Patent Description:
Vesicular-mediated communication between cells appears critical in many biological processes. Small vesicles released from cells have recently emerged as important mediators of inter-cellular communication. These vesicles, that have been termed "extracellular vesicles (EVs)", are inclusive of exosomes released from the endosomal cell-membrane compartment and of microvesicles released from the cell surface by plasma membrane budding. The EV content of proteins, lipids and nucleic acids varies with the cell of origin and, after incorporation into recipient cells, they may transfer information which may change the phenotype and function of recipient cells.

Cellular senescence is a phenomenon by which cells cease to divide. Over the last few decades, this phenomenon has emerged as an important contributor to aging and age-related diseases and conditions. The available evidence suggests that senescence causes a loss of tissue-repair capacity because of cell cycle arrest in progenitor cells. Furthermore, senescent cells produce pro-inflammatory and matrix-degrading molecules in the so-called senescence-associated secretory phenotype (SASP). Accordingly, cellular senescence has become an attractive target for therapeutic exploitation.

Drugs able to kill senescent cells specifically in cell culture, termed senolytics, are able to reduce cellular senescence in vivo and to counteract ageing and age-related diseases and conditions. A number of senolytics are known in the prior art, including HSP90 inhibitors, Bcl-<NUM> family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Furthermore, International patent application <CIT> discloses a method for treating a patient suffering from a disease or condition caused by stem cell dysfunction or increased senescence, comprising administering to the patient a composition comprising extracellular vesicles (EVs) obtained from stem cells of a subject who is younger or healthier than the patient to be treated. <CIT> specifically mentions mesenchymal stem cells as the source for extracellular vesicles.

The present inventors have now surprisingly found that extracellular vesicles derived from human liver stem cells are much more effective in reducing cellular senescence than extracellular vesicles derived from mesenchymal stem cells (MSCs).

Accordingly, the present invention relates to a method of manufacturing a pharmaceutical preparation of extracellular vesicles derived from a cell culture of a non-oval human liver pluripotent progenitor cell line which expresses the hepatic cell marker albumin and the stem cell markers CD44, CD73 and CD90, and does not express the cell markers CD117, CD34 and CD45, the preparation being capable of strongly reducing cellular senescence. The method according to this aspect of the invention comprises the following steps:.

Optionally, the method of the invention further includes pooling two or more of the preparations selected in step (d).

The invention is based on the discovery that extracellular vesicles derived from human liver stem cells are able to reduce cellular senescence, independently of the age of the donor and of the cell passage from which they are obtained. This is a surprising discovery vis-à-vis the teachings of <CIT>, which discloses that conditioned media from young, but not old stem cells, can reverse senescence in fibroblasts and aged stem cells. Moreover, <CIT> mentions mesenchymal stem cells as the EVs source, while the present inventors found that EVs derived from human liver stem cells are much more effective in reducing cellular senescence than extracellular vesicles derived from mesenchymal stem cells (MSCs).

One advantage of using extracellular vesicles as senolytics derives from the fact that extracellular vesicles are natural products. In addition, as shown in the examples, human liver stem cells are easily isolated and extracellular vesicles are readily purified from the isolated human liver stem cells. Extracellular vesicles have been shown to preferentially target damaged, senescent cells, which makes them a particularly effective tool for reducing cellular senescence and treating diseases and conditions caused by cell damage and senescence.

Cellular senescence was described for the first time in <NPL>). Senescent cells do not proliferate despite the presence of nutrients, growth factors and absence of contact inhibition, but remain metabolically active. This phenomenon is known as "replicative senescence" and was mainly attributed to telomere shortening. Further studies have shown that senescence can also be induced by other stimuli, such as oncogenic stress, DNA damage, cytotoxic drugs and irradiation. Under certain circumstances, cell senescence may be beneficial as it acts as a tumor suppressor. However, senescence increases with aging due to the accumulation of cellular damage. Senescent cells secrete cytokines, metalloproteinases and growth factors, which constitute the so-called senescence-associated secretory phenotype (SASP). This age-dependent increase in cellular senescence and SASP contributes to decreased tissue homeostasis and aging. Additionally, the age-dependent increase in senescence burden may be responsible for numerous age-related diseases and conditions.

The relationship between cellular senescence and a number of diseases and conditions is known in the prior art. Known senescence-related diseases and conditions include for example atherosclerosis, diabetes mellitus type <NUM>, asthenia, hair graying, skin-ageing, sarcopenia, age-related adiposity, fibrosis and in particular pulmonary fibrosis, glaucoma, cataracts, diabetic pancreas, osteoarthritis, degenerated intervertebral discs, cancer, pulmonary hypertension, age-related cardiovascular disease, age-related neurodegeneration, age-related cognitive impairment, Alzheimer's disease, Parkinson's disease, macular degeneration, chronic obstructive pulmonary disorder, emphysema, insulin insensitivity (see, inter alia, <NPL>; <NPL>; <NPL>; <NPL>;. <NPL>; <NPL>).

Accordingly, extracellular vesicles derived from human liver stem cells are particularly suitable for reducing cellular senescence and treating the aforementioned senescence-related diseases and conditions.

The type of liver stem cells used as the EVs source within the context of the present invention is the non-oval human liver pluripotent progenitor cell line expressing hepatic cell markers described in International patent application <CIT>. International patent application <CIT> also discloses a method of isolating the aforementioned non-oval human liver pluripotent progenitor cell line.

In a preferred embodiment, the non-oval human liver pluripotent progenitor cell line is capable of differentiating into mature liver cells, insulin producing cells, osteogenic cells and epithelial cells. The non-oval human liver pluripotent progenitor cell line expresses the hepatic cell marker albumin and the stem cell markers CD44, CD73, and CD90 and does not express the cell markers CD117, CD34, and CD45.

Next to the type of stem cell source the age of the patient and the general health status influences the capability of the EV preparation to reduce cellular senescence. While it was shown that EVs taken from a patient at the age of <NUM> are still effective, a higher efficacy is associated with EVs derived from stem cells from patients of a younger age. It is preferred that the EVs are obtained from a stem cell source from patients of up to <NUM> years of age. Furthermore, a higher number of culture passages of the stem cells the EVs are derived from decreases the capability of the EV preparation to reduce cellular senescence. While preparations of EVs taken from a HLSC culture at passage <NUM> still shows a significant senescence-reducing activity, an EV preparation taken from a HLSC culture at less than <NUM> passages is preferred.

Numerous markers are available for measuring cellular senescence, but the current standard for detecting senescent cells is the measurement of a specific β-galactosidase enzymatic activity at pH <NUM> (<NPL>). Advantageously, SA-β-gal is a recognized marker for all types of senescent cells, whereby the SA-β-gal assay can be used for the assessment of the senescence of any type of cells.

According to the general SA-β-galactosidase-based cellular senescence assay, a cell sample to be assayed is incubated in a culture medium, then contacted with a DNA intercalating dye, such as Hoechst dye. SA-β-gal -positive cells are then quantified by routine methods, such as by sCMOS camera detection technology.

As mentioned, the SA-β-galactosidase-based cellular senescence assay can be performed with different cell types. Well recognized cell types for performing the SA-β-gal cellular senescence assay are human IMR90 fibroblasts with etoposide-induced senescence and primary Ercc1-/- murine embryonic fibroblasts (MEFs) with oxidative stress-induced senescence, both described in <NPL>).

In an experimental study carried out using the aforementioned types of SA-β-galactosidase-based cellular senescence assays, the present inventors found that the EVs derived from human hepatic stem cells are particularly effective in reducing the senescence of cells as compared to umbilical cord MSC-derived EVs. The EVs used for in the manufacturing method of the invention are EVs derived from the non-oval human liver pluripotent progenitor cell line expressing hepatic cell markers disclosed in <CIT>.

Furthermore, by using the aforementioned types of the SA-β-galactosidase-based cellular senescence assays, the present inventors obtained a preparation of EVs derived from human hepatic stem cells having a particularly strong senescence-reducing activity. Indeed, the inventors found that such a preparation of EVs is capable of reducing the senescence of a population of senescent cells by about <NUM>% when employed at a concentration of <NUM> x <NUM><NUM> EVs/ml. At a dose of about <NUM> x <NUM><NUM> EVs/ml, the obtained preparation was shown to reduce senescence by about <NUM>%.

Based on the strong senescence-reducing activity of preparations of EVs derived from human hepatic stem cells according to the method of the invention, EV preparations at a concentration of <NUM> x <NUM><NUM> EVs/ml show a reduction of senescence of a population of test senescent cells by at least <NUM> %, or by at least <NUM>%, or by <NUM>%, wherein senescence is measured in a SA-β-galactosidase-based cellular senescence assay using as the control the test senescent cells not treated with EVs. A preparation of a lesser concentration of <NUM> x <NUM><NUM> EVs/ml showed a reduction of senescence of at least <NUM> %, or <NUM> %.

Interestingly, the senescence-reducing activity of the EVs preparation obtained by the method of the invention is present, although with a reduced strength, even when the extracellular vesicles are derived from an old donor (i.e. <NUM> years old) or when the extracellular vesicles are purified from cells kept in culture for an extended period of time, for example for up to passage <NUM>.

HLSCs were isolated from human cryopreserved normal adult hepatocytes (Lonza, Basel, Switzerland) as described before (<NPL>).

Briefly, HLSC6B and HLSC2 were cultured in alpha minimum essential medium (Lonza, Basel, Switzerland) supplemented with L-glutamine (<NUM>), penicillin (<NUM> IU/ml), streptomycin (<NUM>µg/ml) (all from Sigma, St. Louis, MO, USA), <NUM>% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA, USA) and human recombinant epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) (4ng/ml both). Cells were expanded, characterized, and cryo-preserved as described previously. Two days before extracellular vesicles (EVs) isolation, HLSCs were culture in culture medium where FCS was substituted with EVs-depleted FCS to avoid serum EVs contaminations.

Extracellular vesicles (EVs) were obtained from supernatants of HLSC6b and HLSC2 (<NUM> × <NUM> cells/T75 flask) cultured in serum-free Roswell Park Memorial Institute Medium (RPMI) (Euroclone S. A, Italy) for <NUM>. Viability of cells at the time of supernatant collection was <NUM>% as confirmed by Trypan blue exclusion. Briefly, supernatants were centrifuged at <NUM>,<NUM> for <NUM> at <NUM> for the removal of cell debris and apoptotic bodies, followed by ultracentrifugation at <NUM>,<NUM> for <NUM> at <NUM> (Beckman Coulter Optima L-<NUM>, Fullerton, CA, USA). The pellet of EVs obtained was resuspended in RPMI supplemented with <NUM>% dimethyl sulfoxide (DMSO) and stored at -<NUM> until use (<NPL>).

The Ercc1-/- MEFs were isolated as disclosed in <NPL>). The MEF assay was performed essentially as disclosed in the same article. In brief, MEFs (<NUM> × <NUM><NUM>) at <NUM>% O<NUM> were seeded per well in <NUM>-well plates at least <NUM> prior to treatment. Following addition of the EVs, the MEFs were incubated for <NUM> to <NUM> under <NUM>% O<NUM> oxygen conditions. For fluorescence analysis of SA-β-Gal activity, cells were washed <NUM> ×with PBS, C12FDG (<NUM>) was added to the culture medium, and the cells were incubated for <NUM>-<NUM>. Ten minutes prior to analysis, the DNA intercalating Hoechst dye (<NUM>µg/ml) was added to the cells. For quantitative analysis of cell number (Hoechst staining) and number of C12FDG positive, senescent cells, a laser-based line scanning confocal imager IN Cell Analyzer <NUM> with large field-of view sCMOS camera detection technology was used. An acquisition protocol was established using Acquisition software v4. <NUM>, including parameters such as the plates and wells that were imaged, wavelengths, and exposure time. The acquired images were analyzed using the Multi Target Analysis Module that allows the creation of various decision trees and the application of appropriate classification filters to different image stacks. All samples were analyzed in duplicate with <NUM>-<NUM> fields per well and mean values and standard deviations being calculated accordingly.

Human IMR90 lung fibroblasts were obtained from American Type Culture Collection (ATCC) and cultured in EMEM medium with <NUM>% FBS and pen/strep antibiotics. To induce senescence, cells were treated for <NUM> with <NUM> etoposide. Two days after etoposide removal, about <NUM>% of IMR90 cells tested SA-β-Gal positive. Cells were treated for <NUM> with <NUM> <NUM>-DMAG and the percentage of SA-β-Gal-positive cells was determined using C12FDG-based senescence assay. This assay is disclosed in <NPL>).

The experimental studies illustrated above were performed with extracellular vesicles derived from human liver stem cells obtained from a <NUM> years old donor (HLSC6B) and with extracellular vesicles derived from human liver stem cells obtained from a <NUM> years old donor (HLSC2).

The experimental data show that both the HLSC6B-derived EVs and the HLSC2-derived EVs are able to reduce the percent of senescent IMR90 and MEFs cells in a reproducible manner (see <FIG>, B <FIG>). <FIG> shows that a senescence-reducing activity of HLSC6B-derived EVs is already noticeable at a low concentration of <NUM> x <NUM><NUM> EVs/ml. HLSC6B-derived EVs are more effective than HLSC2-derived EVs in reducing fibroblast cellular senescence in both the MIF and the IMR90 assay, but both types of HLSC-derived EVs are in any case more effective than umbilical cord MSC-derived EVs (see Figure A, B and <FIG>).

Furthermore, the experimental show that, although the ability of HLSC-derived EVs to suppress senescence diminishes with passage, this ability is still observed at passage <NUM> and is statistically significant at passage <NUM> both in the case of HLSC6B-derived EVs and in the case of HLSC2-derived EVs (see <FIG>).

Claim 1:
A method of manufacturing a pharmaceutical preparation of extracellular vesicles (EVs) derived from a cell culture of a non-oval human liver pluripotent progenitor cell line which expresses the hepatic cell marker albumin and the stem cell markers CD44, CD73 and CD90, and does not express the cell markers CD117, CD34 and CD45, the method comprising the steps of:
• isolating EVs from the conditioned medium of the cell culture;
• preparing one or more samples from the isolated EVs at a predetermined concentration of EVs;
• testing the activity of each EVs sample in a potency test measuring the reduction of cellular senescence wherein senescence is measured in a SA-β-galactosidase-based cellular senescence assay using as the control the test senescent cells not treated with EVs;
• selecting the preparations in which the reduction of cellular senescence measured exceeds a predetermined threshold;
• optionally pooling two or more selected preparations.