Patent Application
20210253660
The present invention relates to the technical field of recombinant peptides production, particularly biologically active hormones, such as the bovine follicle-stimulating hormone. Additionally, it relates to compositions for veterinary use, particularly with a composition comprising the recombinant bovine follicle-stimulating hormone, and its use for increasing reproduction in bovines and other ungulates.
In the agricultural and livestock industry, improvements in reproductive science and the multiplication of more productive genotypes are directly linked to increases in productivity, which is why it is particularly important to increase reproductive performance in animals of commercial interest, particularly, bovines and ovines. One of way to increase the reproductive performance of these animals is to induce superovulation of females with a desirable genotype. To this end, a hormone treatment with follicle-stimulating hormone (FSH) is used, which induces the ovulation of several oocytes when injected into females. FSH is a glycoprotein hormone that is a heterodimer, a beta, specific for FSH, and an alpha, common to luteinizing hormone (LH) and others.
Currently, FSH is extracted from the pituitary glands of slaughtered pigs, where injectable hormones having this origin such as Folltropin-V™ and Pluset™ are nowadays the most used by the livestock industry. Due to its origin and structure, these exogenous hormones have a number of risks associated with their use, including the risk of contamination with pathogens such as prions or microorganisms, contamination of the hormonal preparation containing LH, since it is purified together with the FSH, and some preparations even declare this effect. The effect of said contamination reduces the process of superovulation, reduces the efficiency of fertilization, and affects the quality of embryos. In addition, due to their animal origin, these hormonal preparations have a circulation half-life of about 5 hours, which requires a considerable amount of injections during the superovulation protocol. Additionally, having a porcine origin, this hormone can induce a humoral immune response when administered in other species, such as bovines.
Due to all the aforementioned problems, the livestock industry has sought to improve the quality of the FSH administered, mainly, by means of molecular techniques where FSH of recombinant origin has been generated. In the state of the art, different alternatives of recombinant bovine FSH have been proposed. Document WO 2008/098169 A2 refers to the production of a recombinant bovine FSH (BoviPure FSH™) where the a subunit is fused with the β subunit using a spacer sequence to generate a unique DNA sequence that encodes this recombinant protein. The spacer sequence corresponds to the Carboxy-terminal peptide of human chorionic gonadotropin (CTP), which generates a linear sequence. Consequently, they generate a recombinant FSH, where it is not explicit whether there is any increase in the half-life of this protein or another similar advantage. The use of CTP as a spacer sequence to generate a recombinant FSH, is also disclosed in document US 2015/0335713 A1, which refers to a method to increase the reproductive performance of female bovines with respect to superovulation and generation of viable embryos using recombinant FSH. This recombinant FSH was generated from the fusion of the β subunit with the a subunit of bovine FSH using CTP as a spacer peptide or repeated G4S in a single amino acid chain.
In the general disclosure document of 2005 (Ruman J I, Pollak S, Trousdale R K, Klein J, Lustbader J W. Effects of long-acting recombinant human follicle-stimulating hormone analogs containing N-linked glycosylation on murine folliculogenesis. Fertil Steril. 2005 April; 83 Suppl 1: 1303-9), the generation of recombinant FSH in a single sequence by the use of the CTP spacer peptide is also promoted.
In the state of the art, in order to increase the half-life and biological activity of FSH, site-directed mutagenesis has been used in both the alpha and beta units of the FSH, as shown in document U.S. Pat. No. 5,338,835, which refers to the production of recombinant FSH, among others, by the generation of a fusion protein where the a subunit common to FSH and LH fuses with the β subunit of FSH, which in turn is coupled in its carboxyl-terminal (C-Ter) to a peptide of 34 CTP amino acids, as a spacer peptide, generating a linear sequence. In this document, recombinant FSH versus native FSH in rats is tested, showing that recombinant FSH has a longer half-life than native FSH.
Also, recombinant FSH have been generated by co-transfection of the alpha and beta subunit in human cell lines such as HEK293, as disclosed in the document WO 2014/183175 A1, which refers to the development of a method for production and/or purification of hormones, focusing on FSH from human or animal origin, using human cells as cell lines for the expression of the same. This recombinant FSH was tested in rats, showing that the recombinant FSH increases the weight of the ovary of the rat after a daily injection of recombinant FSH with human chorionic gonadotropin hormone for a period of 3 days.
In the article of Gifre, L. et al., 2017 (Gifre, L., Aris, À., Bach, A., & Garcia-Fruitós, E. Trends in recombinant protein use in animal production. Microbial cell factories. 2017, 16(1), 40), a comprehensive study was conducted on the market of different hormonal supplements, among them, native and recombinant FSH, showing that the current objective of the market is to seek for a long-lasting recombinant FSH, for its use in the agricultural and livestock market.
Therefore, in order to reduce the number of problems of the porcine FSH that is currently on the market, there is a need to generate a new, improved recombinant FSH, with a longer half-life, to reduce the number of injections and the cost associated thereto.
A first object of the present invention is a synthetic polynucleotide sequence that encodes a single-chain recombinant bovine follicle-stimulating hormone, comprising;
The first mentioned synthetic polynucleotide sequence encoding the beta subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No. 1, includes at its 5′ end a sequence encoding an endogenous secretion signal. On the other hand, the second polynucleotide encoding the alpha subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No. 2, includes at its 3′ end a sequence encoding a multiple histidine region.
In a preferred embodiment, the synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone is the sequence identified as SEQ ID No. 4.
A second object of the present invention is a vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprising a DNA construct containing a promoter sequence operatively linked to:
Particularly, in a preferred embodiment the aforementioned vector is a lentiviral vector.
In a preferred embodiment, the vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprises a DNA construct containing a promoter sequence operatively linked to SEQ ID No. 4 and a cleavage and polyadenylation sequence.
A third object of the present invention is a transduced cell expressing a single-chain recombinant bovine follicle-stimulating hormone, comprising a vector for the expression of a synthetic polynucleotide sequence encoding said single-chain recombinant bovine follicle-stimulating hormone, wherein said vector comprises a DNA construct that contains a promoter sequence operatively linked to:
In a preferred embodiment, the transduced cell is a Chinese hamster ovary cell.
In a preferred embodiment, the cell transduced with the vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprises a DNA construct containing a promoter sequence operatively linked to SEQ ID No. 4 and a cleavage and polyadenylation sequence.
A fourth object of the present invention is a synthetic polypeptide sequence of a single-chain recombinant bovine follicle-stimulating hormone, comprising:
Preferably, the first polypeptide having sequence SEQ ID No. 5 includes at its amino terminal end a signal sequence of endogenous secretion, while the second polypeptide having sequence SEQ ID No. 6 includes at its carboxyl terminal end a multiple histidine region.
In a preferred embodiment, the synthetic polypeptide sequence of a single-chain recombinant bovine follicle-stimulating hormone is the sequence SEQ ID No. 8.
A fifth object of the present invention is a method for obtaining a single-chain recombinant bovine follicle-stimulating hormone, comprising the steps of:
Preferably, the synthetic polynucleotide sequence encoding the single-chain recombinant bovine follicle-stimulating hormone is SEQ ID No. 4, and the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone is SEQ ID No. 8.
In another preferred embodiment, the expression vector used in the aforementioned method is a lentiviral vector.
Preferably, the transduced cells are Chinese hamster ovary cells, in a medium preferably free of serum and free of proteins.
The purification of the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone is performed, in a preferred embodiment, by means of affinity chromatography columns.
A sixth object of the present invention is a pharmaceutical composition containing a synthetic polynucleotide of a single-chain recombinant bovine follicle-stimulating hormone, wherein said hormone has an amino acid sequence comprising:
Preferably, the pharmaceutical composition comprises the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone of SEQ ID No. 8.
A final object of the present invention is a method of treatment for the superovulation of ungulates, comprising:
Preferably, the pharmaceutical composition comprises the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone of SEQ ID No. 8. Accordingly, preferably, the total dose administered ranges from 100 to 1000 μg of the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone of SEQ ID No. 8.
The present invention relates to a synthetic nucleotide sequence encoding a bovine recombinant follicle-stimulating hormone, a synthetic polypeptide sequence of said bovine recombinant follicle-stimulating hormone, a pharmaceutical composition comprising the same, and its use to increase reproduction in bovines and other ungulates. The advantage of the present recombinant hormone is its long half-life demonstrated in vivo, particularly in bovines, which also have a biological response significantly superior to the response that is usually obtained with commercially available hormones to date.
All the technical and scientific terms used to describe the present invention have the same meaning as is understood for a person with basic knowledge in the technical field in question. However, to define more clearly the scope of the invention as defined by the claims, a list of the terms used in this description is included below.
The term “FSH” should be understood as the acronym in English of Follicle-Stimulating Hormone, as a glycoproteic polypeptide that is produced in the pituitary gland whose half-life in its natural state is between 2 to 4 hours. This hormone stimulates follicular growth and development, and it is commonly used to induce superovulation in ungulates.
The term “alpha subunit” corresponds to the alpha subunit of the heterodimer corresponding to the follicle-stimulating hormone, which is common to several proteins such as the luteinizing hormone (LH) or other hormones. In turn, the term “beta subunit” corresponds to the beta subunit of the heterodimer corresponding specifically to the follicle-stimulating hormone.
The term “nucleotide sequence” or “polynucleotides”, should be understood as a double strand of DNA, or a single strand of DNA, natural or synthetic, or products of the transcription of said DNA (for example, RNA molecules). In turn, a “DNA sequence encoding a product of interest”, should be understood as a nucleotide sequence that transcribes a functional RNA molecule, or that encodes a peptide, polypeptide, or functional protein of interest to the present invention. It should be understood that the present invention is not related to genomic nucleotide sequences in their natural state, but refers to nucleotide sequences in an isolated, or purified, or partially purified, synthetic or recombinant state, obtained by any method of genetic engineering known in the state of the art.
The term “peptide sequence” or “polypeptides” should be understood as a sequence of amino acids, natural or synthetic, or products of RNA translation. When these polypeptides have a stable and three-dimensional structure, they are called proteins.
The term “recombinant” refers to any nucleotide (DNA, RNA) or amino acid sequence modified by any method of genetic engineering known in the state of the art, which generates, as a result, a new nucleotide or amino acid sequence different from that found in nature.
The term “identity” or “similarity” between nucleotide sequences should be understood as the percentage of identical nucleotides that the compared sequences share with each other, in a particular comparison window. The percentage of identity can be calculated by using a sequence comparison algorithm or by manual alignment and visual inspection. For example, sequences and identity percentages can be obtained by using computer resources available on the Internet, such as the computer program BLAST (http://blast.ncbi.nlm.nih.gov/) or the computer program FastDB. The identity of sequences can also be determined by means of hybridization assays thereof. The greater the degrees of hybridization astringency used in the assay, the greater the complementarity of the sequences required for them to hybridize. High stringency conditions are described by Sambrook et al. (Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, 1989).
The term “DNA construct” or “expression cassette” should be understood as a nucleotide sequence artificially created by genetic engineering tools, which generally comprises a transcription promoter sequence, operatively linked to a DNA sequence encoding a protein of interest, and adjacent to it a DNA cleavage and polyadenylation sequence. For this DNA construct to be expressed in an appropriate host, in the case of the present invention, a eukaryotic cell, or a higher organism, must be inserted into an expression vector, which contains all of the DNA sequences necessary for the proper expression within said host, together with a series of molecular markers that allow to detect and select those hosts that have been transformed.
The term “spacer sequence” corresponds to a sequence of nucleotides that binds gene sequences encoding multimeric proteins that are bound by non-covalent bonds in nature. This way, the spacer sequence makes it possible to obtain a single-chain multimeric protein without interfering with the biological activity of said protein. The term “spacer sequence” may also refer to the peptide sequence that is encoded by the polynucleotide spacer sequence. In particular for the present invention, the spacer sequence links the beta and alpha sequences of the bovine recombinant follicle-stimulating hormone, maintaining the steric folding properties of both subunits without interfering in the binding of this molecule to the receptor, thus generating a sole amino acid chain.
The term “transduced cell” should be understood as a host cell subject to the transduction process by which an exogenous DNA sequence is incorporated. Said cell can be a eukaryotic somatic cell or an embryo.
The term “ungulates” refer to placental mammals that sustain themselves and walk with the ends of the fingers, that may or may not be covered by a hoof. Some examples of ungulates are animals for cattle such as bovines, ovines, caprines, porcines, equines, deer, among others, not limited to those previously mentioned.
The term “superovulation” should be understood as the increased number of ovulated follicles or the production of multiple embryos by the injection of FSH.
The term “effective amount” should be understood as the amount of FSH that must be administered to produce the desired effect.
As previously described, a first object of the present invention relates to a synthetic polynucleotide sequence that encodes a single-chain recombinant bovine FSH. This sequence was designed without altering the three-dimensional structure of the polypeptide sequence encoded in the synthetic polynucleotide sequence. In order to obtain said sequence that encodes the single-chain recombinant bovine FSH, the alpha chain was linked to the beta chain of the FSH, in such a way that the linking met two fundamental requirements: (1) that both subunits retained sufficient steric freedom so as not to affect its folding and assembly capacity, as it occurs naturally with the heterodimer, and (2) that the peptide resulting from the linker sequence will not affect the interaction of the binding of the recombinant FSH with its receptor. Based on these requirements, a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone was obtained, comprising: (i) a first polynucleotide encoding the beta subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No.1, or a variant thereof with at least 85% sequence identity therewith; covalently linked to (ii) a second polynucleotide encoding the alpha subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No. 2 or a variant thereof with at least 85% sequence identity therewith; and covalently linked to (iii) a third polynucleotide linking said first and second polynucleotides, which encodes a spacer sequence derived from human keratin 10 having sequence SEQ ID No. 3 or a variant thereof with at least 85% identity of sequence therewith. As these three sequences or their variants are covalently linked, a configuration of the subunit type of the FSH (beta)-linker-(alpha) is formed.
While it is mentioned that, preferably, SEQ ID No. 1 corresponds to the beta subunit of the beta subunit sequence of the Bos taurus FSH, the present invention is not limited to the use of said sequence and the sequence of the beta subunit of any other ungulate may be used. Similarly, while the preferred embodiment of the invention is SEQ ID No. 2, corresponding to the alpha subunit of the alpha subunit sequence of the Bos Taurus FSH, the present invention is not limited to the use of said sequence and the alpha subunit sequence of the of any other ungulate may be used. In turn, the spacer sequence SEQ ID No. 3 may come from any other sequence or be any spacer sequence known in the state of the art, with the condition that said sequence is sufficiently flexible to allow the correct folding of the two subunits of the single-chain recombinant bovine FSH.
The sequence variants of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, may have an identity of at least 80% with the original sequence, preferably having a sequence identity of at least 85%, even more preferably at least 95% identity, where, regardless of the variations that the original sequences may suffer, they maintain their biological function.
The first mentioned synthetic polynucleotide sequence encoding the beta subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No. 1, includes at its 5′ end a sequence encoding an endogenous secretion signal, although in another preferred embodiment the polynucleotide sequence could have a heterologous signal secretion. When the polynucleotide sequence of the present invention is transduced into a cell through an expression vector, said signal sequence allows its secretion into the medium to be easily purified. In particular, the nucleotide sequence encoding that endogenous secretion signal corresponds to the first 54 bases of SEQ ID No. 1.
On the other hand, the second polynucleotide encoding the alpha subunit of the follicle-stimulating hormone of the Bos taurus species having sequence SEQ ID No. 2, includes at its 3′ end a sequence encoding a multiple histidine region, although in another preferred embodiment the polynucleotide sequence may not have said region, or have another type of region like that of the Strep-Tag® system. In particular, this region comprises between 6 and 10 histidine, preferably 6 histidine, and it is used for the purification of the single-chain recombinant bovine FSH. Particularly, the nucleotide sequence encoding this region corresponds to the last 27 bases of SEQ ID No. 2.
In a preferred embodiment, the synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone is the sequence identified as SEQ ID No. 4. This sequence is formed by the linking of the nucleotide sequences SEQ ID No. 1, SEQ ID No. No. 2, and SEQ ID No. 3, which include the nucleotide sequence encoding the endogenous secretion signal that forms part of SEQ ID No. 1, and the sequence encoding the histidine region that forms part of SEQ ID No. 2.
A second object of the present invention is a vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprising a DNA construct containing a promoter sequence operatively linked to:
Particularly, in a preferred embodiment the aforementioned vector is a lentiviral vector, but a plasmid vector or any expression vector known in the state of the art for these purposes can be used.
In a preferred embodiment, the vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprises a DNA construct containing a promoter sequence operatively linked to SEQ ID No. 4 and a cleavage and polyadenylation sequence. In a preferred embodiment, the DNA construct also includes a protein for the detection of the expression, preferably in that case the green fluorescent protein (GFP) is used, but not limited to this particular marker.
The nucleotide sequences encoding SEQ ID No. 4 and the sequence encoding the green fluorescent protein are found under the same cytomegalovirus immediate early promoter, although any other suitable promoter from those already known in the art may be used.
In this preferred embodiment, the nucleotide sequence encoding the green fluorescent protein is used only to detect the level of expression, to select by fluorescence those clones of transduced cells that express the highest levels of the protein of interest, in this particular case, the single-chain recombinant bovine FSH having SEQ ID No. 4.
Subsequently, and with standard laboratory methodology, the green fluorescent protein is eliminated in order to obtain only the single-chain recombinant bovine FSH.
A third object of the present invention is a transduced cell expressing a single-chain recombinant bovine follicle-stimulating hormone, comprising a vector for the expression of a synthetic polynucleotide sequence encoding said single-chain recombinant bovine follicle-stimulating hormone, wherein said vector comprises a DNA construct that contains a promoter sequence operatively linked to:
In a preferred embodiment, the transduced cell is a Chinese hamster ovary cell, although the invention is not limited to this host cell. The possibility of transduction of other cell types is considered, such as: HEK 293, NSO, BHK-21, PER.C6 or U937. Moreover, any recombinant protein expression system can be used, such as expression system in insect cells, yeast, algae, genetically modified animals or even in cell-free systems.
In a preferred embodiment, the cell transduced with the vector for the expression of a synthetic polynucleotide sequence encoding a single-chain recombinant bovine follicle-stimulating hormone, comprises a DNA construct containing a promoter sequence operatively linked to SEQ ID No. 4 and a cleavage and polyadenylation sequence. Preferably, the expression vector used for the transduction of the cells is the lentiviral expression vector previously mentioned.
A fourth object of the present invention is a synthetic polypeptide sequence of a single-chain recombinant bovine follicle-stimulating hormone, comprising:
The variants of the sequences SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7, can have an identity of at least 80% with the original sequence, where preferably they have a sequence identity of at least 85%, even more preferably at least 95% identity, where, regardless of the variations that the original sequences suffer, they maintain their biological function.
While it is mentioned that preferably SEQ ID No. 5 corresponds to the beta subunit of the beta subunit sequence of the Bos taurus FSH, the present invention is not limited to the use of said sequence, and the beta subunit sequence of any other ungulate may be used. Similarly, while the preferred embodiment of the invention is SEQ ID No. 6 that corresponds to the alpha subunit of the alpha subunit sequence of the Bos Taurus FSH, the present invention is not limited to the use of said sequence, and the alpha subunit sequence of any other ungulate may be used. In turn, the spacer sequence SEQ ID No. 7 can come from any other sequence or be any spacer sequence known in the state of the art, with the condition that said sequence is sufficiently flexible to allow the correct folding of the two subunits of the single-chain recombinant bovine FSH, and that it contains at least 2 sites of N-glycosylation.
Preferably, the first polypeptide having sequence SEQ ID No. 5 includes at its amino terminal end a signal sequence of endogenous secretion, although the presence of said signal sequence is not necessary. In another embodiment of the present invention, SEQ ID No. 5 may not contain said signal sequence since it is not relevant for obtaining the single-chain recombinant bovine FSH. In turn, the second polypeptide having sequence SEQ ID No. 6 includes at its carboxyl terminal end a multiple histidine region, although in another preferred embodiment the polynucleotide sequence may not have said region or have another type of region, such as that of the system Strep-Tag®, which comprises 8 specific amino acids that give the protein a particular protein purification method.
In a preferred embodiment, the synthetic polypeptide sequence of a single-chain recombinant bovine follicle-stimulating hormone is the sequence SEQ ID No. 8, as shown in
On the other hand, the preferred embodiment of the single-chain recombinant bovine FSH of SEQ ID No. 8 has a three-dimensional structure as observed in
A fifth object of the present invention is a method to obtain a single-chain recombinant bovine follicle-stimulating hormone, comprising the steps of:
Preferably, the synthetic polynucleotide sequence encoding the single-chain recombinant bovine follicle-stimulating hormone is SEQ ID No. 4, and the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone is SEQ ID No. 8.
In another preferred embodiment, the expression vector used in the aforementioned method is a lentiviral vector.
Preferably, the cells that are transduced are Chinese hamster ovary cells, in a medium preferably free of serum and free of proteins.
The purification of the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone is performed, in a preferred embodiment, by means of metal ion affinity chromatography. However, any purification method that is known in the state of the art may be used, without these polypeptide sequences necessarily containing a histidine region at their carboxy terminal end. That is, protein purification techniques such as isoelectric point separation, pH, or an ion exchange column may be used. They can also be separated by size or molecular weight via exclusion chromatography or SDS-PAGE, or be separated by polarity or hydrophobicity through high-performance liquid chromatography or reversed-phase chromatography.
A sixth object of the present invention is a pharmaceutical composition comprising a synthetic polynucleotide of a single-chain recombinant bovine follicle-stimulating hormone, wherein said hormone has an amino acid sequence comprising:
Preferably, the pharmaceutical composition comprises the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone of SEQ ID No. 8.
The pharmaceutically acceptable excipients present in the pharmaceutical composition are carriers that enable the administration of the active compound, in this case the single-chain recombinant bovine FSH, to an ungulate. Said excipients will depend directly on the route of administration used and on the pharmacodynamic and pharmacokinetic properties intended for the composition. As such, a pharmaceutically acceptable excipient can be a binder, diluent, disintegrant, lubricant, or coating compound.
Another object of the present invention is a method of treatment for the superovulation of ungulates that allows to increase the reproduction thereof, comprising the administration of an effective dose of a pharmaceutical composition comprising:
In a preferred embodiment, the pharmaceutical composition comprises the synthetic polypeptide sequence of the single-chain recombinant bovine follicle-stimulating hormone having SEQ ID No. 8.
A final object of the present invention is a method to induce estrus synchronization in ungulates, which comprises the administration of a single dose between 20 and 200 μg of the pharmaceutical composition previously described to the animals.
The pharmaceutical composition of the present invention can be administered by any route known in the state of the art, although it is preferably administered through an intramuscular injection or an intravenous injection.
The methods of treatment of the present invention enable the induction of superovulation and estrus synchronization in ungulates, preferably in cattle, but the method can be used in a large number of species such as cows, sheep, goats, buffaloes, pigs, boars, horses, antelopes, bison, reindeer, llamas, alpacas, vicunas, among others, and not limited the aforementioned antecedents.
The following examples are intended to illustrate the invention and its preferred embodiments, but under no circumstances should they be considered to restrict the scope of the invention, which will be defined by the wording of the claims annexed hereto.
In order to generate the single-chain recombinant bovine follicle-stimulating hormone, sequences comprising the heterodimer of FSH were selected together with a spacer or linker sequence.
The beta subunit of FSH was selected from the amino acid sequence of this Bos taurus protein, which corresponds to sequence SEQ ID No. 5. In its amino terminus, it has an endogenous secretion signal peptide and glycosylation sites. The alpha subunit was also obtained from the sequence of this Bos taurus protein, to which the endogenous secretion signal peptide was removed.
This subunit corresponds to the sequence SEQ ID No. 6. Since the FSH is a heterodimer, in order to select the spacer sequence, it was necessary to consider that both subunits should conserve enough steric release so as not to affect its folding and ability to assemble properly, as the heterodimer does. The spacer sequence should also not affect the interaction of the FSH with the receptor.
For this reason, a portion of the carboxyl terminal of human keratin 10 was selected as the spacer sequence, to which two possible sites of N-glycosylation were added in order to increase the half-life of the hormone; this sequence corresponds to SEQ ID No. 7.
Subsequently, in silico, a linear sequence comprising the beta subunit (SEQ ID No. 5), the spacer sequence (SEQ ID No. 7), and the alpha subunit (SEQ ID No. 6) was generated. With this amino acid sequence, a DNA sequence was generated by means of in silico codons optimization for its expression in human cells. Codon optimization was performed by the company Genscript (http://www.genscript.com/).
The gene sequence resulting from this process corresponds to SEQ ID No. 4, which was synthesized and cloned into the pLWC-LGL expression vector. The map of the resulting vector is shown in
The pLCW-scbFSH vector, which already contains the cloned scbFSH sequence, was cut with the restriction enzymes EcoRl, Sall, Pst, Nael, and NgoMIV, in order to evaluate its correct cloning. In
Having generated this vector, it was transfected in conjunction with 3 commercial helper vectors pLP1, pLP2, and pLPNSVF, in HEK293FT cells in order to generate the lentiviral particles, using cationic polymers (polyethyleneimine, PEI). These particles were extracted from the culture medium at 48 hours post-transfection, concentrated by ultracentrifugation at 23,000 rpm for 1 hour and 30 minutes, and stored at −80° C.
Using the lentiviral particles containing the vector with the sequence for scbFSH, the transduction of the Chinese Hamster Ovary (CHO) cell line was performed, where the transduced cell populations with the highest fluorescence intensity (GFP) were selected through flow cytometry with Cell Sorter. Subsequently, each isolated clone was characterized as to the amount of scbFSH accumulated in its culture medium.
The scbFSH accumulated in the culture medium of each of the preselected clones was determined using the Bovine FSH ELISA Kit (LifeSpan BioSciences, Inc.). The results of the ELISA were taken as a basis to calculate the specific productivity (Qp) of each clone, that is, the amount of scbFSH produced by each cell in a period of 24 hours. The clone that showed better levels of expression was transfected with the CRE recombinase to cleave the coding sequence for the green fluorescent protein and was used as a basis for new rounds of transduction with the pLCW-scbFSH vector.
After three rounds of transduction/recombination, four clones were preselected for scbFSH expression analysis. The specific productivity of each of these clones is shown in Table 1.
The CHO-scbFSH clone was adapted to growth conditions in a serum free HyClone SFM4CHO medium. The culture conditions were established in 250 mL spinner flasks using temperatures between 35 and 37° C. in a SFM4CHO medium supplemented with 4 mM glutamine. The cells adapted to suspension were transferred to a 10-litre scale bioreactor (Winpact fermentation system, FS-07 series). Cultures were kept at 35° C. with an average cell density of 2×106 cells/mL, and a perfusion rate of 1 volume per day.
The collected medium was clarified by means of sequential filtration through 5 and 0.4 micrometres. The clarified medium was stored at −80° C. until further purification by means of metal ion affinity chromatography (I MAC).
The scbFSH contained in the culture medium was purified by means of metal ion affinity chromatography using a Ni Sepharose High Performance matrix, on an XK 50/20 column (GE healthcare). The chromatography was performed on an AKTA PRIME PLUS (GE Healthcare). The input medium was supplemented with 30 mM imidazole and adjusted to pH7.4. In order to separate contaminating proteins, the column was washed with 100 mM imidazole in phosphate buffer. The elution was carried out at 200 mM imidazole.
The purified scbFSH was diafiltered against phosphate buffer, quantified, and lyophilized in glass flasks for later uses.
In correspondence with its amino acid sequence, the scbFSH molecule should have a molecular weight of 26.6 kDa. However, this molecule has a total of six potential N-glycosylation sites. Four of these sites are endogenous, while the remaining two were incorporated as part of the spacer or linker sequence linking the alpha and beta chains. The N-glycans incorporated to these sites are usually of heterogeneous size and composition, providing an additional weight between 3.5 and 5 kDa each. Thus, if it is considered that the 6 N-glycosylation sites have been occupied, then, the sum of the weights contributing as N-glycosylation should oscillate between 21 and 30 kDa, generating a molecule having a heterogeneous molecular weight between 48 and 57 kDa. The SDS-PAGE and Western Blot assays show that, indeed, scbFSH migrates as a diffuse band above 48 kDa, suggesting that the 6 potential N-glycosylation sites could be occupied (
In order to analyse its oligosaccharide profile, the scbFSH molecule was digested with the PNGase F enzyme. The released oligosaccharides were separated by means of normal-phase liquid chromatography (NP-HPLC) on a TSK-Gel Amide-80 column (Tosoh Biosep, Japan). These oligosaccharides were also subjected to digestion with the neuraminidase enzyme in order to remove the terminal sialic acids and thus determine the degree of sialylation of the N-linked sugars.
The current market of the FSH for the agricultural and livestock sector is dominated by two fundamental commercial products: Pluset (25%) and Folltropin-V (70%). However, these products, as it was previously explained, are extracted from the pituitary gland of pigs, with the consequent disadvantages linked to them. Since they are the only products available to conduct the relevant comparative analyses, in order to demonstrate the viability of the scbFSH of the present invention as an alternative, the following studies were carried out:
a) Purity and Electrophoretic Mobility
The purity and electrophoretic mobility of scbFSH was evaluated in comparison with its commercial pair Folltropin-V®. Both molecules were subjected to SDS-PAGE and Western blot assays.
The SDS-PAGE demonstrated that the scbFSH produced by recombinant DNA technology is a highly pure product, which could also be evidenced by means of HPLC gel-filtration chromatography, where a single peak could be observed. In the Western blot assay, Folltropin-V® shows an immunoreactive band of approximately 23 kDa, resulting from the dissociation of the alpha and beta chains, whereas the scbFSH shows an immunoreactive band of around 50 kDa corresponding to the sum of weight weights of the alpha and beta chains of bovine FSH (approximately 26.6 kDa), plus an additional 21 to 30 kDa for N-glycosylation.
b) Biological Activity (In Vitro Maturation of Bovine Oocytes)
The biological activity of scbFSH produced by recombinant DNA technology was evaluated in an in vitro maturation assay of bovine oocytes. In this assay, the commercial counterparts Folltropin-V® and Pluset® were also evaluated. To that end, the oocytes were aspirated from ovaries of cows slaughtered in a slaughterhouse. After centrifuging at 3000 g for 10 minutes, the oocytes with cumulus from at least five cell layers were selected and washed three times in a washing medium (Tyrode's medium with HEPES buffer solution and supplemented with 0.3% BSA, 0.2 mM sodium pyruvate, and 50 μg/mL gentamicin). Once washed, groups of 25 oocytes were transferred to 100 μl drops of maturation medium (SOF supplemented with 0.8% BSA, 0.33 mM sodium pyruvate, 1×MEM essential amino acids, 1×MEM non-essential amino acids, 1 mM of glutamine, 5 μg/mL LH, 1 μg/mL 17b-estradiol, and 50 μg/mL gentamicin), supplemented, or not, with FSH. The oocytes were cultured for 24 hours at 38.5° C., in 5% CO2, and 95% humidity. After this time, the oocytes were analysed by microscopy and the expansion of the cumulus was taken as a FSH-induced maturation criterion.
The expansion of the cumulus-oocyte complexes, as well as the extrusion of the first polar body, were taken as a maturation criterion. This assay showed that all three hormones are biologically active, in terms of their ability to induce in vitro maturation of bovine oocytes (
c) Biological Activity (Induction of Superovulation in Ovines)
The biological activity of scbFSH produced by recombinant DNA technology was also evaluated by means of a superovulation assay in ovines. The commercial counterparts Folltropin-V® and Pluset® were also evaluated in the assay, following consensus protocols for each hormone as described in the table below. The assay was conducted on Corriedale sheep between 50 and 60 kg, which were randomized into three experimental groups according to the three types of hormones evaluated. The different FSH were administered intramuscularly at 12-hour intervals in decreasing doses for 96 hours, starting 24 hours before the removal of the intravaginal sponge containing progestins (60 mg medroxyprogesterone acetate, Progespon®, Syntex, Argentina). At the end of the scheme, transrectal ultrasonography was performed and the follicles larger than 10 mm in diameter were counted. The results shown in the table below demonstrate that, under the conditions evaluated, the scbFSH hormone yields better superovulation levels than its commercial counterparts Folltropin-V® and Pluset®.
17 ± 2.9
The results shown in Table 2 demonstrate that, under the conditions evaluated, the scbFSH hormone yields better superovulation levels than its commercial counterparts Folltropin-V® and Pluset®.
The ability of scbFSH to induce superovulation in bovine cattle was evaluated in five red Holstein Friesian cows older than 5 years of age with monitoring of estrous cycle prior to the field test, determining an average of 20 days for the animals under study, and maintaining ad libitum feeding in clover and ryegrass meadow.
A superovulation scheme with scbFSH of 96 hours was followed, starting on day 4 after implanting a bovine intravaginal device impregnated with progesterone (Syntex). In the superovulation scheme, a total dose of 500 μg scbFSH divided into 8 doses of 62.5 μg each was administered at 12-hour intervals. On day 7, 5 ml of Lutalyse (Zoetis) were administered, and on day 9 transrectal ultrasonography was performed to count the total number of follicles larger than 10 mm in diameter, both on the left ovary (LO) and the right ovary (RO). The results of this assay are shown in Table 3 below, showing the number of follicles per ovary, and per animal 24 hours after completing the hormonal induction.
The superovulation assay in bovines yielded an average ovarian response of 22.6±2.5 follicles larger than 10 mm. This type of response is significantly superior to the response usually obtained with commercial hormones, where the number of follicles ranges between 10 to 17 per animal.
The ovulatory response in bovines also constitutes evidence of the ability of the hormone scbFSH to induce estrus synchronization as a previous step to fixed-time artificial insemination (FTAI), demonstrating the potential of this hormone for assisted reproduction applications where it is necessary to synchronize the period when births occur.
Advantages of the Proposed Technical Solution
The solution proposed by the present invention is a viable FSH alternative for use in bovine cattle, which would complement the products currently on the market, additionally offering the following advantages:
| Number | Date | Country | Kind |
|---|---|---|---|
| 1347-2018 | May 2018 | CL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CL2019/050040 | 5/16/2019 | WO | 00 |
