HLA CLASS I MOLECULES IN IN VITRO FERTILIZATION AND FURTHER MEDICAL IMPLICATIONS

Abstract

The present invention relates to a nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof for use in a method of increasing efficiency of embryonic implantation in an in vitro fertilization programme, (I) wherein the at least one nucleic acid molecule is selected from nucleic acid molecules (a) encoding a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs 1 to 17, (b) comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs 18 to 23, (c) encoding a polypeptide which is at least 85% identical, preferably at least 90% identical, and most preferred at least 95% identical to the amino acid sequence of (a), (d) consisting of a nucleotide sequence which is at least 95% identical, preferably at least 96% identical, and most preferred at least 98% identical to the nucleotide sequence of (b), (e) consisting of a nucleotide sequence which is degenerate with respect to the nucleic acid molecule of (d), (f) consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), said fragment comprising at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) corresponding to the nucleic acid molecule of any one of (a) to (f), wherein T is replaced by U, and (II) the vector comprises the nucleic acid molecule of (I); (III) the host cell is transformed, transduced or transfected with the vector of (II); and (IV) the at least one protein or peptide is selected from proteins or peptides being encoded by the nucleic acid molecule of (I); and wherein the method of increasing embryonic implantation efficiency comprises (i) contacting the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof with the unfertilized, fertilized oocyte, and/or preimplantation embryo prior to the transfer of the fertilized oocyte or preimplantation embryo to the uterus; or (ii) contacting the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof with the uterus prior to, simultaneously with and/or after the transfer of the fertilized oocyte or preimplantation embryo to the uterus; or (iii) systemically administering the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination prior to, simultaneously with and/or after the transfer of the fertilized oocyte or preimplantation embryo to the uterus, preferably via injection, transdermal and/or vaginal administration.

Claims

1. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof for use in a method of increasing efficiency of embryonic implantation in an in vitro fertilization programme, (I) wherein the at least one nucleic acid molecule is selected from nucleic acid molecules (a) encoding a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs 1 to 17, (b) comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs 18 to 34, (c) encoding a polypeptide which is at least 85% identical, preferably at least 90% identical, and most preferred at least 95% identical to the amino acid sequence of (a), (d) consisting of a nucleotide sequence which is at least 95% identical, preferably at least 96% identical, and most preferred at least 98% identical to the nucleotide sequence of (b), (e) consisting of a nucleotide sequence which is degenerate with respect to the nucleic acid molecule of (d), (f) consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), said fragment comprising at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) corresponding to the nucleic acid molecule of any one of (a) to (f), wherein T is replaced by U, and (II) the vector comprises the nucleic acid molecule of (I); (III) the host cell is transformed, transduced or transfected with the vector of (II); and (IV) the at least one protein or peptide is selected from proteins or peptides being encoded by the nucleic acid molecule of (I); and wherein the method of increasing embryonic implantation efficiency comprises (i) contacting the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof with the unfertilized, fertilized oocyte, and/or preimplantation embryo prior to the transfer of the fertilized oocyte or preimplantation embryo to the uterus; or (ii) contacting the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof with the uterus prior to, simultaneously with and/or after the transfer of the fertilized oocyte or preimplantation embryo to the uterus; or (iii) systemically administering the nucleic acid molecule, vector, host cell, or protein or peptide, or any combination prior to, simultaneously with and/or after the transfer of the fertilized oocyte or preimplantation embryo to the uterus, preferably via injection, transdermal and/or vaginal administration.

2. The nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof for use according to claim 1, wherein prior to in vitro fertilization is (i) any time between after the collection of the unfertilized oocyte and directly before the transfer of the fertilized oocyte or preimplantation embryo to the uterus, and (ii) preferably any time between after the fertilization of the oocyte by sperm and directly before the transfer of the fertilized oocyte or preimplantation embryo to the uterus.

3. The nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof for use according to claim 1, wherein after in vitro fertilization is (i) any time between directly after the transfer of the fertilized oocyte or preimplantation embryo to the uterus and 6 days after the transfer of the fertilized oocyte or preimplantation embryo to the uterus, (ii) preferably is any time between directly after the transfer of the fertilized oocyte or preimplantation embryo to the uterus and 4 days after the transfer of the fertilized oocyte or preimplantation embryo to the uterus, and (iii) most preferably is any time between directly after the transfer of the fertilized oocyte or preimplantation embryo to the uterus and 2 days after the transfer of the fertilized oocyte or preimplantation embryo to the uterus.

4. An ex vivo or in vitro method for increasing the likelihood of a fertilized oocyte or preimplantation embryo to become implanted during in vitro fertilization of a female comprising culturing an isolated oocyte or preimplantation embryo in the presence of a nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof as defined in claim 1.

5. The method of claim 4, wherein the isolated oocyte is an unfertilized oocyte or a fertilized oocyte.

6. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof as defined in claim 1 for use in preventing abortion during pregnancy.

7. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof as defined in claim 1 for use in treating or preventing pre-eclampsia in a pregnant female.

8. The nucleic acid molecule, vector, host cell, or protein or peptide, or any combination thereof for use of claim 7, wherein the pregnant female carries a male embryo or foetus.

9. An inhibitor of the nucleic acid molecule or a protein or as defined in claim 1 for use in treating or preventing the HELLP syndrome.

10. The inhibitor for use of claim 9, wherein (i) the inhibitor of the nucleic acid molecule is selected from a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and/or (ii) the binding molecule of the protein, preferably the inhibitor of the protein is selected from a small molecule, an antibody or antibody mimetic, an aptamer.

11. The inhibitor for use of claim 10, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies.

12. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof as defined in claim 1 for use in treating or preventing an autoimmune disease, preferably in a pregnant female, wherein the autoimmune disease is preferably dermatomyositis, Hashimoto's thyroiditis, Sjögren syndrome or sclerodermia.

13. A nucleic acid molecule, a vector, a host cell, or a protein or peptide, or any combination thereof as defined in claim 1 for use in treating or preventing graft versus host disease.

14. The inhibitor for use or the method of any preceding claim, (I) wherein the at least one nucleic acid molecule is selected from nucleic acid molecules (a) encoding a polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs 1 to 6, (b) comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs 18 to 23, (c) encoding a polypeptide which is at least 85% identical, preferably at least 90% identical, and most preferred at least 95% identical to the amino acid sequence of (a), (d) consisting of a nucleotide sequence which is at least 95% identical, preferably at least 96% identical, and most preferred at least 98% identical to the nucleotide sequence of (b), (e) consisting of a nucleotide sequence which is degenerate with respect to the nucleic acid molecule of (d), (f) consisting of a fragment of the nucleic acid molecule of any one of (a) to (e), said fragment comprising at least 150 nucleotides, preferably at least 300 nucleotides, more preferably at least 450 nucleotides, and most preferably at least 600 nucleotides, and (g) corresponding to the nucleic acid molecule of any one of (a) to (f), wherein T is replaced by U, and (II) the vector comprises the nucleic acid molecule of (I); (III) the host cell is transformed, transduced or transfected with the vector of (II); and (IV) the at least one protein or peptide is selected from proteins or peptides being encoded by the nucleic acid molecule of (I).

Description

EXAMPLE 1: SUPPLEMENTATION OF BLASTOCYTE CELL CULTURE MEDIUM WITH HLA CLASS IB AND IW GENES

[0128] This example relates to a procedure to support implantation of an in vitro fertilized embryo by supplementing blastocyte cell culture with synthesised HLA class Ib and Iw molecules or by applying them systemically.

[0129] HLA-G, HLA-H, HLA-J, HLA-L, HLA-V, HLA-V, HLA-Y, HLA-E, and/or HLA-F was added to embryo cell culture. The doses of HLA proteins added to the cell culture was evaluated according to Sjöblom et al. (Sjöblom C1, Wikland M, Robertson SA., Hum Reprod. 1999 December; 14(12):3069-76) and Bhatnagar et al. (Bhatnagar P1, Papaioannou VE, Biggers JD, Development. 1995 May; 121(5):1333-9). Recombinant HLA-G or HLA-H, HLA-J, HLA-L, HLA-V, HLA-V, HLA-Y, HLA-E, HLA-F proteins were generated as state of the art and according to Favier et al (Favier et al., PLoS One. 2011; 6(7): e21011).

[0130] For intravenous applications, recombinant HLA-G, HLA-H, HLA-J, HLA-L, HLA-V, HLA-V, HLA-Y, HLA-E, and/or HLA-F proteins were generated under cGMP conditions. The quality, sterility, endotoxin testing as well as chemical purity are tested according to the monographs from the European Pharmacopeia, V.8.0 (European Pharmacopoeia (Ph. Eur.) Vol 8 (2013-2016) European Directorate of Quality of Medicines).

[0131] In order to compare the effect of the HLA supplementation to implantation and pregnancy rates, a control group was also monitored which did not receive HLA protein supplementation.

[0132] The embryos were transferred at day 5, according to guidelines of the American society for reproductive medicine (Fertil Steril, 2017; 107:882-96). Successful implantation was monitored by measurement of serum levels of free beta-human Choriongonadotropin (β-hCG) and vaginal ultra sound observation.

[0133] It could be observed that implantation rates, as well as successful pregnancies were significantly higher from embryos receiving the HLA supplementation treatment as compared to the control group.

EXAMPLE 2—EXTRACTION AND DETERMINATION OF HLA-CLASS IB AND IW GENES IN BLASTOCYTE CELL culture medium

[0134] In order to evaluate HLA class Ib and Iw expression via quantitative real-time polymerase chain reaction (qRT-PCR) from blastocyte cell culture media RNA has been extracted first. Since blastocytes shed RNA in extracellular vesicles (EVs) (Giacomini et al., Sci Rep. 2017; 7: 5210), RNA was isolated by first collecting EVs in ExoQuick™ (SBI Systems Bioscience Inc. Mountain View Calif., USA) according to protocol from König et al. (König et al., Hum Immunol. 2016 September;77(9):791-9). After the removal of the ExoQuick™ reagent, RNA was extracted according to the protocol from Stratifyer Blood extraction kit. In brief, after proteinase K digestion, lysates were admixed with germanium-coated magnetic particles in the presence of special buffers, which promote the binding of nucleic acids. Purification was carried out by means of consecutive cycles of mixing, magnetization, centrifugation and removal of contaminants. RNA was eluated with 25 μl elution buffer and RNA eluates were then stored at −80° C. until use. All extracts were tested for sufficient high-quality RNA content by quantification with qRT-PCR of the constitutively expressed gene Calmodulin 2 gene (CALM2) which is known as a stable reference/housekeeper gene. For a detailed analysis of gene expression by qRT-PCR methods, primers flanking the region of interest and a fluorescently labeled probe hybridizing in-between were used. RNA-specific primer/probe sequences were used to enable RNA-specific measurements by locating primer/probe sequences across exon/exon boundaries. In case multiple isoforms of the same gene existed, primers were selected to amplify all relevant or selected splice variants as appropriate. All primer pairs were checked for specificity by conventional PCR reactions. Specific primers have been generated against HLA-H, HLA-J, HLA-L, HLA-V, HLA-V, HLA-Y, HLA-E, HLA-F and HLA-G.

[0135] HLA-H, HLA-J, HLA-L, HLA-V, HLA-V, HLA-Y, HLA-E, HLA-F and HLA-G were found to be expressed in blastocyte cell culture.