Patent Application
20220152127
The present invention relates to a microorganism characterized in that it is genetically modified to express the human growth factor of keratinocytes (KGF/FGF7) or its functional orthologues, derivatives and fragments. The present invention finds its primary application in the medical field, in the treatment of vaginal atrophy conditions induced by menopause.
Vaginal atrophy is a frequent problem in postmenopausal women. In fact, this physiological event causes alterations in the vaginal epithelium, characterized by a thinning of the tissue and reduced lubrication, with consequent inflammation of the vaginal mucosa and the final tract of the urinary tract, which occur in over 50% of menopausal women [Pandit and Ouslander, 1997].
Vaginal atrophy can also occur in premenopausal women, for example after surgical removal of an endometrial carcinoma, or after para-aortic pelvic lymphadenectomy or adjuvant vaginal brachytherapy (VBT), interventions performed to reduce the risk of local recurrence and improve survival of cancer patients [Keys et al., 2004].
The lack of estrogens related to menopause induces a progressive vulvar atrophy, characterized by a thinning of the mucosa, with a 30-50% reduction in the levels of vascularization. The treatment of choice is oral hormone replacement therapy (HRT), which however is not recommended in women previously suffering from oncological diseases [Edey et al., 2018]. Furthermore, the use of HRT presents problems also for healthy women, being related to a higher incidence of cancer [Kim et al., 2018].
The keratinocyte growth factor (KGF/FGF7) (NCBI Reference Sequence: Ncbi accession number NG 029159, GenBank amino acid sequence: Accession number AAB21431.1), a member of the fibroblast growth factor (FGF) family, plays a key role in regulating cell proliferation, migration and differentiation during development and in response to tissue repair and injury processes [Finch and Rubin, 2004]. KGF acts by binding to its specific receptor tyrosine-kinase, FGFR2-IIIb/KGFR, generated through an alternative splicing of the FGFR2 gene and mainly expressed on epithelial cells of different organs, in which it plays a key role in the control of epithelial growth and differentiation [Eswarakumar et al., 2005]. The KGF/KGFR pathway is essential for maintaining the integrity and functionality of epithelial tissues in adults, due to the cytoprotective and regenerative activity of KGF. Indeed, the expression of KGF is strongly upregulated following lesions in various epithelial tissues such as skin, kidneys, bladder, pancreas, stomach and intestine [Werner et al., 1992; Marchese et al., 1995; Brauchle et al., 1996; Werner, 1998]. Furthermore, KGF protects the pulmonary epithelium from lesions and improves distal airway repair by stimulating cell proliferation, inhibiting apoptosis and the formation of oxygen free radicals, and mobilizing progenitor epithelial cells [Gomperts et al., 2007]. It has been shown that treatment with recombinant KGF (Palifermina) protects epithelial cells from a variety of lesions, including radiation-induced damage. Therefore, the pharmacological use of KGF has been approved by the FDA for the treatment of severe oral mucositis resulting from radiotherapy or chemotherapy in patients with haematological tumours or head and neck cancers [Spielberger et al., 2004; Beaven and Shea, 2007; Brizel et al., 2008; Barasch et al., 2009]. On the other hand, the vaginal administration of KGF during the neonatal period in murine models determines an oestrogen-independent proliferation in the vaginal epithelium, suggesting a potential link between oestrogen treatment and activation of KGF/KGFR signalling [Masui et al., 2004]. In this context, the present inventors have previously shown that KGF can be used as an alternative treatment to local administration of estrogens (see International patent application WO2014/023773). The previous patent application refers to the use of KGF and its related pharmaceutical compositions in the treatment of vaginal atrophy, dysuria, vaginal pain and/or vaginal dryness induced by a post-menopausal state, by surgery, by a disease and/or by chemotherapy or radiotherapy. Furthermore, in vitro and in vivo results on the local effects of KGF on vaginal epithelium have been published in an international journal [Ceccarelli et al. 2014]. In particular, the effect of local administration of KGF on vaginal atrophy has been evaluated in vivo on an animal model, represented by female CD1 strain ovariectomized mice, using Pluronic F127, a synthetic amphiphilic polymer, as a vehicle to allow the gradual release of KGF. Kinetic studies showed that the release of KGF was almost complete within 24 hours, thus indicating the need for daily treatment.
The administration of therapeutic molecules directly at the mucosa level due to infections or diseases involving that tissue not only increases their effectiveness and specificity, but also contributes to reducing the side effects compared to systemic routes of administration [Neutra and Kozlowski, 2006; Davis, 2001; Holmgren and Czerkinsky, 2005].
The strategy of administration at the level of the mucous membranes is considered better than the systemic one because of easier execution, it does not require the use of needles and syringes and therefore of trained personnel; furthermore, the immunogenicity of soluble proteins is lower when these are administered through the mucous tissue. Probiotics are vital microorganisms that exert beneficial effects on the host when provided in adequate quantities. Probiotics are generally isolated from stool samples from normal individuals, mostly from breastfed babies. Examples of probiotics for human use are those belonging to the Lactobacillus (e.g. L. acidophilus, L. casei), Bifidobacterium (e.g. B. animalis subsp lactis), or Saccharomyces (e.g. S. boulardii, S. florentinus) species. Lactococcus Lactis is an optional non-invasive and mesophilic heterofermentative bacterium (ideal growth temperature around 30° C.), widely used in the dairy industry [Pontes et al., 2011]. It belongs to the family of lactic acid bacteria (LAB), which represents a heterogeneous group of gram-positive microorganisms of great technological importance [del Carmen et al., 2011]. LABs play a key role in maintaining the balance of intestinal and vaginal microflora, and can be isolated from vaginal secretions of healthy women [Fuller, 1989; Reid and Bocking, 2003; Hoesl and Altwein 2005; Todorov et al., 2007]. Thanks to their numerous beneficial properties, and their status as generally recognized as safe (GRAS) organisms, LABs are the most commonly used probiotic microorganisms, and can be defined as “live microorganisms which, when administered in adequate quantities, confer benefits to the host” [FAO/WHO, 2001]. Currently, L. lactis is the best described member of the LAB and is considered the model organism of this group, not only for its economic importance, but also for the following characteristics:
The use of engineered lactobacilli to produce molecules of interest directly in situ is already known.
For example, patent application EP3067058 refers to a method for producing therapeutic cannabinoids characterized by the administration to a host of the subspecies of Lactobacillus Paracasei, subspecies Paracasei F19 probiotic, genetically modified to produce and secrete cannabinoids of Cannabis sativa, in association with the caproic acid, able to establish the enzymatic reactions of the biosynthetic pathway that leads to the production of cannabinoids directly in situ in said host.
Application WO2014066945 refers to genetically modified probiotics expressing recombinant phenylalanine ammonia lyase (PAL) to treat phenylketonuria.
L. lactis can be genetically engineered to efficiently produce and secrete several proteins, a feature recently used by scientists to convey therapeutic proteins directly to mucous membranes, particularly through intranasal, oral or genital mucosal surfaces.
Sufficient data are currently available to support the use of recombinant LABs, in particular L. lactis, to convey therapeutic proteins to mucosal tissues [Bermúdez-Humarán et al., 2004]. Therefore, there is still the need to overcome the above described disadvantages and to improve both the effectiveness of the KGF-based therapy for the treatment of vaginal atrophy and the compliance of patients.
In particular, there is a need to obtain an engineered microorganism capable of directly colonizing the vaginal mucosa, being controllable in the production of KGF by a safe inducible system in patients.
For the above described drawbacks and to improve both the effectiveness of the KGF-based therapy for the treatment of vaginal atrophy and the “compliance” of the patients, the present inventors applied a strategy based on the creation of a genetically modified strain for the KGF production. The present authors used a strain of L. lactis, defined as a GRAS (Generally Recognized As Safe) organism, previously used as a vehicle for various biomedical products, such as vaccines, antigens and other therapeutic agents [Bron and Kleerebezem, 2018] to produce recombinant KGF.
Therefore, the present inventors have genetically modified a strain of L. lactis to make it able to produce KGF in a controlled manner, using a controlled expression system using Nisin (NICE system), and inserting a secretion signal peptide upstream of the KGF gene specific for L. lactis, Usp45. The engineered lactobacillus object of the present invention is able to directly colonize the vaginal mucosa, and its production of KGF can be controlled by the NICE inducible system, through the oral intake of Nisin. Furthermore, the proliferative activity of recombinant KGF produced by L. lactis on primary cells of vaginal epithelium is demonstrated here, thus supporting the therapeutic activity of the microorganism of the invention.
Therefore, it is an object of the present invention a microorganism characterized in that it is genetically modified to express the human growth factor of keratinocytes (KGF/FGF7) or its functional orthologues, derivatives or fragments. Preferably the microorganism is a probiotic. More preferably the microorganism is a GRAS organism.
Preferably, said microorganism or probiotic is a lactic acid bacterium, more preferably of the genus Lactobacillus or Lactococcus.
Preferably, said microorganism belongs to the genus Lactococcus, preferably Lactococcus Lactis. More preferably said Lactococcus Lactis is the NZ3900 strain.
In a preferred embodiment, the microorganism as defined above has been genetically modified with a recombinant polynucleotide comprising a nucleic acid encoding the KGF, its functional orthologues, derivatives or functional fragments and/or said microorganism comprises a plasmid comprising a nucleic acid coding for the human keratinocyte growth factor (KGF/FGF7) or its functional orthologues, derivatives or fragments.
Said nucleic acid encoding the human KGF, its functional orthologues, derivatives or fragments is preferably operatively linked to an expression promoter, preferably inducible, more preferably a nisin-inducible promoter, e.g. the PnisA promoter.
Preferably, said KGF, its functional orthologues, derivatives or fragments are secreted.
Preferably, said KGF, its orthologues, derivatives or functional fragments are expressed as a fusion protein with a secretion signal that works in the microorganism, such as the probiotic, more preferably said signal is Usp45 or PrtP.
Preferably the Usp45 signal has a sequence having at least 80% identity with the SEQ ID NO: 6 or 7. The plasmid described above therefore in a preferred embodiment comprises a sequence having at least 80% identity with the SEQ ID NO: 6.
Preferably the PrtP signal has a sequence having at least 80% identity with the SEQ ID NO: 10. The plasmid described above therefore in a preferred embodiment comprises a sequence coding for a sequence having at least 80% identity with the SEQ ID NO: 10.
Preferably said KGF comprises a sequence having at least 80% identity with SEQ ID NO:2, 3 or with the sequence aa. 56-194 of SEQ ID NO:3.
The nucleic acid encoding the KGF comprises preferably a sequence having at least 80% identity with the SEQ ID NO: 1 or 11.
The plasmid described above therefore in a preferred embodiment comprises a sequence having at least 80% identity with the SEQ ID NO: 1 or 11 or with the sequences encoding the SEQ ID NO: 2, 3 or the sequence aa. 56-194 of SEQ ID NO:3.
Preferably, the microorganism according to the invention is able to colonize the vaginal mucosa. Preferably said microorganism releases in a controlled manner the KGF. The production of KGF can be controlled by an inducible system, e.g. NICE system, through the oral intake of Nisin. The KGF produced by the microorganism of the present invention has effects on primary cells of vaginal epithelium.
Further objects of the invention are a composition comprising said microorganism and at least one excipient and a pharmaceutical composition comprising said microorganism and at least one pharmaceutically acceptable excipient.
The microorganism may be or not in a lyophilized form. Preferably said microorganism is in an amount of 12-24×1011-12-24×1012 cfu per gram of composition.
Another object of the invention is a combination which comprises:
The microorganism or the composition or the combination according to the invention are preferably for medical use, more preferably for use in the treatment of vaginal atrophy, dysuria, vaginal pain and/or vaginal drying induced by a post-menopausal status, by surgery, by a pathology and/or by chemotherapy or radiotherapy.
The microorganism or the composition or the combination according to the invention are preferably for use in the production of human KGF or its functional orthologues, derivatives or fragments directly in situ in a host, more preferably in the human vaginal mucosa. Preferably the microorganism is administered topically on the vagina mucosa, preferably by introduction in the vaginal cavity, for example by hydrogels, vaginal tablets, suppositories, particulate systems and intravaginal rings. Even more preferably the microorganism has been genetically modified with a recombinant polynucleotide comprising a nucleic acid encoding the human KGF, its functional orthologues, derivatives or functional fragments and/or said microorganism comprises a plasmid comprising a nucleic acid coding for the human keratinocyte growth factor (KGF/FGF7) or its functional orthologues, derivatives or fragments. More preferably the nucleic acid encoding the human KGF, its functional orthologues, derivatives or fragments is operatively linked to an inducible expression promoter.
The inducible expression promoter is preferably a nisin-inducible promoter, preferably the PnisA promoter.
Preferably, an inducer of the expression promoter, preferably nisin, is also administered. More preferably the promoter is the PnisA promoter. Preferably the expression promoter inducer is administered orally.
A further object of the present invention is a combination which comprises the composition as defined above and an inducer of the expression promoter, where the composition can be administered on the vaginal mucosa for example by hydrogels, vaginal tablets, suppositories, particulate systems and intravaginal rings, while the expression promoter inducer can be administered orally.
For “Keratinocyte growth factor” or “KGF/FGF7” or “KGF” it is intended the entire wild type protein KGF (NCBI Reference Sequence: NP 002000.1; GenBank amino acid sequence: CAG46799.1):
or the amino acid sequence:
or functional allelic variants, or orthologues, fragments, mutants, derivatives or analogues thereof.
In the present invention, functional variants, orthologues, fragments, mutants, derivatives or analogues possess the same pharmacological activity as the KGF protein.
The object of the present invention is therefore a microorganism, able to integrate into the bacterial flora of the host, and comprising a plasmid containing the KGF gene.
The microorganism according to the invention is preferably administered in a single administration by vaginal route, preferably in a dose of at least 12×1011 colony forming units (cfu). The inducer of KGF secretion, for example nisin, is preferably administered at least once a day.
The microorganism according to the invention or the composition comprising the same can be administered by hydrogels, vaginal tablets, suppositories, particulate systems and intravaginal rings.
Preferably the present composition and the present microorganism are for use in the treatment of conditions of vaginal atrophy and/or vaginal atrophy related to dysuria and/or vaginal atrophy related to vaginal pain and/or dryness induced by a chemotherapy treatment. Preferably said orthologues, derivatives and fragments possess the same therapeutic properties as KGF. Preferably, the chemotherapeutic agent is tamoxifen or an anticancer drug belonging to the family of selective oestrogen receptor modulators. Preferably, the microorganism according to the invention was previously genetically modified by transformation with a nucleotide vector comprising a nucleic acid bearing the KGF gene, or functional allelic variants, orthologues, fragments, mutants, derivatives or analogues thereof.
Preferably the above described microorganism is authorized for use in food for humans or animals. Preferably the microorganism according to the invention is incapsulated.
Preferably the microorganism or composition for use according to the invention is administered in a dose of 12-24×1011-12-24×1012 cfu.
Preferably, the microorganism or composition according to the invention are formulated as a medicament or nutraceutical. Preferably, the composition according to the invention is administered by hydrogels, vaginal tablets, suppositories, particulate systems or intravaginal rings. The inducer, such as nisin, will be administered preferably orally. A further object of the invention is a method for producing the microorganism as defined above comprising the genetic modification of at least one Lactococcus Lactis cell by means of a recombinant polynucleotide comprising the KGF to obtain a KGF-expressing microorganism. To engineer L. lactis, the present inventors used the NICE system (nisin-controlled expression system) which was tested and demonstrated its versatility in other LAB, such as Leuconostoc lactis, Lactobacillus helveticus, Streptococcus sp., Bacillus sp., Enterococcus sp. [Eichenbaum et al., 1998] and Lactobacillus plantarum [Pavan et al., 2000]. Nisin is a food preservative and a natural, toxicologically safe antibacterial. It is considered natural because it is a polypeptide produced by some strains of L. lactis for food use [Delves-Broughton et al., 1996]. All the bacteria in which this system was tested showed a dose-response profile for nisin for beta-glucuronidase activity, characterized by a level of undetectable protein activity in non-inducing conditions and an increase in levels (from 10 to 60 times) induced by the external addition of sub-inhibitory quantities of nisin. This feature allowed to exploit the NICE system for the use of recombinant LABs as systems for administering vaccines and biotherapeutics at the level of the intestinal mucosa [Poelvoorde et al., 2007]. The success of the Phase I clinical trial of an interleukin-10 L. lactis secreting strain for the treatment of Crohn's disease has opened new horizons for the use of genetically modified LAB as a therapeutic vehicle [Bermúdez-Humarán, 2009].
In the context of the present invention, when reference is made to specific DNA sequences, it is understood that even the RNA molecules identical to the said polynucleotides, except for the fact that the RNA sequence contains uracil instead of thymine and the skeleton of the RNA molecule contains ribose instead of deoxyribose, RNA sequences complementary to the sequences described therein, functional fragments, mutants and their derivatives, proteins encoded by them, functional fragments, mutants and their derivatives are included in the invention. Also included in the present invention are nucleic acid or amino acid sequences derived from the nucleotide or amino acid sequences shown in the present invention, e.g. functional fragments, mutants, derivatives, analogues and sequences with a % of identity of at least 70% with the above mentioned sequences. The term “complementary” sequence refers to a polynucleotide which is not identical to the sequence but has a basic sequence complementary to the first sequence or encodes the same amino acid sequence of the first sequence. A complementary sequence may include DNA and RNA polynucleotides. The term “functional” can be understood as being able to maintain the same activity. The term “fragment” refers to polynucleotides which preferably have a length of at least 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides, 1500 nucleotides, etc. or to polypeptides which preferably have a length of at least 10aa, 20aa, 30aa, 40aa, 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa., etc. “Derivatives” can be recombinant or synthetic. In the context of the present invention, the term “derivatives” when referred to protein indicates a chemically modified protein or an analogue thereof, where at least one substituent is not present in the unmodified protein or an analogue thereof, i.e. a protein that is covalently modified. Typical modifications are ammuines, carbohydrates, alkyl groups, acyl groups, esters and the like. As used herein, the term “derivatives” also refers to longer or shorter sequences of polynucleotides/proteins and/or having, for example, an identity percentage of at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably at least 99%, with the sequences here described. In the present invention, “at least 70% identity” indicates that the identity can be a sequence identity of at least 70%, or 75%, or 80%, or 85%, or 90%, or 95% or 100% compared to the indicated sequences. This applies to all the aforementioned % of identity. Preferably, the % of identity concerns the entire length of the indicated sequence. The alignment finalized to determine the percent amino acid sequence identity can be achieved in various ways that are within the skill in the art knowledge, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign software (DNASTAR). The present invention finds application both in the treatment of human beings and in the veterinary sector. The derivative of the invention also includes “functional mutants” of the polypeptides, which are polypeptides that can be generated by mutating one or more amino acids in their sequences and that maintains their activity. In fact, the polypeptide of the invention, if required, can be modified in vitro and/or in vivo, for example by glycosylation, myristylation, amidation, carboxylation or phosphorylation, and can be obtained, for example, by known synthetic or recombinant techniques in the art. In the present invention “functional” is intended for example as “maintaining its activity” e.g. immunomodulatory activity or anti-inflammatory activity. Also part of the invention are polynucleotides that have the same nucleotide sequences as a polynucleotide exemplified herein except for nucleotide substitutions, additions or deletions within the polynucleotide sequence, as long as these variant polynucleotides substantially retain the same relevant functional activity as the polynucleotides specifically exemplified herein (for example, they code a protein having the same amino acid sequence or the same functional activity encoded by the exemplified polynucleotide). Therefore, the polynucleotides described herein should be understood to include mutants, derivatives, variants and fragments, as discussed above, of specifically exemplified sequences. The present invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences of the invention so as to allow hybridization with this sequence under stringent standard conditions and standard methods (Maniatis, T. et al, 1982). The polynucleotides described herein can also be defined in terms of more particular identity and/or similarity intervals with those here exemplified. The identity of the sequence typically will be greater than 60%, preferably greater than 75%, more preferably greater than 80%, still more preferably greater than 90% and may be greater than 95%. The identity and/or similarity of a sequence can be of 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% or higher with respect to a sequence here exemplified. In the context of the present invention, the microorganism as defined above has been genetically modified by transformation. Each transformation method known to the expert in the art can be used, for example by chemical (CaCl2) or physical (electroporation) methods. The term “plasmid” as used herein refers to any molecule capable of autonomous replication that is suitable for transforming a receiving bacterial strain and containing DNA sequences that direct and/or control the expression of heterologous DNA sequences inserted. Different types of plasmids can be used, such as those with low or high number of copies, expression plasmids and cosmids. The microorganism according to the invention can also be defined as “recombinant”. The present microorganism can colonize the vaginal mucosa thus presenting the advantage of limiting repeated administrations of the microorganism. The inducible KGF expression system inserted in a microorganism which is able to colonize the vaginal mucosa, according to the invention, allows a controlled and prolonged release of KGF, thus ameliorating the therapeutic effect of KGF ad favouring patients compliance. Moreover, the inducer that is administered is preferably a food supplement as nisin.
The present invention will now be depicted with non-limiting examples, with reference to the following figures.
Materials and Methods
KGF Gene Amplification
The DNA sequence encoding the KGF was obtained from GenBank (access number NC_000015.10).
KGF Gene Sequence:
KGF is a protein of 29 KDa and 194 amino acids.
KGF Protein Sequence:
The KGF cDNA was amplified by human fibroblasts using Pfu DNA polymerase (Promega) and the following primers: F: 5′-ggatcctgcaatgacatgactccagagc-3′ (SEQ ID NO:4); R: 5′-gagctcttaagttattgccataggaagaaag-3′ (SEQ ID NO:5). The amplification was performed according to the following program: denaturation at 95° C. for 45 seconds, pairing at 59° C. for 30 seconds, extension at 72° C. for 80 seconds; 35 PCR cycles were conducted. The PCR product was visualized by electrophoresis on 1% agarose gel.
Cloning of the KGF Gene in the pET30a Vector
Purification of PCR product from the agarose gel was performed using the Wizard Gel extraction kit and the PCR Clean-up (Promega) system. The purified KGF was cloned in the vector PJet1.2/blunt (Fermentas) by T4 DNA ligase (Fermentas) and the ligated products were transformed into E. coli DH5a cells. Plasmid DNA isolation was performed using the Pure Yield Plasmid Mini Prep (Promega) system as described by the manufacturer. The recombinant plasmids were controlled by digestion with restriction enzymes and DNA sequencing. The plasmid DNA was then digested by the restriction enzymes BAMHI and SacI (New England Biolabs) and transferred into an expression vector pET30a (Novagen, cod. 69909-3) using T4 DNA ligase (Fermentas). The ligated products were transformed into cells of E. coli TOP10 (Invitrogen). The recombinant plasmids were extracted and confirmed by digestion with restriction enzymes and DNA sequencing.
Construction of the Usp45-KGF Plasmid
Usp45 Gene Sequence:
Usp45 Protein Sequence:
A synthetic sequence of Usp45 including restriction enzyme recognition sites was designed by pairing the following primers: F: 5′-atcttcatgaaaaaaaagattatctcagctattttaatgtctacagtgatcttaagtgctgcagccccgttgtcaggtgtttacgc tgatg-3′(SEQ ID NO:8); R: 5′-gatccatcagcgtaaacacctgacaacggggctgcagcacttaagatcactgtagacttaaaatactgagat aatcttttttttcatgaa-3′ (SEQ ID NO:9). The DNA fragments of Usp45 were phosphorylated and bound by T4 DNA ligase (Fermentas) to a pET30a-KGF expression vector digested with the restriction enzymes BamHI and BglII and dephosphorylated. In this way a recombinant plasmid was obtained in which the Usp45 sequence is joined to the KGF coding sequence by a 6 nucleotide linker containing a cutting site for the restriction enzyme BamHI. The ligated products were transformed into E. coli TOP10 cells, and the recombinant plasmids were confirmed by digestion with appropriate restriction enzymes. Due to the presence of a cutting site of the BamHI restriction enzyme between the sequence of Usp45 and KGF, the final protein encoded by the pET30a-Usp45-KGF expression plasmid differed from the original KGF sequence for to the presence of 3 more amino acids. For this reason, the correct KGF protein sequence was obtained by substitution with an in vitro synthesized sequence in which the nucleotides coding for the three additional amino acids were removed. The Usp45-KGF insert was first transferred to the pGL3-basic vector [Promega, cat. N. E1751], and therefore the new synthetic sequence was inserted in the correct position through the digestion with restriction enzymes Bpu10I and PstI, followed by ligation. The insert was then confirmed by DNA sequencing.
Construction of the Recombinant L. lactis Strain NZ3900/pNZ8149-Usp45-KGF for Food Use
A strain of L. lactis capable of secreting KGF was generated by transformation of the strain NZ3900 of L. lactis (MoBiTec-Molecular Biotechnology), for food use, with an expression plasmid containing KGF fused to the Usp45 secretion signal. The L. lactis strains were grown at 30° C. in liquid M17 medium supplemented with lactose or glucose at 0.5% and 10 μg/ml of chloramphenicol or erythromycin. The NZ3900 strain is a standard strain for food selection based on the ability to grow on lactose. This strain is a progeny of NZ3000, a strain in which the lactose operon, which is generally present on the plasmids, has been integrated into the chromosome, and the lacF gene has been eliminated. The lacF gene deletion renders this strain incapable of growing on lactose, unless the lacF is supplied on a plasmid [de Ruyter et al., 1996]. Thus, the host strain of L. lactis NZ3900 can grow on glucose, but in the presence of the lacF gene on a plasmid, it can also grow on lactose. Therefore, the commercial vector for L. lactis pNZ8149 (MoBiTec) [Mierau et al., 2005], which contains lacF as a food selection marker, was chosen for the expression of KGF so that the transformed cells can be selected for the ability to grow on lactose. The Elliker medium (tryptone, yeast extract, sodium chloride, sodium acetate, ascorbic acid, agar) was used for the selection of the Lac+colonies. On this rich medium both Lac+ and Lac-cells can grow, but when lactose is added as the sole source of carbon, the lactose fermenting cells provide yellow colonies. To generate the plasmid pNZ8149-Usp45-KGF (for brevity indicated from now on as PNZKGF), the plasmids pNZ8149 from L. lactis and pGL3 basic-Usp45-KGF were digested with the restriction enzymes NcoI and SacI and then ligated. The resulting plasmid was transferred into competent cells of L. lactis by electroporation. In short, the competent NZ3900 cells of L. lactis were prepared by two successive sub-inoculations in M17 medium containing 0.5% glucose (GM17 medium). Then 5 mL of culture medium were transferred to 50 ml of GM17 medium and incubated for about 3 hours at 30° C., until an OD600 nm of 0.3 was reached. The cells were collected by centrifugation (6000 rpm, 20 min, 4° C.), subsequently washed in 400, 200, 100 and then 40 mL of sterile buffer (0.5 M sucrose, 10% glycerol) and centrifuged again (6000 rpm, 20 min, 4° C.). The cells were finally resuspended in 4 mL of sterile buffer and stored at 4° C. Then, 40 μL of cells were placed in a pre-chilled electroporation cuvette with 1 μL of DNA (pNZKGF, in TE buffer) which was kept on ice during electroporation (2000 V, 25 μF, 200Ω and a pulse from 4.5 to 5 ms). After electroporation, 1 ml of GM17 medium, CaCl2 (2 mM) and MgCl2 (20 mM) were added to the cuvette, then incubated on ice for 5 minutes and then at 30° C. for 1.5 hours. After electroporation, the bacteria were plated on Elliker agar (for the selection of Lac+transformants) and incubated for 1 or 2 days at 30° C.
Induction of KGF Protein Expression and Secretion
KGF expression was induced by the addition of sub-inhibitory amounts of nisin to the culture medium. Briefly, 5 ml of L. lactis NZ3900 cells transformed with the pNZKGF plasmid were grown overnight at 30° C., then diluted 1/25 in 2×10 ml of fresh medium and grown at 30° C. until reaching an OD600 nm equal to 0.4. 10 ml of culture were induced with 1 ng/ml of nisin, while the other 10 ml of culture, not induced, were used as a negative control. The cells were incubated for 3 hours and the OD600 nm was measured to monitor the growth of induced and non-induced cultures. The cells were collected by centrifugation at 6.000 rpm for 5 minutes. Both cell lysates and supernatants were obtained and tested for the presence of the KGF protein.
KGF Secretion Optimization
The optimization of the KGF secretion was performed by modifying various parameters, such as the collection time after Nisin induction and the Nisin concentration, to find the optimal conditions for the expression and secretion of the recombinant protein. Different concentrations of Nisin (0.5, 0.75 and 1 ng/ml) and different collection times after induction (3, 12 and 24 hours) were examined.
KGF Protein Expression
The cell lysates of L. lactis, induced or not with 0.5 ng/ml of Nisin for 3, 12 or 24 hours, were analyzed at the protein level by SDS-PAGE. 15 μL of each sample were loaded onto 15% polyacrylamide gel. The proteins were visualized by staining with blue coomassie.
ELISA Assay
The supernatants of L. lactis cells were collected, induced or not with 0.5, 0.75 or 1 ng/ml of Ni sin for 3, 12 or 24 hours. The concentration of KGF in the induced and non-induced L. lactis supernatants was measured using a standard ELISA kit (R&D Systems), as recommended by the manufacturer. The sensitivity of the ELISA test is ≥10 pg/mL. The values are presented in graph and table as mean±standard deviation.
Western Blot Analysis
Primary cultures of cells of the human vaginal mucosa (HVM) have been established starting from 1 cm2 of full-thickness biopsy of the vaginal mucosa, as previously reported [Panici et al., 2007] [informed consent from patients was obtained], and maintained in basal medium for keratinocytes added with growth factor aliquots (KGM, Lonza), with change of medium twice a week. The cells were treated for 30 minutes with recombinant human KGF (Upstate Biotechnology), as a positive control, or with Nisin-induced L. lactis supernatants for 12 or 24 hours. The cells were lysed in the RIPA buffer. Total proteins (50-150 ng) were run under reducing conditions by SDS-PAGE on 10% polyacrylamide gel and transferred to Immobilon-FL membranes (Merck Millipore). The membranes were blocked in TBS buffer containing 0.1% Tween 20 (TBS-T) and 5% milk for 1 hour at 25° C. and then incubated overnight at 4° C. with the following primary antibodies: anti-phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology) and anti-ERK2 (Santa Cruz Biotechnology). The membranes were then incubated with secondary antibody conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich) for 1 hour at 25° C. The bound antibody was detected by enhanced chemiluminescent detection reagents (Pierce Biotechnology Inc), according to the manufacturer's instructions. Densitometric analysis was performed with the Quantity One program (Bio-Rad Laboratories).
Results
KGF Gene Amplification and Cloning
The KGF gene was amplified from human fibroblasts by PCR using appropriate primers, and visualized on a 1% agarose gel, in which a 500-bp product is shown, compatible with the KGF gene dimensions (
Construction of the Usp45-KGF Plasmid
In order to obtain the extracellular secretion of KGF in L. lactis cells, it was necessary to insert a specific secretion signal peptide for L. lactis upstream of the KGF sequence. The signal peptides mainly used for protein secretion in L. lactis are:
(1) the signal peptide of the main secreted lactococcal proteins, Usp45 and
(2) the proteinase signal peptide associated with the PrtP cell wall [sequence aa: MQRKKKGLSFLLAGTVALGALAVLPVGEIQAKA (SEQ ID NO: 10)]. Generally, the signal peptide of Usp45 provides better results and is more widespread than that of PrtP. Thus, inventors have cloned the synthetic gene sequence of Usp45 upstream of the KGF gene in the intermediate vector pGL3-basic (
Construction of Recombinant L. lactis Food Use Able to Inducibly Secrete KGF
The goal was to obtain a L. lactis strain able to secrete KGF inducibly, in order to obtain a prolonged release of KGF at the level of the vaginal mucosa. To this end, the Nisin-controlled gene expression system (NICE), developed by NIZO Food Research, was used. Briefly, the Usp45-KGF insert was extracted from pGL3-basic by digestion with the restriction enzymes NcoI and SacI, and cloned in a pNZ8149 vector digested with the same enzymes (
KGF Protein Expression and Secretion
The L. lactis NZ3900 cells transformed with the pNZKGF plasmid were induced or not with 0.5 ng/ml of nisin for 3, 12 or 24 hours, and then collected by centrifugation. KGF protein expression was studied by SDS-PAGE and subsequent Comassie blue staining. In the induced cells a band of 18 kDa was identified, corresponding to the molecular weight of the KGF (
Effectiveness of KGF Secreted by L. lactis
The functionality of KGF secreted by the induced L. lactis cells was evaluated by analysing its ability to induce activation of the ERK pathway in vaginal mucosa cells (HVM). Inventors therefore analyzed ERK phosphorylation in HVM cells after treatment with L. lactis supernatants at 12 or 24 hours of induction with 1 ng/ml of Nisin. Non-induced cell supernatants were used as a negative control, while human recombinant KGF was used as a positive control. Using Western blot, we showed an activation of MAPK ERK1 and 2 after 30 minutes of treatment with both induced supernatants, comparable to that observed after treatment with KGF (
The present authors have engineered a lactobacillus compatible with the vaginal microenvironment (L. lactis) to produce KGF inducibly. The procedure used is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 102019000003115 | Mar 2019 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/055705 | 3/4/2020 | WO | 00 |
