, SLIRP polypeptides can be synthesized directly or obtained by chemical or mechanical disruption of larger molecules, fractioned and then tested for one or more activity of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Functional or non-functional variants may also be produced by Northern blot analysis of total cellular RNA followed by cloning and sequencing of identified bands derived from different tissues/cells, or by PCR analysis of such RNA also followed by cloning and sequencing. Thus, synthesis or purification of an extremely large number of functional or non-functional variants is possible using the information contained herein.
SLIRP polypeptide variants also include fusion proteins, for example, where another peptide sequence is fused to the polypeptide of interest to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), hexahistidine, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will hinder SRA binding activity.
SLIRP polypeptides may also include conjugated proteins. In this regard, a protein may be modified by attachment of a moiety (e.g. a fluorescent, radioactive, or enzymatic label, or an unrelated sequence of amino acids to make a fusion protein) that does not correspond to a portion of the peptide in its native state. Thus, SLIRP peptides may also comprise chimeric proteins comprising a fusion of an isolated peptide with another peptide. For example, a peptide capable of targeting the isolated peptide to a cell type or tissue type, enhancing stability of the isolated peptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein. A moiety fused to an isolated peptide or a fragment thereof also may provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labelling such as green fluorescent protein. Purified isolated peptides include peptides isolated by methods including, but are not limited to, immunochromotography, HPLC, size-exclusion chromatography, ion-exchange chromatography and immune-affinity chromatography.
The polypeptides herein can be conjugated by well-known methods, including bifunctional linkers, formation of a fusion polypeptide, and formation of biotin/streptavidin or biotin/avidin complexes by attaching either biotin or streptavid in/avid in to the peptide and the complementary molecule. Depending upon the nature of the reactive groups in - 1 9 - an isolated peptide and a targeting agent, a conjugate can be formed by simultaneously or sequentially allowing the functional groups of the above-described components to react with one another. Numerous art-recognized methods for forming a covalent linkage can be used. See, e.g., March, J., Advanced Organic Chemistry, 4th Ed., New York, N.Y., Wiley and Sons, 1985), pp.326-1 120.
In general, conjugated SLIRP peptides may be prepared by using well-known methods for forming amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective conjugated peptide components. As would be apparent to one of ordinary skill in the art, reactive functional groups that are present in the amino acid side chains of the peptide preferably are protected, to minimize unwanted side reactions prior to coupling the peptide to the derivatizing agent and/or to the extracellular agent. As used herein, "protecting group" refers to a molecule which is bound to a functional group and which may be selectively removed therefrom to expose the functional group in a reactive form. Preferably, the protecting groups are reversibly attached to the functional groups and can be removed therefrom using, for example, chemical or other cleavage methods. Thus, for example, the peptides described herein can be synthesized using commercially available side-chain-blocked amino acids (e.g., FMOC-derivatized amino acids from Advanced Chemtech Inc., Louisville, Ky.). Alternatively, the peptide side chains can be reacted with protecting groups after peptide synthesis, but prior to the covalent coupling reaction. In this manner, conjugated peptides can be prepared in which the amino acid side chains do not participate to any significant extent in the coupling reaction of the peptide to the other agent, such as a cell-type-specific targeting agent.
If a peptide does not have a free amino- or carboxyl-terminal functional group that can participate in a coupling reaction, such a group can be introduced, e.g., by introducing a cysteine (containing a reactive thiol group) into the peptide by synthesis or site directed mutagenesis. Disulfide linkages can be formed between thiol groups in, for example, the peptide and the targeting compound. Alternatively, covalent linkages can be formed using bifunctional cross linking agents, such as bismaleimidohexane (which contains thiol-reactive maleimide groups and which forms covalent bonds with free thiols). See also the Pierce Co. Immunotechnology Catalogue and Handbook Vol. 1 for a list of exemplary homo- and hetero-bifunctional cross linking agents, thiol-containing amines and other molecules with reactive groups.
For peptides that exhibit reduced activity in a conjugated form, the covalent bond between the peptides and its conjugate is selected to be sufficiently labile (e.g., to enzymatic cleavage) so that it is cleaved following transport to its target, thereby - 20 - releasing the free peptides at the target. Biologically labile covalent linkages, e.g., imino bonds, and "active" esters can be used to form prodrugs where the covalently coupled peptides are found to exhibit reduced activity in comparison to the activity of the peptides alone.
It will be appreciated that the amino acids in the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 that are required for activity may be incorporated into larger peptides and still maintain their function. Preferably, the amino acids required for SRA binding include at least amino acids 21 to 26 or 60 to 67 or are other contiguous sequences of between about 5 and 20 amino acids and more preferably between about 6 and 15 amino acids.
Preferably, the isolated peptides are non-hydrolyzable in that the bonds linking the amino acids of the peptide are less readily hydrolyzed than peptide bonds formed between L-amino acids. To provide such peptides, one may select isolated peptides from a library of non-hydrolyzable peptides, such as peptides containing one or more D- amino acids or peptides containing one or more non-hydrolyzable peptide bonds linking amino acids.
Alternatively, one can select peptides that are optimal for a preferred function in suitable assay systems and then modify such peptides as necessary to reduce the potential for hydrolysis by proteases. For example, to determine the susceptibility to proteolytic cleavage, peptides may be labelled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis, e.g., by sequencing peptides and proteolytic fragments. Alternatively, potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of an isolated peptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds that are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide.
Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Non-hydrolyzable bonds include - psi[CH2NH]~ reduced amide peptide bonds, -psi[COCH2]~ ketomethylene peptide bonds, -psi[CH(CN)NH]~ (cyanomethylene)amino peptide bonds, -psi[CH2CH(OH)]~ hydroxyethylene peptide bonds, -psi[CH20]~ peptide bonds, and -psi[CH2S]- thiomethylene peptide bonds. Likewise, various changes may be made including the addition of various side groups that affect the manner in which the peptide functions, or which favourably affect the manner in which the peptide functions for the purposes of the present invention. Such changes may involve adding or subtracting charge groups, - 21 - substituting amino acids, adding lipophilic moieties that may or may not affect binding but that affect the overall charge characteristics of the molecule facilitating a specific outcome with a physiological benefit. For each such change, no more than routine experimentation is required to test whether the molecule functions according to the invention. One simply makes the desired change or selects the desired peptide and applies it in a fashion as described in detail herein.
The peptides herein may also be linked to a variety of polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG). Replacement of naturally occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify peptides. Another approach is to use bifunctional crosslinkers, such as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl 6-[3-(2 pyridyldithio) propionamido] hexanoate, and sulfosuccinimidyl 6-[3- (2 pyridyldithio) propionamido]hexanoate.
It may be desirable to use derivatives of SLIRP peptides that are conformationally constrained. Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide. Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.
The active conformation of the peptide may be stabilized by a covalent modification, such as cyclization or by incorporation of gamma-lactam or other types of bridges. For example, side chains can be cyclized to the backbone to create a L-g am ma- lactam moiety on each side of the interaction site. Cyclization also can be achieved, for example, by formation of cystine bridges, coupling of amino and carboxy terminal groups of respective terminal amino acids, or coupling of the amino group of a lysine residue or a related homolog with a carboxy group of aspartic acid, glutamic acid or a related homolog. Coupling of the alpha-amino group of a polypeptide with the epsilon- amino group of a lysine residue, using iodoacetic anhydride, can be also undertaken. Another approach is to include a metal-ion complexing backbone in the peptide structure. Typically, the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the coordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a coordination number of four to six. The nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulphur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, - 22 - or carboxyl functionalities. In addition, the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino. The peptide construct can be either linear or cyclic, however a linear construct is typically preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly that has four nitrogens (an N4 complexation system) in the backbone that can complex to a metal ion with a coordination number of four.
Other methods for identifying variants of the isolated peptides herein rely upon the development of amino acid sequence motifs to which potential epitopes may be compared. Each motif describes a finite set of amino acid sequences in which the residues at each (relative) position may be (a) restricted to a single residue, (b) allowed to vary amongst a restricted set of residues, or (c) allowed to vary amongst all possible residues. For example, a motif might specify that the residue at a first position may be any one of valine, leucine, isoleucine, methionine, or phenylalanine; that the residue at the second position must be histidine; that the residue at the third position may be any amino acid residue; that the residue at the fourth position may be any one of the residues valine, leucine, isoleucine, methionine, phenylalanine, tyrosine or tryptophan; that the residue at the fifth position must be lysine, and so on. The motifs in SEQ ID No:2 at amino acids 21 -26 and 60-67 provide further assistance to those skilled in the art as search, evaluation, or design criteria for functional variants of the polypeptides disclosed herein.
Thus, the present invention also provides methods for identifying functional variants of an isolated polypeptide. In general, the methods include selecting an isolated peptide, such as the isolated peptide identified herein as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Then a first amino acid residue of the isolated peptide is mutated to prepare a variant peptide. In one embodiment, the amino acid residue can be selected and mutated as indicated by a computer model of peptide conformation. Peptides bearing mutated residues that maintain a similar conformation (e.g. secondary structure) can be considered potential functional variants that can be tested for function using the assays described herein. Any method for preparing variant peptides can be employed, such as synthesis of the variant peptide, recombinantly producing the variant peptide using a mutated nucleic acid molecule, and the like. The properties of the variant peptide in relation to the isolated peptides described previously are then determined according to standard procedures as described herein. - 23 -
Variants of the isolated peptides prepared by any of the foregoing methods can be sequenced, if necessary, to determine the amino acid sequence and thus deduce the nucleotide sequence which encodes such variants.
The present invention also provides fragments of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 comprising at least about 10, 20, 30, 50 or 100 amino acid residues. In this context "about" includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either extreme or at both extremes. For instance, about 40-90 amino acids in this context means a polypeptide fragment of 40 plus or minus several, a few, 5, 4, 3, 2 or 1 amino acid residues to 90 plus or minus several a few, 5, 4, 3, 2 or 1 amino acid residues. Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes. Particularly highly preferred are the recited ranges plus or minus as many as 3 amino acids at either or at both the recited extremes. Especially particularly highly preferred are ranges plus or minus 1 amino acid at either or at both extremes of the recited ranges with no additions or deletions. Preferably, the fragments include at least one biological activity of the polypeptide from which they are fragmented, such as SRA binding affinity and/or ability to bind an antibody to the full polypeptide. Alternatively, according to the present invention, the fragment may not have SRA binding affinity.
Fragments or portions of SLIRP polypeptides may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
Other SLIRP fragments may comprise an epitope-bearing portion of a polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Preferably, the epitope is an immunogenic or antigenic epitope of the polypeptide. An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope."
As to the selection of fragments bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence check Z-1 of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e. immunogenic epitopes) nor to the amino or carboxyl terminals. Antigenic epitope- - 24 - bearing peptides and polypeptides may be contiguous or conformational epitopes and are useful to raise antibodies, including monoclonal antibodies that bind specifically to a SLIRP polypeptide. The epitope-bearing fragments of the invention may be produced by any conventional means apparent to those skilled in the art.
The present invention also includes non-peptide mimetics. A wide variety of techniques may be used to elucidate the precise structure of a peptide. These techniques include amino acid sequencing, x-ray crystallography, mass spectroscopy, nuclear magnetic resonance spectroscopy, computer-assisted molecular modelling, peptide mapping, and combinations thereof. Structural analysis of a peptide provides a large body of data that comprise the amino acid sequence of the peptide as well as the three-dimensional positioning of its atomic components. From this information, non-peptide peptidomimetics may be designed that have the required chemical functionalities for therapeutic activity but are more stable, for example less susceptible to biological degradation.
Thus, variants of the present invention also include mimetics. Nonpeptide analogs of peptides, such as those that provide a stabilized structure or lessened biodegradation, may be employed in the methods and uses of the present invention. Peptide mimetic analogs can be prepared based on a selected peptide by replacement of one or more residues by nonpeptide moieties. Preferably, the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, conformation such as a conformation that is not able to bind SRA. Thus, the present invention also provides for the use of a polypeptide described herein for designing a mimetic thereof such as a non-peptide peptidomimetic.
Selective Binding Agents
As used herein, the term "selective binding agent" refers to a molecule which has specificity for the polypeptides described herein. Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable selective binding agents may be prepared using methods known in the art. An exemplary selective binding agent of the present invention is capable of binding a portion of the polypeptides thereby inhibiting or enhancing the binding of the polypeptides to other molecules. In an example, amongst others, of the present invention, a selective binding agent is capable of binding a portion of the SLIRP polypeptides thereby inhibiting the binding of the SLIRP polypeptides to SRA.
Selective binding agents such as antibodies and antibody fragments that bind polypeptides herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, may be - 25 - used in the methods and uses of the present invention. The antibodies may be polyclonal including monospecific polyclonal, monoclonal (MAbs), recombinant, chimeric, humanized such as CDR-grafted, human, single chain, and/or bispecific, as well as fragments, variants or derivatives thereof. Antibody fragments include those portions of the antibody that bind to an epitope on the polypeptide. Examples of such fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full- length antibodies. Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
Polyclonal antibodies generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of the polypeptide and an adjuvant. It may be useful to conjugate the polypeptide to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for antibody titre.
Monoclonal antibodies are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et at., Nature, 256:495-497 (1975) and the human B-cell hybridoma method, Kozbor, J. Immunol., 133:3001 (1984);(1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987). Also provided by the invention are hybridoma cell lines that produce monoclonal antibodies reactive with polypeptides herein.
Monoclonal antibodies of the invention may be modified for use as therapeutics. One embodiment is a "chimeric" antibody in which a portion of the heavy and/or light chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies, so long as they exhibit the desired biological activity.
In another embodiment, a monoclonal antibody of the invention is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using - 26 - methods described in the art (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting at least a portion of a rodent complementarity-determining region (CDR) for the corresponding regions of a human antibody.
Also encompassed by the invention are human antibodies that bind polypeptides herein. Using transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunization with a polypeptide antigen (i.e., having at least 6 contiguous amino acids), optionally conjugated to a carrier. In one method, such transgenic animals are produced by incapacitating the endogenous loci encoding the heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof. Partially modified animals, that is, those having less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies with human (rather than e.g., murine) amino acid sequences, including variable regions that are immunospecific for these antigens. See PCT application nos. PCT/US96/05928 and PCT/US93/06926. Additional methods are described in U.S. Patent No. 5,545,807, PCT application nos. PCT/US91/245, PCT/GB89/01207, and in EP 546073B1 and EP 546073A1 . Human antibodies may also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
In an alternative embodiment, human antibodies can be produced from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991 );(1991 ) and Marks et al., J. Mol. Biol., 222:581 (1991 )). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT Application No. PCT/US98/17364, which describes the isolation of high affinity and functional agonistic antibodies.
Chimeric, CDR grafted, and humanized antibodies are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein. In a preferred embodiment, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein. The antibodies of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and - 27 - immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)) for the detection and quantitation of polypeptides. The antibodies will bind polypeptides with an affinity that is appropriate for the assay method being employed.
For diagnostic applications, in certain embodiments, antibodies may be labelled with a detectable moiety. The detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3H, 4C, 32P, 35S, or 25l; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.
Competitive binding assays rely on the ability of a labelled standard (e.g., a polypeptide described herein or an immunologically reactive portion thereof) to compete with the test sample (a candidate polypeptide) for binding with a limited amount of antibody. The amount of the candidate polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibody. To facilitate determining the amount of standard that becomes bound, the antibodies typically are insolubilized before or after the competition, so that the standard and candidate polypeptide that are bound to the antibodies may conveniently be separated from the standard and candidate polypeptide which remain unbound.
Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and/or quantitated. In a sandwich assay, the test sample (analyte) is typically bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex. The second antibody may itself be labelled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labelled with a detectable moiety (indirect sandwich assays). For example, one type of sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.
The selective binding agents, including antibodies, are also useful for in vivo imaging. An antibody labelled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labelled antibody in the host is assayed. The antibody may be labelled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art. - 28 -
Selective binding agents which may be used in the methods and uses of the invention, including antibodies, may be used as therapeutics. These therapeutic agents are generally agonists or antagonists, in that they either enhance or reduce, respectively, at least one of the biological activities of a polypeptide herein, including SRA binding. In one embodiment, antagonist antibodies of the invention are antibodies or binding fragments thereof which are capable of specifically binding to a polypeptide herein and which are capable of inhibiting or eliminating the functional activity of the polypeptide in vivo or in vitro. In preferred embodiments, the selective binding agent, e.g., an antagonist antibody will inhibit the functional activity of the polypeptide by at least about 50%, and preferably by at least about 80%. In another embodiment, the selective binding agent may be an antibody that is capable of interacting with SRA or some other binding partner (a ligand or receptor) of the polypeptides described herein thereby inhibiting or eliminating SRA binding activity in vitro or in vivo. Selective binding agents, including agonist and antagonist like antibodies, are identified by screening assays that are well known in the art.
The invention also relates to a kit comprising selective binding agents (such as antibodies) and other reagents useful for detecting the levels of the polypeptides described herein in biological samples. Such reagents may include, a detectable label, blocking serum, positive and negative control samples, and detection reagents.
The invention further provides a method for assessing the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal;
(ii) identifying the nucleotide sequence encoding a SLIRP polypeptide as described herein in the one or more cells;
(iii) analysing the nucleotide sequence for one or more mutations which will reduce the fertility of the male animal.
The one or more cells may be isolated from any one of blood, sputum, or semen from the male animal. Moreover, the one or more cells may be quantified to assess the fertility. Sequencing using techniques known in the art may be employed to identify said nucleotide sequence.
In this respect, the invention also provides a kit for assessing the fertility of a male animal, the kit using the preceding method and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations. - 29 -
Alternatively, a kit is also provided for assessing the fertility of a male animal, the kit also using the preceding method and comprising at least one allele-specific oligonucleotide probe specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
The polypeptides of the present invention can be used to clone its binding partners, using an expression cloning strategy. Radiolabeled ( 25-lodine) polypeptide or affinity/activity-tagged polypeptide (such as an Fc fusion or an alkaline phosphatase fusion) can be used in binding assays to identify a cell type or cell line or tissue that expresses the receptor(s). RNA isolated from such cells or tissues can be converted to cDNA, cloned into a mammalian expression vector, and transfected into mammalian cells (such as COS or 293 cells) to create an expression library. A radiolabeled SLIRP polypeptide can then be used as an affinity ligand to identify and isolate from this library the subset of cells which express the receptor(s). DNA can then be isolated from these cells and transfected into mammalian cells to create a secondary expression library in which the fraction of cells expressing receptor(s) is many-fold higher than in the original library. This enrichment process can be repeated iteratively until a single recombinant clone containing the receptor is isolated. Isolation of the receptor(s) is useful for identifying or developing novel agonists and antagonists of the polypeptide signalling pathway. Such agonists and antagonists include soluble polypeptide receptor(s), receptor antibodies, small molecules, proteins, peptides, carbohydrates, lipids, or antisense oligonucleotides, and they may be used for treating, preventing, or diagnosing one or more disease or disorder, such as sperm dysfunction or dysmotility.
In particular, antibodies may be used to detect polypeptides of the invention present in biological samples by a method that comprises:
(i) providing an antibody of the invention;
(ii) incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and
(iii) determining whether antibody-antigen complex comprising said antibody is formed. Suitable samples include extracts of tissues such as brain, breast, ovary, lung, colon, pancreas, testes, liver, muscle, prostate and bone tissues or from neoplastic growths derived from such tissues.
Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like. - 30 -
SRA Binding protein binding assays
One type of assay for identifying substances that bind to the polypeptides of the present invention involves contacting a SRA binding protein comprising a SLIRP polypeptide described herein, which is immobilised on a solid support, with a non-immobilised candidate substance determining whether and/or to what extent the SLIRP polypeptide and candidate substance bind to each other. Alternatively, the candidate substance may be immobilised and the SLIRP polypeptide non-immobilised.
In a preferred assay method, the SLIRP polypeptide is immobilised on beads such as agarose beads. Typically this is achieved by expressing the component as a GST- fusion protein in bacteria, yeast or higher eukaryotic cell lines and purifying the GST- fusion protein from crude cell extracts using glutathione-agarose beads. As a control, binding of the candidate substance, which is not a GST-fusion protein, to the immobilised SLIRP polypeptide is determined in the absence of the SLIRP polypeptide. The binding of the candidate substance to the immobilised SLIRP polypeptide is then determined. This type of assay is known in the art as a GST pulldown assay. Again, the candidate substance may be immobilised and the SLIRP polypeptide non-immobilised.
It is also possible to perform this type of assay using different affinity purification systems for immobilising one of the components, for example Ni-NTA agarose and hexahistidine-tagged components.
Binding of the SLIRP polypeptide to the candidate substance may be determined by a variety of methods well-known in the art. For example, the non-immobilised component may be labelled (with for example, a radioactive label, an epitope tag or an enzyme- antibody conjugate). Alternatively, binding may be determined by immunological detection techniques. For example, the reaction mixture can be Western blotted and the blot probed with an antibody that detects the non-immobilised component. ELISA techniques may also be used.
Candidate substances are typically added to a final concentration of from 1 to 1000 nmol/ml, more preferably from 1 to 100 nmol/ml. In the case of antibodies, the final concentration used is typically from 100 to 500 μg ml, more preferably from 200 to 300 ng/ml.
Another type of in vitro assay involves determining whether a candidate substance modulates binding of a protein/agent known to interact with SLIRP, such as SRA. Such an assay typically comprises contacting SLIRP protein with the known interacting protein in the presence or absence of the candidate substance and determining if the candidate substance has an effect on SLIRP binding to the known interacting protein. This - 31 - candidate may be used according to the present invention to competitively bind SLIRP thereby inhibiting SLIRP binding to SRA, or may be a modified form of the candidate where the candidate is modified according to any of the methods described herein.
Whole cell assays
Candidate substances may also be tested on whole cells for their effect on sperm cell function and/or motility. Preferably the candidate substances have been identified by the above-described in vitro methods. Alternatively, rapid throughput screens for substances capable of increasing sperm cell motility and/or function may be used as a preliminary screen and then used in the in vitro assay described above to confirm that the affect is on SLIRP.
The candidate substance, i.e. the test compound, may be administered to the cell in several ways. For example, it may be added directly to the cell culture medium or injected into the cell. Alternatively, in the case of polypeptide candidate substances, the cell may be transfected with a nucleic acid construct which directs expression of the polypeptide in the cell. Preferably, the expression of the polypeptide is under the control of a regulatable promoter.
Typically, an assay to determine the effect of a candidate substance identified by the method of the invention on sperm cell motility and/or function comprises administering the candidate substance to a cell and determining whether the substance affects either motility and/or function within the cell.
The concentration of candidate substances used will typically be such that the final concentration in the cells is similar to that described above for the in vitro assays.
In a preferred embodiment, the candidate substance is administered to the cell together with functional SLIRP. A substance that enhances SLIRP may serve to increase sperm cell function and/or motility.
Thus, this invention is also particularly useful for screening compounds by using the SLIRP polypeptide or fragment thereof in any of a variety of drug screening techniques to identify candidate substances that enhance SLIRP function.
The SLIRP polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, for the formation of - 32 - complexes between a SLIRP polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a SLIRP polypeptide or fragment and a known ligand is interfered with by the agent being tested.
Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with a SLIRP polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the SLIRP polypeptide or fragment, or (ii) for the presence of a complex between the SLIRP polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding assays the SLIRP polypeptide or fragment is typically labelled. Free SLIRP polypeptide or fragment is separated from that present in a protein protein, protein:RNA or protein:DNA complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to SLIRP or its interference with SLIRP:ligand binding, respectively.
Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the SLIRP polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on Sep. 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with SLIRP polypeptide and washed. Bound SLIRP polypeptide is then detected by methods well known in the art.
Purified SLIRP can be coated directly onto plates for use in the aforementioned drug screening techniques. However, antibodies to the polypeptide can be used to immobilize the SLIRP polypeptide on the solid phase.
Polynucleotides
The present invention further provides a method for modulating the motility and/or function of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or - 33 -
(vi) a functional variant of any one of (i) to (v).
The present invention further provides the preceding method wherein the polypeptide is encoded by a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19. These polynucleotide sequences are species specific and encode the polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, respectively.
The above are referred herein as SLIRP polynucleotides and may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti- sense strand.
The human SLIRP gene, also known as DC50; PD04872; C14orf156, is present on chromosome 14 (position 14q24.3). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl /SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA.
Reference to "isolated" SLIRP polynucleotide(s) means a polynucleotide, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts of DNA molecules. Isolated SLIRP polynucleotides further include such molecules produced synthetically.
SLIRP polynucleotides include those that comprise a nucleotide sequence different to those specifically described herein but which, due to the degeneracy of the genetic code, still encode the same polypeptide. The genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants of SLIRP polynucleotides such as SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
The present invention also provides fragments of SLIRP polynucleotides. Preferred fragments comprise at least 10, 20, 30, 40, 50, 60 or 70 contiguous nucleotides. Other preferred fragments encode SLIRP polynucleotides with at least one important property of the full length polypeptide or epitope bearing portions of the larger polypeptide. Methods for determining fragments would be readily apparent to one skilled in the art and are exemplified in more detail below. - 34 -
SLIRP polynucleotides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
The present invention may also use isolated polynucleotides that selectively hybridize with at least a portion of a SLIRP polynucleotide. As used herein to describe nucleic acids, the term "selectively hybridize" excludes the occasional randomly hybridizing nucleic acids under at least moderate stringency conditions. Thus, selectively hybridizing polynucleotides preferably hybridize under at least moderate stringency conditions and more preferably under high stringency conditions. The hybridising polynucleotides may be used, for example, as probes or primers for detecting the presence of SLIRP polynucleotides encoding polypeptides in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 such as cDNA or mRNA.
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55 'C, can be used, e.g., 5x SSC, 0.1 % SDS, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5x or 6x SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5x (or less) SCC. SSC Buffer Concentrate (20X) contains 3M sodium chloride and 0.3M sodium citrate (pH 7.0).
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived and are known to those skilled in the art. For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity. Preferably a minimum length for a hybridizable nucleic acid is at - 35 - least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20, 30 or 40-70 nucleotides.
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as a 3' terminal poly(A) tail of a SLIRP polynucleotide), or to a complementary stretch of T (or U) residues, would not be included as a selectively hybridizable SLIRP polynucleotide, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
Using the nucleic acid sequences taught herein and relying on cross-hybridization, one skilled in the art can identify SLIRP polynucleotides in other species that encode SLIRP polypeptides. If used as primers, the invention provides compositions including at least two nucleic acids that selectively hybridize with different regions of the target nucleic acid so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity.
The selectively hybridisable SLIRP polynucleotides described herein or more particularly portions thereof can be used to detect SLIRP nucleic acid in samples by methods such as the polymerase chain reaction, ligase chain reaction, hybridization, and the like. Alternatively, these sequences can be utilized to produce an antigenic protein or protein portion, or an active protein or protein portion.
In addition, portions of the selectively hybridisable SLIRP polynucleotides described herein can be selected to selectively hybridize with homologous SLIRP polynucleotides in other organisms. These selectively hybridisable polynucleotides can be used, for example, to simultaneously detect related sequences for cloning of homologues of SLIRP polynucleotides.
As indicated above, SLIRP polynucleotides that encode a SLIRP polypeptide include, but are not limited to, those encoding the amino acid sequences of the polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Rather, when employed in the methods and uses of the present invention, SLIRP polynucleotides may comprise the coding sequence for the polypeptide and additional sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA - 36 - processing, including splicing and polyadenylation signals, for example ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. SLIRP polynucleotides also include those encoding a polypeptide, such as the entire protein, lacking the N terminal methionine.
Thus, SLIRP polynucleotides include those with a sequence encoding a SLIRP polypeptide fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. The "HA" tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein. Furthermore, the "GFP" tag is another peptide useful for purification and a reporter of expression which corresponds to an epitope for a derived from the jellyfish Aequorea victoria.
The methods and uses of the present invention may use variants of SLIRP nucleic acid molecules which encode variants of SLIRP polypeptides. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Non-naturally occurring variants may be produced using mutagenesis techniques known to those in the art.
Such variants include those produced by nucleotide substitutions, deletions or additions that may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the encoded polypeptide. Also especially preferred in this regard are conservative substitutions.
The present invention may also use isolated SLIRP polynucleotides comprising a nucleotide sequence at least 60, 70, 80 or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence encoding the polypeptide having the complete amino acid sequence in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
For the purposes of the present invention a nucleotide sequence that is 95% identical to a reference sequence is identical to the reference sequence except that it may include - 37 - up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at least 60, 70, 80, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence encoding a polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 5371 1 ). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981 ), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 60, 70, 80, 90, 95, 96, 97, 98 or 99 percent identical to the nucleic acid sequence of the polypeptides in SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 will encode a SLIRP polypeptide. In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison.
It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having one or more properties of the full polypeptide such as SRA binding. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid). - 38 -
Antisense Nucleic Acids and Ribozymes
The present invention also extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of amino acid sequences of SLIRP polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 at the translational level. This approach utilises antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
In this respect, the present invention further provides a method for modulating the motility and/or function of a sperm cell, wherein antisense nucleic acids or siRNAs are used to reduce or eliminate the expression of a SLIRP polynucleotide.
The present invention further provides a method for sterilising a male animal including the step of reducing levels or reducing activity of SLIRP polypeptide within the cell, for example, amongst others, using antisense nucleic acids or siRNAs to reduce or eliminate the expression of a SLIRP polynucleotide in the sperm cells of the male animal. As expression of the SLIRP polynucleotide may be restored upon removal of, for example, the antisense nucleic acids, the sterilising of the male animal can be reversed.
Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule [See: Weintraub, (1990) Sci. Am., 262:40-46; Marcus-Sekura, (1988) Anal. Biochem., 172:289-295]. In the cell, they hybridise to that mRNA, forming a double-stranded molecule. The cell does not translate an mRNA complexed in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridise to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into target cells. Antisense methods have been used to inhibit the expression of many genes in vitro [Hambor et at., (1988) J. Exp. Med., 168:1237-1245]. Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognise specific nucleotide sequences in an RNA molecule and cleave it [Cech, (1988) J. Am. Med. Assoc., 260:3030-3034]. Because they are sequence-specific, only mRNAs with particular sequences are inactivated. - 39 -
Investigators have identified two types of ribozymes, Tetrahymena-type and "hammerhead"-type. Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead"-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species and eighteen base recognition sequences are preferable to shorter recognition sequences.
The SLIRP polynucleotide sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave, mRNAs for amino acid sequences of SLIRP polypeptides of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, thus inhibiting expression of the SLIRP polynucleotide sequences.
Screening Methods
The SLIRP polynucleotides herein may be used to screen for mutations in a gene encoding a SLIRP polypeptide and/or to secure expression of SLIRP polypeptides described herein with SRA binding activity or another biological activity of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Alternatively, such mutations that result in the expression of SLIRP polypeptides described herein which prevent or alter SRA binding activity may also be screened for.
Such mutations may affect expression of a SLIRP polynucleotide and can have an effect on SLIRP polypeptide expression in sperm cells causing dysmotile or dysfunctional sperm. Thus, use of SLIRP polynucleotides and identification of mutations in SLIRP polynucleotides may be used as a biomarker of dysmotile or dysfunctional sperm
A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
cDNA or genomic libraries of various types may be screened as natural sources of SLIRP polynucleotides, or such polynucleotides may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR. The choice of cDNA libraries normally corresponds to a tissue source that is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences. - 40 -
The nucleic acid sequences used in the methods and uses of the invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a polynucleotide sequence of interest.
Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 : "Molecular Cloning: a laboratory manual. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Coldspring Harbour Laboratory Press, Coldspring Harbour, NY or Ausubel et al., 1992 Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.G. and Struhl, K. (1987). John Wiley and Sons, NY. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce SLIRP polypeptides may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridisation conditions are well known in the art.
Detectably labelled nucleic acid molecules hybridisable to a DNA molecule of the invention are also provided and include nucleic acid molecules hybridisable to a non-coding region of a nucleic acid encoding a polypeptide of the present invention, which non-coding region is selected from the group consisting of an intron, a 5' non-coding region, and a 3' non- coding region.
Polynucleotide polymorphisms associated with alleles of the SLIRP polynucleotides of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19 which predispose to certain disorders such as dysmotile or dysfunctional sperm can be detected by hybridisation with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under stringent to moderately stringent hybridisation and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, stringent conditions will be used. Hybridisation stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out - 41 - nonspecific/adventitious bindings, that is, which minimize noise. Since such indications identify neutral DNA polymorphisms as well as mutations, these indications need further analysis to demonstrate detection of a disorder susceptible allele.
Probes for the alleles may be derived from the sequences of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19 or its corresponding gene. The probes may be of any suitable length, which span all or a portion of the gene or SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and which allow specific hybridisation to a region of interest. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridises to the target sequence with the requisite specificity.
The probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g. Sambrook et al., 1989 : "Molecular Cloning: a laboratory manual. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Coldspring Harbour Laboratory Press, Coldspring Harbour, NY or Ausubel et al., 1992 Current Protocols in Molecular Biology. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.G. and Struhl, K. (1987). John Wiley and Sons, NY. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change ligand-binding affinities, interchain affinities, or the polypeptide degradation or turnover rate.
Probes comprising synthetic oligonucleotides or other SLIRP polynucleotides may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labelled by nick translation, Klenow fill-in reaction, or other methods known in the art.
Portions of the SLIRP polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1 .0 kb, from a polynucleotide sequence encoding a SLIRP polypeptide are preferred as probes. The probes may also be used to determine whether mRNA - 42 - encoding the polypeptide is present in a cell or tissue and whether the genomic organisation of the genes locus is deleted or otherwise damaged.
A variety of DNA technologies may thus be used to identify mutant alleles in a range of individuals. A number of these alleles may comprise minor alterations to the genomic sequence, such as point mutations including insertions deletions and/or substitutions. Fragments of nucleic acid which comprise these mutations may be used in diagnostic screening as described below. Accordingly, the present invention provides one or more SLIRP polynucleotides or fragments thereof as described herein comprising mutations with respect to the wild type sequence. In a further embodiment, the present invention provides a plurality of SLIRP polynucleotides or fragments thereof as described herein for use in screening the DNA of an individual for the presence of one or more mutations/polymorphisms. The plurality of sequences is conveniently provided immobilized to a solid substrate as is described below.
Nucleic acid arrays
SLIRP polynucleotides of the invention, including probes that may be used to detect both normal (wild type) and abnormal SLIRP sequences in nucleic acid samples, may be immobilized to a solid phase support. The probes will typically form part of a library of DNA molecules that may be used to detect simultaneously a number of different genes in a given genome.
Techniques for producing immobilised libraries of DNA molecules have been described in the art. Generally, most prior art methods describe the synthesis of single-stranded nucleic acid molecule libraries, using for example masking techniques to build up various permutations of sequences at the various discrete positions on the solid substrate. U.S. Patent No. 5,837,832, the contents of which are incorporated herein by reference, describes an improved method for producing DNA arrays immobilised to silicon substrates based on very large scale integration technology. In particular, U.S. Patent No. 5,837,832 describes a strategy called "tiling" to synthesize specific sets of probes at spatially-defined locations on a substrate which may be used to produce the immobilised DNA libraries of the present invention. U.S. Patent No. 5,837,832 also provides references for earlier techniques that may also be used. Thus, nucleic acid probes may be synthesised in situ on the surface of the substrate.
Alternatively, single-stranded molecules may be synthesised off the solid substrate and each pre-formed sequence applied to a discrete position on the solid substrate. For example, nucleic acids may be printed directly onto the substrate using robotic devices equipped with either pins or pizo electric devices. - 43 -
The library sequences are typically immobilised onto or in discrete regions of a solid substrate. The substrate may be porous to allow immobilisation within the substrate or substantially non-porous, in which case the library sequences are typically immobilised on the surface of the substrate. The solid substrate may be made of any material to which polypeptides can bind, either directly or indirectly. Examples of suitable solid substrates include flat glass, silicon wafers, mica, ceramics and organic polymers such as plastics, including polystyrene and polymethacrylate. It may also be possible to use semi-permeable membranes such as nitrocellulose or nylon membranes, which are widely available. The semi-permeable membranes may be mounted on a more robust solid surface such as glass. The surfaces may optionally be coated with a layer of metal, such as gold, platinum or other transition metal. A particular example of a suitable solid substrate is the commercially available BiaCore™ chip (Pharmacia Biosensors™).
Preferably, the solid substrate is generally a material having a rigid or semi-rigid surface. In preferred embodiments, at least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different polymers with, for example, raised regions or etched trenches. It is also preferred that the solid substrate is suitable for the high density application of DNA sequences in discrete areas of typically from 50 to 100 μηη, giving a density of 10000 to 40000 cm 2.
The solid substrate is conveniently divided up into sections. This may be achieved by techniques such as photoetching, or by the application of hydrophobic inks, for example teflon-based inks (Cel-line™, USA).
Discrete positions, in which each different member of the library is located may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
Attachment of the nucleic acid sequences to the substrate may be by covalent or non- covalent means. The nucleic acid sequences may be attached to the substrate via a layer of molecules to which the library sequences bind. For example, the nucleic acid sequences may be labelled with biotin and the substrate coated with avidin and/or streptavidin. A convenient feature of using biotinylated nucleic acid sequences is that the efficiency of coupling to the solid substrate can be determined easily. Since the nucleic acid sequences may bind only poorly to some solid substrates, it is often necessary to provide a chemical interface between the solid substrate (such as in the case of glass) and the nucleic acid sequences. Examples of suitable chemical interfaces include hexaethylene glycol. Another example is the use of polylysine coated glass, the polylysine then being chemically modified using standard procedures to - 44 - introduce an affinity ligand. Other methods for attaching molecules to the surfaces of solid substrate by the use of coupling agents are known in the art, see for example W098/49557.
Binding of complementary nucleic acid sequence to the immobilised nucleic acid library may be determined by a variety of means such as changes in the optical characteristics of the bound nucleic acid (i.e. by the use of ethidium bromide) or by the use of labelled nucleic acids, such as polypeptides labelled with fluorophores. Other detection techniques that do not require the use of labels include optical techniques such as optoacoustics, reflectometry, ellipsometry and surface plasmon resonance (SPR) - see W097/49989, incorporated herein by reference.
Thus, the present invention provides a solid substrate having immobilized thereon at least one SLIRP polynucleotide, preferably two or more different SLIRP polynucleotides, for example two or more different polynucleotides corresponding to different alleles. In a preferred embodiment the solid substrate further comprises polynucleotides derived from genes other than the SLIRP gene.
High throughput expression profiling has a broad range of applications with respect to SLIRP polypeptides, including, but not limited to: the identification and validation of disease-related genes as targets for therapeutics; molecular toxicology of polypeptides of the invention and inhibitors thereof; stratification of populations and generation of surrogate markers for clinical trials; and enhancing polypeptide related small molecule drug discovery by aiding in the identification of selective compounds in high throughput screens (HTS), particularly with respect to disorders affecting the function and activity of sperm affecting fertility.
Vectors and Host Cells
The step of increasing SLIRP polypeptide levels within the sperm or other cell may be accomplished by plasmid mediated transfection of the cell with vectors including SLIRP polynucleotides that are able to express SLIRP polypeptides within the cell.
Plasmid mediated transfection of target cells has been used to increase wild type and amino and carboxy terminal tagged SLIRP over expression. This has been achieved using CMV promoter driven vectors eg pFLAG-CMV™-5a (carboxy tag), p3xFLAG- CMV™-7.1 (amino tag), both from Sigma-Aldrich™. Also pcDNA4/TO derivative vectors from Invitrogen™ to express carboxy terminal tagged SLIRP.
The step of decreasing SLIRP polypeptide levels within the cell may be accomplished using siRNA technologies to deplete expression of SLIRP polypeptides of the invention from the cell. - 45 -
SLIRP levels have been depleted using siRNA technologies as described in Hatchell et al., 2006 using reagents supplied by Dharmacon, Inc.™, specific for the SLIRP gene.
A nucleic acid molecule encoding the amino acid sequence of a SLIRP polypeptide may be inserted into an appropriate expression vector using standard ligation techniques. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur). A nucleic acid molecule encoding the amino acid sequence of polypeptide herein may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems), and/or eukaryotic host cells. Selection of the host cell will depend in part on whether the polypeptide is to be post-translationally modified (e.g., glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable.
Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" in certain embodiments, will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.
Optionally, the vector may contain a "tag"-encoding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another "tag" such as FLAG, HA (hemaglutinin influenza virus) or myc for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified polypeptide by various means such as using certain peptidases for cleavage.
Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source) or synthetic, or the flanking sequences may be native sequences that normally function to - 46 - regulate polypeptide expression. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.
The flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein other than the gene flanking sequences will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
Where all or only a portion of the flanking sequence is known, it may be obtained using PCR and/or by screening a genomic library with suitable oligonucleotide and/or flanking sequence fragments from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for the optimal expression of the polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).
A transcription termination sequence is typically located 3' of the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination - 47 - sequence in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.
A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.
Other selection genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes which are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The mammalian cell transformants are placed under selection pressure which only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to the amplification of both the selection gene and the DNA that encodes a polypeptide described herein. As a result, increased quantities of the polypeptide are synthesized from the amplified DNA.
A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. The Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth herein and used in a prokaryotic vector.
A leader, or signal, sequence may be used to direct the polypeptide out of the host cell. Typically, a nucleotide sequence encoding the signal sequence is positioned in the coding region of the nucleic acid molecule encoding the polypeptide, or directly at the 5' end of the polypeptide coding region. Many signal sequences have been identified, and any of those that are functional in the selected host cell may be used in conjunction with - 48 - the nucleic acid molecule. Therefore, a signal sequence may be homologous (naturally occurring) or heterologous to the gene or cDNA encoding the polypeptide. Additionally, a signal sequence may be chemically synthesized using methods described herein. In most cases, the secretion of the polypeptide from the host cell via the presence of a signal peptide will result in the removal of the signal peptide from the secreted polypeptide. The signal sequence may be a component of the vector, or it may be a part of the nucleic acid molecule that is inserted into the vector.
Included within the scope of this invention is the use of either a nucleotide sequence encoding a native signal sequence joined to a polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence joined to a polypeptide coding region. The heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell. For prokaryotic host cells that do not recognize and process the native polypeptide signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders. For yeast secretion, the native polypeptide signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders. In mammalian cell expression the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.
In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various presequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add presequences, which also may affect glycosylation. The final protein product may have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incidental to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the N- terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.
In many cases, transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is particularly true where a polypeptide is produced in eukaryotic host cells, especially mammalian host cells. The introns used may be naturally occurring within the gene, especially where the gene used is a full length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the gene (as for most cDNAs), the intron(s) may be obtained from - 49 - another source. The position of the intron with respect to flanking sequences and the gene is generally important, as the intron must be transcribed to be effective. Thus, when a cDNA molecule is being transcribed, the preferred position for the intron is 3' to the transcription start site, and 5' to the polyA transcription termination sequence. Preferably, the intron or introns will be located on one side or the other (i.e., 5' or 3') of the cDNA such that it does not interrupt the coding sequence. Any intron from any source, including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell(s) into which it is inserted. Also included herein are synthetic introns. Optionally, more than one intron may be used in the vector.
The expression and cloning vectors that may be used in the methods and uses of the present invention will each typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the polypeptide. Promoters are untranscribed sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes, inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, initiate continual gene product production; that is, there is little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding the polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. The native gene promoter sequence may be used to direct amplification and/or expression of a nucleic acid molecule. A heterologous promoter is preferred, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter, and if it is compatible with the host cell system that has been selected for use.
Promoters suitable for use with prokaryotic hosts include the beta- lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence(s), using linkers or adapters as needed to supply any useful restriction sites. - 50 -
Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, e.g., heat-shock promoters and the actin promoter.
Additional promoters which may be of interest in controlling gene transcription include, but are not limited to: the SV40 early promoter region; the CMV promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus; the herpes thymidine kinase promoter, the regulatory sequences of the metallothionine gene, prokaryotic expression vectors such as the beta-lactamase promoter; or the tac promoter. Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region which is active in pancreatic acinar cells; the insulin gene control region which is active in pancreatic beta cells; the immunoglobulin gene control region which is active in lymphoid cells; the mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells; the albumin gene control region which is active in liver; the alphafetoprotein gene control region which is active in liver; the alpha 1 -antitrypsin gene control region which is active in the liver; the beta-globin gene control region which is active in myeloid cells; the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain; the myosin light chain-2 gene control region which is active in skeletal muscle; and the gonadotropic releasing hormone gene control region which is active in the hypothalamus.
An enhancer sequence may be inserted into the vector to increase the transcription of a DNA encoding a SLIRP polypeptide by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto- protein and insulin). Typically, however, an enhancer from a virus will be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically located at a site 5' from the promoter. - 51 -
Expression vectors may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the desired flanking sequences are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
Preferred vectors for practicing this invention are those that are compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, Carlsbad, CA), pBSII (Stratagene Company, La Jolla, CA), pET15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacll; Invitrogen), pDSR-alpha (PCT Publication No. WO 90/14363) and pFastBacDual (Gibco/BRL, Grand Island, NY).
Additional suitable vectors include, but are not limited to, cosmids, plasmids or modified viruses, but it will be appreciated that the vector system must be compatible with the selected host cell. Such vectors include, but are not limited to, plasmids such as
®
Bluescript plasmid derivatives (a high copy number ColE1 -based phagemid, Stratagene Cloning Systems Inc., La Jolla CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products {e.g., TOPO™ TA Cloning® Kit, pCR2.1 ® plasmid derivatives, Invitrogen, Carlsbad, CA), and mammalian, yeast, or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives, Clontech, Palo Alto, CA). Further suitable vectors include inducible vectors, for example, amongst others, heavy metal, tetracycline or estrogen inducible vectors. [SC: please include specific examples you have used or could be used inc company info]
After the vector has been constructed and a nucleic acid molecule encoding the polypeptide has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for a polypeptide into a selected host cell may be accomplished by well known methods including transfection, infection, calcium chloride, calcium phosphate, electroporation, microinjection, lipofection or the DEAE- dextran method or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et at., supra. - 52 -
Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell). The host cell, when cultured under appropriate conditions, synthesizes the polypeptide that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity, such activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.
A number of suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 201 10-2209. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61 ); CHO DHFR-cells (Uriaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)); human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573); or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, product production and purification are known in the art. Other suitable mammalian cell lines, are the monkey COS-1 (ATCC No. CRL1650) and COS-7cell lines (ATCC No. CRL1651 ) cell lines, and the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which are available from the ATCC. Each of these cell lines is known by and available to those skilled in the art of protein expression.
Similarly useful as host cells suitable for the methods and uses of the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101 , (ATCC No. 33694) DH5a, DH10, and MC1061 (ATCC No. 53338)) are well-well known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the like may also be employed in this method.
Many strains of yeast cells known to those skilled in the art are also available as host cells for the expression of SLIRP polypeptides. Preferred yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris. - 53 -
Additionally, where desired, insect cell systems may be utilized in the methods of the present invention. Such systems are described for example in Kitts et at., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. {J. Virol., 67:4566-4579 (1993). Preferred insect cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).
One may also use transgenic animals to express glycosylated SLIRP polypeptides or variants thereof. For example, one may use a transgenic milk-producing animal (a cow or goat, for example) and obtain the present glycosylated SLIRP polypeptide in the animal milk. One may also use plants to produce polypeptides. However, in general, the glycosylation occurring in plants is different from that produced in mammalian cells, and may result in a glycosylated product which is not suitable for human therapeutic use.
Pharmaceutical Compositions
Therapeutic compositions are within the scope of the methods and uses of the present invention. Such compositions may comprise a therapeutically effective amount of a SLIRP polypeptide or SLIRP polynucleotide including those described herein in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration. Pharmaceutical compositions may also comprise a therapeutically effective amount of one or more selective binding agents described herein in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
Benefits of methods using such pharmaceutical compositions described herein may include increasing SLIRP levels within a sperm cell in able to restore function and motility to the sperm cell.
The pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colouring, - 54 - flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.
The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the active agent.
The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. In one embodiment of the present invention, pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the polypeptide product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
The pharmaceutical compositions can be capable of parenteral delivery. Alternatively, the compositions may be capable of inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. - 55 -
When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired polypeptide or nucleotide in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the active agent is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid, acid or polyglycolic acid), or beads or liposomes, that provides for the controlled or sustained release of the product which may then be delivered as a depot injection. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
In one embodiment, a pharmaceutical composition may be formulated for inhalation. For example, a polypeptide or nucleotide may be formulated as a dry powder for inhalation. The polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery. In yet another embodiment, solutions may be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. It is also contemplated that certain formulations may be administered orally. In one embodiment of the present invention, SLIRP polypeptides or SLIRP nucleotides that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the active agent. Diluents, flavourings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
Another pharmaceutical composition may involve an effective quantity of the polypeptide or nucleotide in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. - 56 -
Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving polypeptides in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 that describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Additional examples of sustained-sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, ethylene vinyl acetate or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.
The pharmaceutical composition to be used for in vivo administration typically must be sterile. This may be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using these methods may be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
The effective amount of the active agent in the pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the active agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titre the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.1 μg kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 μg kg up to about 100 mg/kg; or 1 μg kg up to about 100 mg/kg; or 5 μg kg up to about 100 mg/kg. - 57 -
The frequency of dosing will depend upon the pharmacokinetic parameters of the active agent and the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implants. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
Alternatively or additionally, the composition may be administered locally via implantation of a membrane, sponge or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, such as the scrotum, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
In some cases, it may be desirable to use the pharmaceutical compositions herein in an ex vivo manner. In such instances, cells, tissues, or organs such as the testis that have been removed from the patient are exposed to the pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient.
Gene/Cell Therapy
An active agent herein such as a polypeptide or selective binding agent can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide or selective binding agent. Such cells may be animal or human cells, and may be autologous, heterologous, or xenogenic. Optionally, the cells may be immortalized. In order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. The encapsulation materials are typically biocompatible, semipermeable polymeric enclosures or membranes that allow the release of the protein - 58 - product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
Additional embodiments of the methods and uses of the present invention relate to cells and methods (e.g., homologous recombination and/or other recombinant production methods) for both the in vitro production of therapeutic polypeptides and for the production and delivery of therapeutic polypeptides by gene therapy or cell therapy. Homologous and other recombination methods may be used to modify a cell that contains a normally transcriptionally silent gene encoding a polypeptide described herein, or an under expressed gene, and thereby produce a cell which expresses therapeutically efficacious amounts of the polypeptides.
This may assist to restore function and/or motility to sperm cell which are, for example, amongst others, affected by low levels or mutated SLIRP polypeptides.
Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes. The basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome or to correct specific mutations within defective genes. Through homologous recombination, a given DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to targeting DNA. The targeting DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process.
It is a general property of DNA that has been inserted into a cell to hybridize, and therefore, recombine with other pieces of endogenous DNA through shared homologous regions. If this complementary strand is attached to an oligonucleotide that contains a mutation or a different sequence or an additional nucleotide, it too is incorporated into the newly synthesized strand as a result of the recombination. As a result of the proofreading function, it is possible for the new sequence of DNA to serve as the template. Thus, the transferred DNA is incorporated into the genome. Attached to these pieces of targeting DNA are regions of DNA that may interact with or control the expression of a polypeptide herein, e.g., flanking sequences. For example, a promoter/enhancer element, a suppresser or an exogenous transcription modulatory element is inserted in the genome of the intended host cell in proximity and orientation sufficient to influence the transcription of DNA encoding the desired polypeptide. The control element controls a portion of the DNA present in the host cell genome. Thus, the expression of the desired SLIRP polypeptide may be achieved not by transfection of - 59 -
DNA that encodes the polypeptide itself, but rather by the use of targeting DNA (containing regions of homology with the endogenous gene of interest), coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals for transcription of the gene encoding the polypeptide.
In an exemplary method, the expression of a desired targeted gene in a cell (i.e., a desired endogenous cellular gene) is altered via homologous recombination into the cellular genome at a preselected site, by the introduction of DNA that includes at least a regulatory sequence, an exon and a splice donor site. These components are introduced into the chromosomal (genomic) DNA in such a manner that this, in effect, results in the production of a new transcription unit (in which the regulatory sequence, the exon and the splice donor site present in the DNA construct are operatively linked to the endogenous gene). As a result of the introduction of these components into the chromosomal DNA, the expression of the desired endogenous gene is altered.
Altered gene expression, as described herein, encompasses activating (or causing to be expressed) a gene which is normally silent (unexpressed) in the cell as obtained, as well as increasing the expression of a gene which is not expressed at physiologically significant levels in the cell as obtained. The embodiments further encompass changing the pattern of regulation or induction such that it is different from the pattern of regulation or induction that occurs in the cell as obtained, and reducing (including eliminating) the expression of a gene which is expressed in the cell as obtained.
One method by which homologous recombination can be used to increase, or cause production of a SLIRP polypeptide described herein from a cell's endogenous gene involves first using homologous recombination to place a recombination sequence from a site-specific recombination system (e.g., Cre/loxP, FLP/FRT) (see, Sauer, Current Opinion In Biotechnology, 5:521 -527, 1994; and Sauer, Methods In Enzymology, 225:890-900, 1993) upstream (that is, 5' to) of the cell's endogenous genomic polypeptide coding region. A plasmid containing a recombination site homologous to the site that was placed just upstream of the genomic polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme. This recombinase enzyme causes the plasmid to integrate, via the plasmid's recombination site, into the recombination site located just upstream of the genomic polypeptide coding region in the cell line (Baubonis and Sauer, Nucleic Acids Res., 21 :2025-2029, 1993; and O'Gorman et al., Science, 251 : 1351 -1355, 1991 ). Any flanking sequences known to increase transcription (e.g., enhancer/promoter, intron or translational enhancer), if properly positioned in this plasmid, would integrate in such a - 60 - manner as to create a new or modified transcriptional unit resulting in de novo or increased polypeptide production from the cell's endogenous gene.
A further method to use the cell line in which the site-specific recombination sequence has been placed just upstream of the cell's endogenous genomic polypeptide coding region is to use homologous recombination to introduce a second recombination site elsewhere in the cell line's genome. The appropriate recombinase enzyme is then introduced into the two-recombination-site cell line, causing a recombination event (deletion, inversion or translocation) (Sauer, Current Opinion In Biotechnology, supra, 1994 and Sauer, Methods In Enzymology, supra, 1993) that would create a new or modified transcriptional unit resulting in de novo or increased polypeptide production from the cell's endogenous gene.
Another approach for increasing, or causing, the expression of the polypeptide from a cell's endogenous gene involves increasing, or causing, the expression of a gene or genes (e.g., transcription factors) and/or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner which results in de novo or increased polypeptide production from the cell's endogenous gene. This method includes the introduction of a non-naturally occurring polypeptide (e.g., a polypeptide comprising a site-specific DNA binding domain fused to a transcriptional factor domain) into the cell such that de novo or increased polypeptide production from the cell's endogenous gene results.
The present invention may further employ DNA constructs useful in the method of altering expression of a target gene. In certain embodiments, the exemplary DNA constructs comprise: (a) one or more targeting sequences; (b) a regulatory sequence; (c) an exon; and (d) an unpaired splice-donor site. The targeting sequence in the DNA construct directs the integration of elements (a)-(d) into a target gene in a cell such that the elements (b)-(d) are operatively linked to sequences of the endogenous target gene. In another embodiment, the DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)-(f) such that the elements of (b)-(f) are operatively linked to the endogenous gene. The targeting sequence is homologous to the preselected site in the cellular chromosomal DNA with which homologous recombination is to occur. In the construct, the exon is generally 3' of the regulatory sequence and the splice-donor site is 3' of the exon.
If the sequence of a particular gene is known, such as the nucleic acid sequence of the polypeptides presented herein, a piece of DNA that is complementary to a selected - 61 - region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest. This piece serves as a targeting sequence(s) upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be incorporated into the newly synthesized daughter strand of DNA. The methods and use of the present invention, therefore, includes nucleotides encoding a polypeptide, which nucleotides may be used as targeting sequences.
Polypeptide cell therapy, e.g., the implantation of cells producing polypeptides described herein, is also contemplated. This embodiment involves implanting cells capable of synthesizing and secreting a biologically active form of the polypeptide. Such polypeptide-producing cells can be cells that are natural producers of the polypeptides or may be recombinant cells whose ability to produce the polypeptides has been augmented by transformation with a gene encoding the desired polypeptide or with a gene augmenting the expression of the polypeptide. Such a modification may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion. In order to minimize a potential immunological reaction in patients being administered a polypeptide, as may occur with the administration of a polypeptide of a foreign species, it is preferred that the natural cells producing polypeptide be of human origin and produce human polypeptide. Likewise, it is preferred that the recombinant cells producing polypeptide be transformed with an expression vector containing a gene encoding a human polypeptide.
Implanted cells may be encapsulated to avoid the infiltration of surrounding tissue. Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or in membranes that allow the release of polypeptide, but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue. Alternatively, the patient's own cells, transformed to produce polypeptides ex vivo, may be implanted directly into the patient without such encapsulation.
Techniques for the encapsulation of living cells are known in the art, and the preparation of the encapsulated cells and their implantation in patients may be routinely accomplished. For example, Baetge et al. (WO 95/05452 and PCT/US94/09299) describe membrane capsules containing genetically engineered cells for the effective delivery of biologically active molecules. The capsules are biocompatible and are easily retrievable. The capsules encapsulate cells transfected with recombinant DNA - 62 - molecules comprising DNA sequences coding for biologically active molecules operatively linked to promoters that are not subject to down-regulation in vivo upon implantation into a mammalian host. The devices provide for the delivery of the molecules from living cells to specific sites within a recipient. A system for encapsulating living cells is described in PCT Application no. PCT/US91/00157 of Aebischer et al. See also, PCT Application no. PCT/US91/00155 of Aebischer et al..; Winn et al., Exper. Neurol., 1 13:322-329 (1991 ), Aebischer et al., Exper. Neurol., rM :269-275 (1991 ); and Tresco et al., ASAIO, 38:17-23 (1992).
In vivo and in vitro gene therapy delivery of SLIRP polypeptides are also part of the methods and use of the present invention. One example of a gene therapy technique is to use the gene (either genomic DNA, cDNA, and/or synthetic DNA) encoding a SLIRP polypeptide described herein that may be operably linked to a constitutive or inducible promoter to form a "gene therapy DNA construct". The promoter may be homologous or heterologous to the endogenous gene, provided that it is active in the cell or tissue type into which the construct will be inserted. Other components of the gene therapy DNA construct may optionally include, DNA molecules designed for site-specific integration (e.g., endogenous sequences useful for homologous recombination); tissue-specific promoter, enhancer(s) or silencer(s); DNA molecules capable of providing a selective advantage over the parent cell; DNA molecules useful as labels to identify transformed cells; negative selection systems, cell specific systems; cell-specific binding agents (as, for example, for cell targeting); cell-specific internalization factors; and transcription factors to enhance expression by a vector, as well as factors to enable vector manufacture.
A gene therapy DNA construct can then be introduced into cells (either ex vivo or in vivo) using viral or non-viral vectors. Certain vectors, such as retroviral vectors, will deliver the DNA construct to the chromosomal DNA of the cells, and the gene can integrate into the chromosomal DNA. Other vectors will function as episomes, and the gene therapy DNA construct will remain in the cytoplasm.
In yet other embodiments, regulatory elements can be included for the controlled expression of the gene in the target cell. Such elements are turned on in response to an appropriate effector. In this way, a therapeutic polypeptide can be expressed when desired. One conventional control means involves the use of small molecule dimerizers or rapalogs (as described in WO 9641865 (PCT/US96/099486); WO 9731898 (PCT/US97/03137) and W09731899 (PCT/US95/03157) used to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating biological process, such as a DNA-binding protein or a transcriptional activation - 63 - protein. The dimerization of the proteins can be used to initiate transcription of the transgene.
An alternative regulation technology uses a method of storing proteins expressed from the gene of interest inside the cell as an aggregate or cluster. The gene of interest is expressed as a fusion protein that includes a conditional aggregation domain that results in the retention of the aggregated protein in the endoplasmic reticulum. The stored proteins are stable and inactive inside the cell. The proteins can be released, however, by administering a drug (e.g., small molecule ligand) that removes the conditional aggregation domain and thereby specifically breaks apart the aggregates or clusters so that the proteins may be secreted from the cell.
Another control means uses a positive tetracycline-controllable transactivator. This system involves a mutated tet repressor protein DNA-binding domain (mutated tet R- 4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline) linked to a polypeptide that activates transcription.
In vivo gene therapy may be accomplished by introducing the gene encoding a polypeptide into cells via local injection of a nucleic acid molecule or by other appropriate viral or non-viral delivery vectors. For example, a nucleic acid molecule encoding a SLIRP polypeptide may be contained in an adeno-associated virus (AAV) vector for delivery to the targeted cells (e.g., Johnson, International Publication No. WO95/34670; and International Application No. PCT/US95/07178). The recombinant AAV genome typically contains AAV inverted terminal repeats flanking a DNA sequence encoding a polypeptide operably linked to functional promoter and polyadenylation sequences.
Alternative suitable viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma virus vectors. U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector. U.S. Patent No. 5,399,346 provides examples of a process for providing a patient with a therapeutic protein by the delivery of human cells that have been treated in vitro to insert a DNA segment encoding a therapeutic protein. Additional methods and materials for the practice of gene therapy techniques are described in U.S. Patent No. 5,631 ,236 involving adenoviral vectors; U.S. Patent No. 5,672,510 involving retroviral vectors; and U.S. 5,635,399 involving retroviral vectors expressing cytokines. - 64 -
Nonviral delivery methods include, but are not limited to, liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene gun). Gene therapy materials and methods may also include the use of inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site- specific integration, DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture. Such additional methods and materials for the practice of gene therapy techniques are described in U.S. Patent No. 4,970,154 involving electroporation techniques; WO96/40958 involving nuclear ligands; U.S. Patent No. 5,679,559 describing a lipoprotein-containing system for gene delivery; U.S. Patent No. 5,676,954 involving liposome carriers; U.S. Patent No. 5,593,875 concerning methods for calcium phosphate transfection; and U.S. Patent No. 4,945,050 wherein biologically active particles are propelled at cells at a speed whereby the particles penetrate the surface of the cells and become incorporated into the interior of the cells.
It is also contemplated that gene therapy or cell therapy can further include the delivery of one or more additional SLIRP polypeptide(s) in the same or a different cell(s), particularly sperm cells. Such cells may be separately introduced into the patient, or the cells may be contained in a single implantable device, such as the encapsulating membrane described above, or the cells may be separately modified by means of viral vectors.
A means to increase endogenous polypeptide expression in a cell via gene therapy is to insert one or more enhancer element into the polypeptide promoter, where the enhancer element(s) can serve to increase transcriptional activity of the gene. The enhancer element(s) used will be selected based on the tissue in which one desires to activate the gene(s); enhancer elements known to confer promoter activation in that tissue will be selected. Here, the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing the polypeptide promoter (and optionally, inserted into a vector and/or 5' and/or 3' flanking sequence(s), etc.) using standard cloning techniques. This construct, known as a "homologous recombination construct", can then be introduced into the desired cells either ex vivo or in vivo.
Gene therapy also can be used to decrease polypeptide expression by modifying the nucleotide sequence of the endogenous promoter(s). Such modification is typically - 65 - accomplished via homologous recombination methods. For example, a DNA molecule containing all or a portion of the promoter of the gene selected for inactivation can be engineered to remove and/or replace pieces of the promoter that regulate transcription. For example the TATA box and/or the binding site of a transcriptional activator of the promoter may be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity thereby repressing the transcription of the corresponding gene. The deletion of the TATA box or the transcription activator binding site in the promoter may be accomplished by generating a DNA construct comprising all or the relevant portion of the polypeptide promoter(s) (from the same or a related species as the polypeptide gene to be regulated) in which one or more of the TATA box and/or transcriptional activator binding site nucleotides are mutated via substitution, deletion and/or insertion of one or more nucleotides. As a result, the TATA box and/or activator binding site has decreased activity or is rendered completely inactive. The construct will typically contain at least about 500 bases of DNA that correspond to the native (endogenous) 5' and 3' DNA sequences adjacent to the promoter segment that has been modified. The construct may be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described herein. Typically, the integration of the construct into the genomic DNA of the cells will be via homologous recombination, where the 5' and 3' DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.
Methods of Treatment
The identification and characterisation of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 as a corepressor of SRA mediated nuclear receptor coactivation and the recognition of the role of SLIRP in sperm motility and function means that there are a range of diseases and disorders, including those associated with fertility that may be treated using therapies based on the polypeptides described herein. This includes either reducing SLIRP levels or using a SLIRP antagonist to reduce fertility and function as a contraceptive, as well as increasing SLIRP levels or using an agonist to enhance fertility and the chances of successful conception.
Thus, the present invention provides a method for treating a disorder associated with an undesirable level of function and/or motility of sperm cells, the method comprising the step of administering an effective amount of an isolated polypeptide comprising:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2; - 66 -
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or
(vi) a functional variant of any one of (i) to (v).
Therapeutic methods in accordance with the present invention will vary depending on whether the intention is to increase, decrease or remove the amount and/or physiological effects of the SLIRP polypeptides described herein, such as SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Hereunder is a range of methods that may be used to increase, decrease or remove the effects of the polypeptide as deemed appropriate by a clinician with a view to achieving a therapeutic outcome.
Thus, the present invention also provides a method of treating a subject suffering from a disorder associated with undesirable physiological levels of a SLIRP polypeptide including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 comprising the step of manipulating the physiological levels of the polypeptide. This manipulation may be intended to affect the activity of downstream signalling pathways and thereby the function and/or motility within sperm cells of the subject being treated.
The physiological levels of the polypeptide can be increased or decreased as required to treat particular disorders. These increases or decreases can be achieved using the polypeptides, polynucleotides and/or selective binding agents described herein. These agents are capable of increasing or decreasing the endogenous production of the polypeptide or can be administered directly to increase or decrease the physiological levels of the polypeptide using the methods described herein. For example, selective binding agents, such as antibodies could be administered to decrease the physiological levels of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 by binding the polypeptide to prevent binding with SRA.
It may be preferable to administer the SLIRP polypeptides, polynucleotides or binding agents of the present invention in combination with other therapeutic agents that are useful for treating a given disease or disorder. Such combinations could use conjugates comprising the polypeptide or the therapy could be concomitant or involve the sequential administration of the agents. For example, amongst others, chemotherapeutic agents and/or radiotherapy may be administered in combination with polypeptides, polynucleotides or binding agents of the present invention.
The polypeptides and/or polynucleotides herein may be used in combination with one or more agents that are adapted to interfere with the natural action of androgens. These - 67 - agents include the androgen receptor antagonists such as flutamide and bicalutamide. Other combination partners include agents that block other receptors on cancer cells that may be responsible for cell proliferation e.g. agents that block erbB-2 or EGF- receptor. Estrogen antagonists are known to those skilled in the art and may be selected from the group consisting of: SERMs and ICI 182,780 (also Faslodex, AstraZeneca). Other inhibitors of estrogen production include aromatase inhibitors (e.g. Letrozole). These may also represent good combination partners for therapy according to the present invention.
Other potential therapies that could be combined with SLIRP based therapies include inhibitors of the tyrosine kinase signalling pathway (EGF-receptor monoclonal antibodies) or small molecule inhibitors such as Iressa®. Inhibitors of the erbB-2 pathway may also be used (e.g. Herceptin®)
The effect of the administered therapeutic composition can be monitored by standard diagnostic procedures. For example, the patient outcomes from the administration of a therapeutic composition can be monitored by assessing any one or more of the recognised markers for disease progression.
The polypeptides may be administered as a therapeutic or a prophylactic depending on the particular circumstances and as deemed appropriate by a medical practitioner.
In another embodiment of the invention, fertility treatment for a male subject may comprise enhancing the expression of SLIRP polypeptides in sperm cells of the subject ex vivo. This could be achieved by transiently expressing SLIRP in sperm obtained from infertile men prior to its use in a range of in vitro fertilization techniques (eg ICSI, intracytoplasmic sperm injection).
Diagnostics/Prognostics
The present invention also provides the use of a polypeptide as a biomarker for identification of dysmotile and/or dysfunctional sperm, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and - 68 -
(vi) a functional variant of any one of (i) to (v).
In another embodiment, the present invention provides the use of a polypeptide as a biomarker for identification of energy production in a sperm cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ϋ) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
Microsatellite marker information (against which primers can be developed and comparative analysis can occur) can be obtained from the Genethon Genetic Linkage Map (http://www.genethon.fr) and the Johns Hopkins University Biolnformatics Genome Database (http://www.bis.med.jhmi.edu/). Their relative order can be determined via The Center of Medical Genetics Database
(http://www2.marshfieldclinic.org/RESEARCH/GENETICS/).
The present invention also provides a method for performing a diagnosis on a patient for a disorder associated with undesirable levels of motility and/or function of the sperm of the patient, comprising:
(i) determining the concentration of a polypeptide described herein in a biological sample, taken from the patient;
(ii) comparing the level determined in step (i) to the concentration range of the polypeptide known to be present in normal subjects; and
(iii) diagnosing whether the patient has the disorder based on the comparison in step (ii).
Diagnostic information may be provided for a male subject wherein, for example, decreased expression of SLIRP polypeptides including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 in the subject's sperm cells, as compared to expression levels in normally functioning sperm cells, may indicate that the subject's sperm have reduced motility and/or function. - 69 -
The diagnostic method of the present invention can be used for any disorder associated with undesirable levels of motility and/or function of the sperm of a patient. Preferably, step (a) above is performed using a binding agent described herein.
The diagnostic method may be applied to patients known to be suffering from a disorder associated with undesirable levels of motility and/or function of the patient's sperm with a view to assessing their response to treatment for the disorder by modulation of the levels of the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. One method for monitoring levels involves using antibodies including monoclonal antibodies to the polypeptide according to SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 as described herein.
Furthermore, the present invention may be applied to assess the prognosis of a patient with undesirable levels of motility and/or function of the patient's sperm. Thus, the present invention also provides a method for prognostic evaluation of a patient comprising:
(i) determining the concentration of a polypeptide described herein in a biological sample, taken from the patient;
(ii) comparing the level determined in step (i) to the concentration range of the polypeptide known to be present in normal subjects; and
(iii) evaluating the prognosis of said patient based on the comparison in step (ii). According to the present invention, expression levels of SLIRP polypeptides including a polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 may be used to provide prognostic information such as future fertility. In individual patient samples quantitative histology may be used to score SLIRP polypeptide levels to provide prognostic information.
Furthermore, microarray data may be used to observe expression levels of nucleic acids of the invention in sperm cells of a subject to provide prognostic information as described above.
Transgenics
The methods and use of the present invention may also employ non-human animals such as mice, rats, or other rodents, rabbits, dogs, goats, sheep, cows, horses, pigs, or other farm animals, in which the gene encoding the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or a variant thereof has been disrupted ("knocked out") such that the level of expression of this gene or genes is(are) significantly decreased or completely abolished. Such animals may be prepared using techniques and methods - 70 - such as those described in U.S. Patent No. 5,557,032. Increased apoptotic activity may be observed in such animals.
The methods and use of the present invention may also employ non-human animals such as mice, rats, or other rodents, rabbits, dogs, goats, sheep, cows, horses, pigs, or other farm animals, in which either the native form of the gene encoding the polypeptide of SEQ ID No: 2, 4, 6, 8, 1 0, 1 2, 14, 16, 1 8, or 20 or variant thereof for that animal or a heterologous gene is over-expressed by the animal, thereby creating a "transgenic" animal. Such transgenic animals may be prepared using well known methods such as those described in U.S. Patent No. 5,489,743 and PCT Application No. W094/28122. Decreased apoptotic activity may be observed in such animals.
The methods and use of the present invention may also employ non-human animals in which the promoter for one or more of the polypeptides of the present invention is either activated or inactivated (e.g., by using homologous recombination methods) to alter the level of expression of one or more of the native polypeptides that can result in the modulation of apoptotic activity.
These non-human animals may be used for drug candidate screening. In such screening, the impact of a drug candidate on the animal may be measured. For example, drug candidates may decrease or increase the expression of the gene encoding the polypeptide. In certain embodiments, the amount of the polypeptide, which is produced may be measured after the exposure of the animal to the drug candidate. Additionally, in certain embodiments, one may detect the actual impact of the drug candidate on the animal. For example, the overexpression of a particular gene may result in, or be associated with, a disease or pathological condition. In such cases, one may test a drug candidate's ability to decrease expression of the gene or its ability to prevent or inhibit a pathological condition such as a condition in a subject resulting in decreased and/or dysmotile sperm. In other examples, the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition. In such cases, one may test a drug candidate's ability to decrease the production of such a metabolic product or its ability to prevent or inhibit a pathological condition.
Set out hereunder is a general approach to the production of transgenic non-human animals.
A. Preparation of Constructs for Transformation
1 . Selection of Transgene - 71 -
The transgene of interest encoding a SLIRP polypeptide or variant thereof is selected. The simultaneous use of more than one transgene for insertion into a single embryo is within the scope of this invention. The structural gene may be obtained from any source, if obtained from vertebrate mammals, the structural gene may be from a homologous source (i.e., a gene from one mouse implanted into another mouse), or from a non-homologous source (i.e., a structural gene from rabbit implanted into a mouse). The transgene may have additional effects on the phenotype of the transgenic mammal, and these effects may be related or unrelated to the physiological action of the polypeptides herein. Preferred transgenes for use in the present invention include myc, myb, E2F (Nevins, J. R., Science 258:424-429 [1992]), abl, ras, pim.1 , src, E1 A (Nevins, supra), HPVE7 (human papilloma virus E7, Nevins, supra), and SV40 early region, SV40 large T antigen, SV40 large T antigen tsA58 mutant, and mutants and fragments thereof. More preferred genes include myc, E2F, SV40 early region, and the SV40 early region tsA58 mutant. The most preferred gene is SV40 early region tsA58 mutant.
2. Selection of Promoter
Promoters useful in practicing this invention are those that are highly regulated with respect to activity, both temporally and spatially. Thus, the promoters of choice are those that are active only in particular tissues or cell types. The source of the promoter may be from any prokaryotic or eukaryotic organism, any vertebrate or invertebrate, or any plant. Where the promoter is obtained from a mammal, the mammal may be homologous (the same species as the mammal to be transfected) or non-homologous (a different species).
3. Other Vector Components
In addition to a promoter and one or more structural genes, the vectors of this invention preferably contain other elements useful for optimal functioning of the vector in the mammal into which the vector is inserted. These elements are well known to those of ordinary skill in the art, and are described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, 1989.
4. Construction of Vectors
Vectors used for transforming mammalian embryos are constructed using methods well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, plasmid and DNA and RNA purification, DNA sequencing, and the like as described, for example in Sambrook, Fritsch, and Maniatis, eds., Molecular Cloning: A Laboratory Manual., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]). - 72 -
B. Production of Transgenic Mammals
The specific lines of any mammalian species used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryos, and good reproductive fitness. For example, when transgenic mice are to be produced, lines such as C57/BL6 x DBA2 F1 cross, or FVB lines are used (obtained commercially from Charles River Labs).
The age of the mammals that are used to obtain embryos and to serve as surrogate hosts is a function of the species used, but is readily determined by one of ordinary skill in the art. For example, when mice are used, pre-puberal females are preferred, as they yield more embryos and respond better to hormone injections.
Similarly, the male mammal to be used as a stud will normally be selected by age of sexual maturity, among other criteria.
Administration of hormones or other chemical compounds may be necessary to prepare the female for egg production, mating, and/or reimplantation of embryos. The type of hormones/cofactors and the quantity used, as well as the timing of administration of the hormones will vary for each species of mammal. Such considerations will be readily apparent to one of ordinary skill in the art.
Typically, a primed female (i.e., one that is producing eggs that can be fertilized) is mated with a stud male, and the resulting fertilized embryos are then removed for introduction of the transgene(s). Alternatively, eggs and sperm may be obtained from suitable females and males and used for in vitro fertilization to produce an embryo suitable for introduction of the transgene.
Normally, fertilized embryos are incubated in suitable media until the pronuclei appear. At about this time, exogenous nucleic acid comprising the transgene of interest is introduced into the female or male pronucleus. In some species such as mice, the male pronucleus is preferred.
Introduction of nucleic acid may be accomplished by any means known in the art such as, for example, microinjection. Following introduction of the nucleic acid into the embryo, the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention. One common method is to incubate the embryos in vitro for 1 -7 days and then reimplant them into the surrogate host.
Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos - 73 - implanted into a particular host will vary, but will usually be comparable to the number of offspring the species naturally produces.
Transgenic offspring of the surrogate host may be screened for the presence of the transgene by any suitable method. Screening is often accomplished by Southern or Northern analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening. Typically, the tissues or cells believed to express the transgene at the highest levels are tested, although any tissues or cell types may be used for this analysis.
Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular markers or enzyme activities, and the like. Blood cell count data is useful for evaluation of thrombocytopenia.
Progeny of the transgenic mammals may be obtained by mating the transgenic mammal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic mammal. Where in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Where mating is used to produce transgenic progeny, the transgenic mammal may be backcrossed to a parental line. SLIRP transgenic mice may also be crossed with other transgenic or knock out lines to further highlight SLIRP's effects. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
The transformed mammals, their progeny, and cell lines of the present invention provide several important uses that will be readily apparent to one of ordinary skill in the art. The mammals and cell lines are particularly useful in screening compounds that have potential as prophylactic or therapeutic treatments for diseases associated with undesirable levels of the polypeptide of SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 or a variant thereof.
In the case of transgenic mammals, screening of candidate compounds is conducted by administering the compound(s) to be tested to the mammal, over a range of doses, and evaluating the mammal's physiological response to the compound(s) over time. Administration may be oral, or by suitable injection, depending on the chemical nature of the compound being evaluated. In some cases, it may be appropriate to administer the compound in conjunction with co-factors that would enhance the efficacy of the compound. - 74 -
General
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 the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.
The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.
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 integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
EXAMPLES
Example 1 - SLIRP is present within the testis and sperm cells
In studies of wild type mice it was observed that SLIRP is abundantly expressed in the cytoplasm of the spermatids and tails of spermatozoa (Figure 1 ). The latter observation is consistent with the high concentration of mitochondria in the mid-tail portion of sperm.
SLIRP is abundantly expressed in the testis (Figure 2). In a Northern blot of human RNA from various tissues, SLIRP is abundantly expressed in testis tissue further supporting a role for its function within this tissue. - 75 -
SLIRP is predominantly mitochondrial. Figure 3 shows that although SLIRP is a nuclear receptor corepressor of nuclear receptor action (Figure 3A), and is closely associated with promoter DNA (as evidenced by ChIP studies, Hatchell et al, 2006, Figure 3B this document), the majority of SLIRP resides in the mitochondria (Figure 3C). HeLa cells were stained with antibodies against the mitochondrial protein Hsp 60 (red) and SLIRP (green) and the resultant merge (yellow) demonstrates colocalization of each protein.
In a small cohort of men with infertility with sperm dysfunction and abnormal sperm shape (teratozoospermia), the level of SLIRP expression was markedly reduced in -80% men compared to men with normal fertility. Figure 4 shows that in men with infertility and teratozoospermia (dysfunctional sperm) SLIRP levels are significantly reduced in the majority of men compared to normal.
SLIRP has been evidenced in the head and annulus of human sperm indicating that morphology and motility could be influenced by a loss of SLIRP (Figure 5). This suggests that SLIRP is important for maintaining sperm function.
Example 2 - Preparation of siRNAs to reduce human SLIRP gene expression
Expression of the SLIRP gene can be selectively diminished by the use of short interfering RNA molecules (siRNAs). siRNAs are double stranded, complementary RNA molecules generally composed of a 19 nucleotide sequence having exact homology with a specific sequence within a target mRNA with a limited number of nucleotides, such as a diuridine sequence, at its 3' end. When introduced into cells, siRNAs will reduce the expression of a given target gene by a combination of enhancing the rate at which a target mRNA is degraded and inhibition of its translation. siRNAs may be used individually, in combination with one another or with siRNAs directed against other targets (ie two or more genes may be knocked down simultaneously).
Table 1 . Target sequences used to prepare siRNAs to reduce human SLIRP gene expression siRNA SLIRP mRNA target Dharmacon catalogue N ° sequence
SLIRP siRNA #1 cgagucagcugaaagaaca D-014696-01
SLIRP siRNA #2 ucaaucagccgguugcuuu D-014696-02
SLIRP siRNA #3 gcacaguucggccauguca D-014696-03 - 76 -
Figure imgf000078_0001
*Note, target sequence defined in D-014696-04 is the same as that for J-014696-09.
SiRNAs directed against human SLIRP mRNA target sequences we have used are listed in table 1 and have been used to deplete SLIRP in a range of cell lines including HeLa (cervical), SW620 (colon), MCF-7 (breast) LnCAP (prostate) cell lines. Typical knockdowns produced by the first 4 siRNAs listed in Table 1 are presented in Figure 6.
The ability of individual and pooled mixtures of siRNAs to deplete cells of SLIRP protein were compared by western analysis. Hela cells were transfected with either Lipofectamine alone (LF), individual siRNAs (si#1 -4), a combination of the 4 SLIRP directed siRNAs or a non-specific (non-SLIRP targeting) siRNA to a final concentration of 20uM. Three days post transfection, lysates from duplicate knockdown treatments were prepared and the expression of SLIRP and β-actin assessed by western analysis. Blots demonstrate that each of the individual siRNAs and a pool of all four were able to deplete SLIRP protein levels in Hela cells without affecting β-actin expression when compared to LF and NS treated cultures. Individual siRNAs as listed in Table 1 corresponding to catalogue numbers D-014696-01 to 04.
SiRNAs targeted to the sequences as in Table 1 or others regions within the SLIRP mRNA may be introduced into tissues by different methods. These include, chemically synthesised molecules transfected into cells via cationic lipid transfer e.g. using Lipofectamine 2000 (Invitrogen®) or electroporation of similar molecules. Cells or tissue may also be transfected with viruses expressing the siRNA of interest or a plasmid directing their expression. For example, subcloning of a sequence encoding the expression of an siRNA directed against the same target as that of SLIRP siRNA#3 (see Table 1 ) into pSuperior.neo + GFP (Oligoengine, catalogue EC-PBS-0005/0006) and subsequent transfection into HeLa cells resulted in sustained depletion of SLIRP protein as assessed by western analysis (Figure 7). - 77 -
Hela cells were transfected with either empty pSuperior.neo+GFP or vector containing a sequence that directs the production of siRNA as described for SLIRP siRNA #3 (pSupSLIRP) as listed in Table 1 . Figure 7 shows SLIRP levels are decreased in pSupSLIRP transfected cells following FACS sorting of GFP positive cells and 7 days post transfection compared with the empty vector, β-actin expression assessed as loading control.
Example 3 - Strategies for generating a SLIRP KO mouse
The normal expression of the SLIRP gene in the mouse may be disrupted using knockout technologies. Multiple strategies may be utilised to achieve altered SLIRP expression in cell lines and/or whole animals. The following strategy provides one such approach.
The mouse SLIRP gene, also known as RIKEN cDNA 1810035L17 gene, is present at chromosome 12 (position 12E). It is composed of 4 exons and is flanked by NRP/Alkbh1 and snwl /SKIP. Data obtained from data bases maintained by National Center for Biotechnology Information and National Library of Medicine, USA. In one nucleotide listing, the SLIRP exon is present on mouse Chromosome 12 in the region 88,784,866-88,790,828 as defined by Genbank Mus musculus Build 37.1 (Figure 8).
This gene is composed of 4 exons, the initiating methionine being in exon 1 . To disrupt the normal expression of SLIRP within the mouse, its genome may be genetically reengineered (Figure 9) to place DNA recombinase recognition sites such as the loxp domain recognised by ere recombinase or Flippase Recognition Target (FRT) sites as recognised by Flippase either side of the region of the gene to be removed. Typically the region to be removed would include the initiating methionine of the gene or a region critical to the normal function of the gene to be targeted. The preparation of DNA constructs, generation of stem cells containing reengineered genes flanked with recombination recognition sequences and ultimately the production of mice capable of transferring a reengineered gene to its offspring are well known to persons skilled in the art. In the example where all or part of the SLIRP gene is flanked by loxp sites, referred to as "floxed SLIRP", mice may either have one (heterozygous) or both (homozygous) copies of the wild type gene replaced with the floxed allele. A floxed gene will express the same protein as a wild type animal, in a manner highly similar to or identical with the wild type animal. This may be achieved by having floxed gene transcription driven by the wild type promoter region.
In an alternate strategy, the genome of a mouse may be reengineered such that all or part of the SLIRP gene is floxed and when exposed to ere a region of the DNA - 78 - removed such that transcripts initiated from the endogenous promoter are targeted for rapid destruction as a result of splicing to an RNA destabilisation sequence. For example, transcripts initiated within exon 1 may be spiced to include a c-fos 3' untranslated region containing signals resulting in the rapid destruction of the mRNA resulting from the recombination event. Similarly, an exon encoding a protein degradation signal e.g. a ubiquitination site that when spliced in frame to a transcript from an endogenous promoter the protein resulting post recombination will be rapidly destroyed thereby minimising its expression.
To generate mice that lack one or have no wild type SLIRP loci, heterozygous and homozygous floxed SLIRP mice respectively may be bred with mice that express a suitable recombinase e.g. ere. By breeding ere expressing mice with a floxed SLIRP animal, the region of DNA between the recognition sites will be removed in cells where ere is expressed. Cre expression will depend upon the promoter used to direct its expression. For example, in the case of actin-cre, the recombinase is present in all tissues where actin is present and considered ubiquitously expressed. When an actin- cre and floxed SLIRP mouse are crossed, wild type expression of SLIRP will be lost from all tissues. However, if floxed SLIRP mice were crossed with villin-cre mice, the SLIRP gene would only be deleted from gastrointestinal epithelium.
Isolated tissues may also be exposed to a suitable recombinase to assess the effects of SLIRP removal via for example transfection with a virus or plasmid expressing the required recombinase.
Example 4 - SLIRP KO influences fertility - localised in areas of mitochondrial energy, and morphology.
A global SLIRP knockout mouse (SLIRPk0/k0) was generated to provide an in vivo model to investigate SLIRP's role in male fertility and spermatogenesis. Although SLIRPwt/k0 male mice are fertile, SLIRPk0/k0 males were shown to have reduced fertility (Figure 10). The SLIRPk0/k0 animal is viable, however when homozygous knockout males were crossed with wild type (wt) females the resultant litter size was reduced by -30% (ko/ko x wt/wt, 4.8 pups/litter) compared with those produced by wt males with comparable females (wt/wt x wt/wt, 6.6).
5l_I Ppko/ko mjce ^ave |3een fou nc| t0 have testes anc| epididymides that are macroscopically normal, and spermatogenesis and sperm production levels that appear quantitatively normal (ie. all germ cell types are present and in normal numbers). S Ll Rpwt/wt & sLipjpko ko testicular tissue was compared by tunnel assay for apoptosis, but - 79 - no differences were identified suggesting alterations in mitochondrial function, rather than increased cell death, as the likely cause of reduced fertility in these animals.
Sperm motility was then assayed using Computer Assisted Sperm Analysis (CASA) and video capture (method as described in Gibbs GM, et al. (201 1 ) Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function. Proc Natl Acad Sci U S A 108(17)7034-7039). Comparison of sperm from the SLIRPk0/k0 animals identified that they have a profound reduction in progressive motility (18.8% v 34.3% at 15 min, * = p<0.01 ) compared to SLIRP^ mice (Figure 1 1 ). Preliminary Transmission Electron Microscopy (TEM) studies of sperm from these animals showed that the annulus is absent, and mitochondrial packing may be disorganised (Figure 12, methods as adapted from Arsov T, et al. (2006) Fat aussie~a new Alstrom syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis. /Wo/ £ndocr/no/ 20(7):1610-1622). In addition, initial JC-1 staining studies were performed on murine sperm from SLIRPwt/wt and SLIRPk0/k0 animals (provides a guide to mitochondrial membrane potential, ΔΨη). It was found that an altered mitochondrial ΔΨη in the SLIRPk0/k0 mice compared to SLIRPwt/wt mice, consistent with SLIRP's putative role in regulating mitochondrial function.

Claims

- 80 -THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1 . A method for modulating the motility of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
2. A method for modulating the function of a sperm cell, the method including the step of increasing or reducing levels of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
3. A method for modulating the motility of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and - 81 -
(vi) a functional variant of any one of (i) to (v).
4. A method for modulating the function of a sperm cell, the method including the step of increasing or reducing the activity of a polypeptide within the cell, as compared to normal levels of the polypeptide within the cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
5. A method for increasing expression of a polypeptide in a sperm cell to improve the function of the cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
6. A method for increasing expression of a polypeptide in a sperm cell to improve the motility of the cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v). - 82 -
7. A method for treating a disorder associated with an undesirable level of function and/or motility of sperm cells, the method comprising the step of administering an effective amount of an isolated polypeptide, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
8. A method for modulating the motility of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or
(vi) a functional variant of any one of (i) to (v).
9. A method for modulating the function of a sperm cell, the method including the step of increasing or reducing expression of a polynucleotide encoding a polypeptide within the sperm cell, the polypeptide being selected from any one or more of the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; or
(vi) a functional variant of any one of (i) to (v). - 83 -
A method according to either of claims 8 or 9, wherein the polypeptide is encoded by a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
A method according to either of claims 8 or 9, wherein the polypeptide is encoded by a polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
A method according to claim 1 1 , wherein the polynucleotide that selectively hybridises to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, comprises a nucleotide sequence 95% to 99% identical to a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
A method according to either of claims 8 or 9, wherein one or more promoters are used to increase the expression of a polynucleotide encoding the polypeptide.
A method according to either of claims 8 or 9, wherein antisense nucleic acids or siRNAs are used to reduce or eliminate the expression of a polynucleotide encoding the polypeptide.
A method for sterilising a male animal comprising a method according to any of claims 1 , 2, 3, 4, 8, 9, or 14, including the step of reducing levels or reducing activity of the polypeptide within the cell.
A method according to claim 15, wherein the sterilising of the male animal is reversible.
Use of a polynucleotide as a biomarker for identification of dysmotile or dysfunctional sperm, the polynucleotide being any one or more selected from the group consisting of SEQ ID No: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19.
A use according to claim 17, wherein a microarray is used to identify the presence of mutations in the polynucleotide.
Use of a polypeptide as a biomarker for prediction of dysmotile or dysfunctional sperm, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2; - 84 -
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
20. Use of a polypeptide as a biomarker for prediction of energy production in a sperm cell, the polypeptide being any one or more selected from the group consisting of:
(i) SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20;
(ii) amino acids 27 to 109 of SEQ ID No: 2;
(iii) amino acids 22 to 109 of SEQ ID No: 2;
(iv) amino acids 21 to 91 of SEQ ID No: 2;
(v) amino acids 21 -26 and/or 60-67 of SEQ ID No: 2; and
(vi) a functional variant of any one of (i) to (v).
21 . A method or use according to any one of claims 1 to 7, wherein the polypeptide comprises a portion of a fusion protein.
22. A method or use according to any one of claims 1 to 7, wherein the polypeptide is a non-peptide mimetic of the polypeptide.
23. A use according to either of claims 19 or 20, wherein a selective binding agent is used to detect the presence or levels of the polypeptide.
24. A use according to claim 23, wherein the selective binding agent is an antibody to the polypeptide.
25. A use according to claim 24, wherein the antibody is a polyclonal antibody.
26. A use according to claim 24, wherein the antibody is a monoclonal antibody.
27. A use according to claim 24, wherein the antibody is a labelled antibody.
28. A use according to any one of claims 23 to 27, wherein the presence or levels of the polypeptide are detected using western blot analysis or enzyme-linked immunosorbent assay (ELISA).
29. A method for assessing the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal;
(ii) identifying the nucleotide sequence encoding a polypeptide according to - 85 - claim 1 in the one or more cells;
(iii) analysing the nucleotide sequence for one or more mutations which will reduce the fertility of the male animal.
30. The method of claim 29, wherein the one or more cells are isolated from any one of blood, sputum, or semen from the male animal.
31 . The method of either of claims 29 or 30, wherein the nucleotide sequence encoding a polypeptide according to claim 1 in the one or more cells is quantified to assess the fertility.
32. The method of any one of claims 29 to 31 , wherein nucleotide sequencing is used for identifying the nucleotide sequence.
33. A kit for assessing the fertility of a male animal, the kit using the method of any one of claims 29 to 32 and comprising at least one oligonucleotide primer specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
34. A kit for assessing the fertility of a male animal, the kit using the method of any one of claims 29 to 32 and comprising at least one allele-specific oligonucleotide probe specific for one or more mutations in a polynucleotide selected from the group comprising SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, or 19, and instructions for use of the kit to detect the one or more mutations.
35. A method for predicting the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal;
(ii) sequencing the SLIRP gene from nucleotides present in the one or more cells; and
(iii) analysing the nucleotide sequence of the SLIRP gene for one or more mutations which will reduce the fertility of the male animal; wherein the presence of the one or more mutations is predictive of a reduced fertility of the male animal when compared to a male animal without the one or more mutations.
36. The method of claim 35, wherein the presence of the one or more mutations is predictive that the male animal is infertile.
37. The method of either of claims 35 or 36, wherein the mutations are within any one or more of the coding region of the exons, non-coding exonic regions, - 86 - intronic regions, or flanking regions of the SLIRP gene.
38. The method according to any one of claims 35 to 37, wherein the one or more cells are taken from blood, epithelial cells, semen, or genetic-bearing material, from the male animal.
39. A method for predicting the fertility of a male animal, comprising the steps:
(i) isolating one or more cells from a male animal; and
(ii) measurement of SLIRP gene product levels from nucleotides present in the one or more cells;
wherein the presence of reduced SLIRP gene product levels is predictive of a reduced fertility of the male animal when compared to SLIRP gene product levels in one or more cells from a normal male animal.
40. A method according to claim 39, wherein SLIRP gene product levels are measured using quantitative reverse transcriptase PCR.
41 . A method according to either of claims 39 or 40, wherein the one or more cells are isolated from semen or ejaculate from the male animal.
PCT/AU2012/000166 2011-02-23 2012-02-23 Therapeutic uses of slirp WO2012113022A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021011930A1 (en) * 2019-07-18 2021-01-21 Chondrial Therapeutics, Inc. Slirp fusion proteins and use thereof for treating leigh syndrome

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009194A1 (en) * 2005-07-22 2007-01-25 The University Of Western Australia Sra binding protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009194A1 (en) * 2005-07-22 2007-01-25 The University Of Western Australia Sra binding protein

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COLLEY, S. M. ET AL.: "Loss of the Nuclear Receptor Corepressor SLIRP Compromises Male Fertility In Vivo", ENDOCR REV, vol. 32, no. 03, June 2011 (2011-06-01), pages PL-31 *
LANZ, R. B. ET AL.: "Steroid Receptor RNA Activator Stimulates Proliferation as Well as Apoptosis In Vivo", MOL. CELL. BIOL., vol. 23, no. 20, 2003, pages 7163 - 7176 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021011930A1 (en) * 2019-07-18 2021-01-21 Chondrial Therapeutics, Inc. Slirp fusion proteins and use thereof for treating leigh syndrome

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