Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-31
2004-11-30
Wax, Robert A. (Department: 1653)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06825329
ABSTRACT:
DESCRIPTION
The invention relates to the human PEM polypeptide, which plays an important role for the maturation of sperm and the nucleic acid that codes for them. The invention comprises the use of PEM as a target in male birth control and for the treatment and diagnosis of male infertility and Alzheimer's disease. The invention also includes a selection process for PEM antagonists as well as the production of binding molecules, which specifically detect PEM. In addition, genes that are regulated by the PEM gene are part of this invention.
The intention to use proteins of the male reproductive tract or sperm proteins as a target group for non-hormonal contraception has been known for several decades. For example, a project with the name “Vaccines for Fertility Regulation” was supported by the World Health Organization (WHO) (P. D. Griffin, Hum. Reprod., 1991, 6: 166-172). Various sperm proteins such as, e.g., PH-20, SP-10, FA-1, FA-2, CS-1, NZ-1, NZ-2 and lactate-dehydrogenase C4 were proposed as candidates for immunocontraception (R. K. Naz, Immunol. Rev., 1999, 171: 193-202). Immunization tests with PH-20 showed that both male and female animals are thus completely and reversibly infertile (P. Primakoff et al., Nature, 1988, 335: 543-546). The use of the intra-acrosomal sperm protein SP-10 as an antigen caused an immunological response in women that reduces fertility (R. W. Wright et al., Biol. Reprod., 1990, 42: 693-701). Active immunization of animals with FA-1 produces a lasting and reversible inhibition of fertility (R. K. Naz and X. Zhu, Biol. Reprod., 1998, 59: 1095-1100).
PEM is a transcription factor that includes the Homeobox family. The corresponding cDNA was cloned from the mouse (M. F. Wilkinson et al., Dev. Biol., 1990, 141: 451-455) and from the rat (S. Maiti et al., J. Biol. Chem., 1996, 271: 17536-17546). PEM transcripts are expressed abundantly and selectively in the male genital tract. In the mouse, the PEM expression was mainly detected in the testes, while in the rat, PEM can mainly be found in the epididymis (K. A. Sutton et al., J. Androl., 1998, 19: 21-30). The in vivo expression of the PEM gene is regulated in these organs by androgens. In addition, PEM transcripts were described in the muscle and in macrophages, but in these cases, the PEM expression does not seem to be regulated by androgens, which can be attributed to the use of different promoters (S. Maiti et al., J. Biol. Chem., 1996, 271: 17536-17546). Despite the unremarkable phenotype of the PEM-knock-out mouse (J. L. Pitman et al., Dev. Biol., 1998, 202: 196-214), it can be assumed that the human PEM plays an essential role in spermatogenesis and/or in sperm maturation. PEM is the sole known transcription factor whose expression is regulated by androgens (S. Maiti et al., J. Biol. Chem., 1996, 271: 17536-17546).
No one has yet found the human PEM ortholog; this suggests a low sequence conservation in different organisms, as can already be determined by the weak identity (73%) between mouse PEM and rat PEM (S. Maiti et al., Genomics, 1996, 34: 304-316).
The invention relates to the identification of human PEM. Both the complete coding PEM-cDNA sequence and the structure of the PEM gene could be determined. The human PEM-amino acid sequence has only 30% identity with the sequence from the mouse and only 32% identity with the sequence from the rat. The human genomic locus could be defined in Xq 25-26.
The identified cDNA sequence is shown in SEQ ID No. 1, and the protein-coding sequence is shown in SEQ ID No. 2. The genomic sequence could also be identified and is shown in SEQ ID No. 3 (corresponding to a cross-section of nucleotides 16000-170967 from Gene Bank Accession No. AC005023). The initial exon extends from nucleotide 168 439 to 168 042. An internal exon extends from nucleotide 165 491 to 165 446, and the terminal exon extends from nucleotide 161 927 to 161 817 (111 nucleotides). In the range of nucleotides 161 698 to 161 693, there is a polyadenylating signal.
The human PEM is preferably coded by (a) the coding area of the nucleic acid sequence shown in SEQ ID No. 1, (b) one of the sequences according to (a) against the backdrop of the degeneracy of the genetic code and/or (c) one of the nucleic acid sequences that hybridize under stringent conditions with the sequences according to (a) and/or (b). The human PEM especially preferably has the amino acid sequence shown in SEQ ID No. 2 or an amino acid sequence that is at least 80%, preferably at least 90%, identical to it.
The term “stringent hybridization” according to this invention is used in this case as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, Laboratory Press (1989), 1.101-1.104). Accordingly, we speak of hybridization under stringent conditions, if after washing for one hour with 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. and especially preferably at 68° C., especially for one hour with 0.2×SSC and 0.1% SDS at 55° C., preferably at 62° C. and especially preferably at 68° C., a positive hybridization signal is still observed. A sequence that hybridizes under such washing conditions with a nucleotide sequence that is shown in SEQ ID No. 1 or a nucleotide sequence that thus corresponds against the backdrop of the degeneration of the genetic code is detected by this invention.
In particular, this invention detects natural, allelic variations of PEM, in which these are optionally also functional mutations. Moreover, recombinant variants, for example functional partial fragments (such as, for example, the “Divergent Paired Class” homeodomains as described for the mouse of Rayle (Develop. Biol. 146 (1991), 255-257)), are also detected by this invention.
Especially preferably, the human PEM has the amino acid sequence that is shown in SEQ ID No. 2 or a sequence that is at least 80%, and especially at least 90%, identical to it. The 1% identity is in this case calculated according to the following formula:
I=n/L×
100%,
whereby n stands for the number of identical amino acids of the two sequences that are compared to one another and L stands for the length of the sequence section used for comparison.
The inhibition of PEM can result in the inhibition of sperm development or maturation and thus represents a novel approach for contraceptive preparations. In addition, the screening for functional mutations in the PEM gene can be used as a diagnostic agent for determining the causes of infertility. By restoring PEM function (e.g., by gene therapy), patient fertility can also be restored.
The subject of the invention is thus the use of human PEM and/or a nucleic acid that codes for this as a target substance for the production of an agent for birth control.
An inhibition of human PEM can be used for inhibiting fertility and especially for inhibiting spermatogenesis in male mammals. This is of great importance in human contraception, but also in veterinary medicine for population control. The inhibition of PEM can be carried out by expression reduction by means of antisense-nucleic acids or ribozymes or on the protein level by using inhibitors such as anti-PEM-antibodies or low-molecular antagonists. The production of antisense molecules and ribozymes can be carried out, for example, as described in Sczakiel (Antisense Nucleic Acid Drug Dev. 7 (1997), 439-444, Lavrovsky et al. (Biochem. Mol. Med. 62 (1997), 11-22) and Thompson (Methods Enzymol. 306 (1999), 241-260). Polyclonal antibodies against human PEM can be carried out by immunization of test animals with human PEM or fragments thereof, optionally on a vehicle such as keyhole-limpet-hemocyanin and recovery of the resulting antibodies from the immunized test animal. Monoclonal antibodies can be obtained by, for example, fusion of spleen cells of the immunized test animal with myeloma cells according to the method of Köhler and Milstein or further developments thereof. Low-molecular inhibitors of PEM can be identified by a screening process as explained in more detail below.
By contrast, an activation of human PEM t
Geserick Christoph
Haendler Bernard
Weiss Bertram
Millen White Zelano & Branigan P.C.
Mondesi Robert B
Schering Aktiengesellschaft
Wax Robert A.
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