WO2011148025A1

WO2011148025A1 - Biomarker for cartilaginous human cells

Info

Publication number: WO2011148025A1
Application number: PCT/ES2011/070376
Authority: WO
Inventors: Antonio CAMPOS MUÑOZ; Miguel Alaminos Mingorance; Pedro HERNÁNDEZ CORTES; Alvaro Morales Villaescusa
Original assignee: Servicio Andaluz De Salud; Universidad De Granada
Priority date: 2010-05-25
Filing date: 2011-05-25
Publication date: 2011-12-01
Also published as: ES2370794A1; ES2370794B1

Abstract

The present invention relates to the use of a biomarker in the selection of cells intended for transplants, specifically, a biomarker for cartilage precursor cells that includes at least one expression product of the susd-2 gene and the derivatives thereof.

Description

HUMAN CARTILAGIN CELL BIOMARKER

The present invention falls within the field of biomedicine and, more specifically, of tissue engineering and regenerative medicine. Specifically, the present invention relates to the use of a biomarker in the selection of cells intended for transplantation.

STATE OF THE PREVIOUS TECHNIQUE Cartilage is a specialized connective tissue, which has a firm flexible matrix that resists mechanical stresses. Participates in the support of the body, for being intimately associated with the skeletal system. The cartilage has cells called chondrocytes, which occupy small cavities called lagoons within the extracellular matrix that they secrete. The extracellular matrix of the cartilage is not vascularized or innervated, and does not have lymphatic vessels; however, the cells receive their nutrition from the blood vessels of the surrounding connective tissues and the synovial fluid by diffusion through the matrix. The extracellular matrix is composed of glycosaminoglycans and proteoglycans, intimately associated with collagen and elastic fibers embedded in the matrix (Deiters and Prehm, Arthritis Res Ther. 2008; 10 (1): R8).

As the fibers are in the extracellular matrix, there are three types of cartilage (Finn and Geneser, 3rd ed . , 2000):

• Hyaline Cartilage: The extracellular matrix is flexible and semi-translucent, blue-gray in color, rich in type II collagen. It is the most frequent in the body. It is found in the nose, larynx, ventral ends of the ribs (at the sites where they are connected with the sternum), and in the tracheal and bronchial rings. This cartilage forms the cartilaginous model of many of the bones during embryonic development, and constitutes the epiphyseal plates of the growing bones. Elastic Cartilage: Elastic cartilage is found in ears, external and internal auditory ducts, epiglottis and larynx (cuneiform cartilage). In almost all aspects, elastic cartilage is identical to hyaline cartilage, and is often related to it. The outer fibrous layer of perichondrium (irregular dense connective tissue, responsible for the growth and regeneration of cartilage) is rich in elastic fibers. The matrix of the elastic cartilage has abundant branched elastic fibers that vary between thin and thick, interposed with bundles of type II collagen fibers, providing greater flexibility than the hyaline cartilage matrix. Chondrocytes of elastic cartilage are more abundant and larger than those of hyaline cartilage.

Fibrocartilage: It is found in intervertebral discs, pubic symphysis and joint discs, and inserted into the bone. It is associated with hyaline cartilage and dense connective tissue. Unlike the other two types of cartilage, fibrocartilage does not have perichondrium, has a small amount of extracellular matrix rich in chondroitin sulfate and dermatan sulfate and has bundles of type II collagen. Chondrocytes are usually aligned in parallel parallel rows with thick bundles of collagen. Numerous pathologies can affect human cartilage, with autoimmune, infectious, traumatic or degenerative diseases being very frequent, with osteoarthritis, arthritis and joint surface ulcers standing out. All these diseases have a very high incidence and represent a great health expenditure in our environment. Conventional treatments for the repair of articular cartilage include microfractures, mosaicoplasties, and the use of autografts and heterologous transplants. Autologous chondrocyte transplantation has emerged as an alternative to the conventional clinical treatment of cartilaginous defects, in many cases it is possible to reduce the pain and improve the patient's joint function by means of the autologous chondrocyte implant. So far, research in this field is based on the culture of chondrocytes for clinical application as suspension cells (ACI, autologous chondrocyte implant) or, more recently, for culture on biocompatible membranes, applying the patient as a membrane on which cultivates a monolayer of cartilaginous cells (MACI, membrane autologous chondrocyte implant).

Since 1987, multiple researchers have developed clinical culture and implant protocols for laboratory-grown chondrocytes in more than 12,000 patients (Marlovits et al., Eur J Radiol. 2006,57 (1): 24-31), the results being very variable from one study to another. While some trials suggest that the use of these techniques can solve joint problems very effectively, most studies, including meta-analysis and review work, show that the usefulness of the autologous chondrocyte implant is very limited. This is probably due to the low density of the cartilaginous tissue formed or the insufficient quantity and quality of the cells implanted in the patient. In general, the cartilage generated after the autologous chondrocyte implant is rich in type I collagen, with poor biochemical and biomechanical characteristics and very different from those in the native cartilage, rich in type II collagen (Tuli et al., Arthritis Res Ther 2003.5 (5): 235-238). This could be due, with high probability, to the fact that the cells used for the cartilage cell therapy could have low rates of cell viability or by the use of cells other than well-differentiated and functional chondrocytes. Numerous researchers have shown that human adult cells tend to dedifferentiate and lose functionality when they are maintained in culture for a long time (Rodríguez-Morata et al., Ann Vasc Surg. 2008,22 (3): 440-448; Alaminos et al. , J Cell Physiol. 2007,21 1 (3): 692-698). In the same way, it is very common to find contaminating or connective mesenchymal cells of chondrocyte cultures, especially fibroblasts from the perichondrium. For that reason, it is important to develop methods to control the levels of differentiation of cultured cells to ensure the use of well differentiated cells in cartilage cell therapy and avoid the use of contaminating cells. Despite this, to date no sufficiently specific markers of human chondrocytes have been described, so far using purely morphological criteria together with the expression of collagen II or aggrecan, compounds existing in the extracellular matrix of mature hyaline cartilage.

The sushi domain containing protein-2 gene (susd-2; GenBank accession number AK026431), is conserved in mammals (chimpanzee, dog, cow, mouse, rat) birds (chicken) fish (zebrafish) and invertebrates (fly of the fruit, mosquito and C. elegans). In humans, the susd-2 gene is located on chromosome 22q1 1 -q12. The expression of SUSD2 has not been determined to date in human or animal cartilage cells. The prediction of expression levels can be carried out using computer programs such as e-Northern (virtual Northern-Blot), which indicates that mRNA levels could be high in kidney and lung, but not in cartilage (Gene-Cards , http://www.genecards.org/cgi-bin / carddisp.pl? gene = SUSD2 & search = SUSD2).

To ensure the success of cell therapy with autologous chondrocytes, it would be necessary to have a specific marker of the mature chondrocyte that is expressed in its surface or in the cytoplasm of asthma cells maintained in culture. In addition, the technical field lacks a biocompatible platform that provides the optimal conditions for the regeneration of cartilaginous tissue.

EXPLANATION OF THE INVENTION

The present invention provides a new chondrocyte biomarker in culture that allows the specific identification of cartilage lineage cells against other mesenchymal or connective lineage cells present in the culture. The use of the biomarker of the present invention together with a specific growth platform for chondrocytes optimizes the regeneration of cartilaginous tissue.

The inventors of the presence presently show that, surprisingly, the susd-2 gene is expressed in human chondrocytes maintained in culture and therefore can be used to differentiate these from other types of mesenchymal and connective cells also present.

Thus, a first object of the present invention is a biomarker of cartilage precursor cells, hereinafter biomarker of the invention, characterized in that it comprises at least one expression product of the susd-2 gene or some fragment of at least one of them. .

In this report, SUSD2 is understood as the expression product of the sushi domain containing protein-2 gene (susd-2 or SUSD-2; GenBank accession number AK026431). It is conserved in mammals (chimpanzee, dog, cow, mouse, rat) birds (chicken) fish (zebrafish) and invertebrates (fruit fly, mosquito and C. elegans). In humans, the SUSD-2 gene is located on chromosome 22q1 1 -q12. The expression of SUSD2 has not been determined to date in human or animal cartilage cells. In the context of the present invention, SUSD2 is also defined by a nucleotide or poly inucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 2, and which would comprise various variants from:

a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

b) nucleic acid molecules whose complementary hybrid chain under astringent conditions with the polynucleotide sequence of (a),

c) nucleic acid molecules whose sequence differs from (a) or (b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence with an identity of at least one

80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2, and in which the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the SUSD2 protein .

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the SUSD2 protein. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, methylation phosphorylation or acylation. As used herein, a protein is "substantially homologous" to the SUSD2 protein when its amino acid sequence exhibits a good alignment with the amino acid sequence SEQ ID NO: 2; that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 1 of at least 50%, typically of at least 80%, advantageously of at least 85 %, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99%. The sequences homologous to the SUSD2 protein can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences. The term "functionally equivalent", as used herein, means that the proteins or fragment / s of the protein / s in question essentially maintain the biological properties described herein. Said capacity can be determined by conventional methods.

The present invention facilitates the selection of chondrocytes in culture by analyzing the expression levels of the susd-2 gene. The expression products of said gene may be its messenger RNA (mRNA; hereinafter also SEQ ID NO: 1) or the protein it encodes, called sushi domain containing 2 (SUSD2; hereafter also SEQ ID NO: 2 ). Accordingly, in a preferred embodiment, the biomarker of the present invention comprises SEQ ID NO: 1 or some fragment thereof. In another preferred embodiment, the fragment is the nucleotide sequence between position 2106 and position 2640 of SEQ ID NO: 1. In another preferred embodiment, the fragment is the nucleotide sequence between position 2765 and position 3143 of SEQ ID NO: 1.

Also, in another preferred embodiment, the biomarker of the present invention comprises SEQ ID NO: 2 or some fragment thereof.

To determine the expression of the biomarker of the present invention, techniques widely known in the art are used. For example, the expression of the susd-2 gene in its form of SEQ ID NO: 1 can be analyzed by quantitative RT-PCR or RNA microarrays. While to analyze the expression in its form of SEQ ID NO: 2 immunohistochemical, western-blot or microarray techniques are used. proteins

As mentioned above, the usefulness of the biomarker of the present invention is defined by providing discriminatory capacity when selecting the cells of a culture that will actually give rise to mature and functional chondrocytes once transplanted. Therefore, another aspect of the present invention relates to the use of the biomarker of the present invention in the selection of in vitro cells intended for transplantation. In a preferred embodiment, the biomarker of the present invention is used in the selection of cells in vitro for the preparation of a cellular implant. In another preferred embodiment, the biomarker of the present invention is used in the selection of cells in vitro for the preparation of an artificial tissue. More preferably, the biomarker of the present invention is used in an in vitro preparation method of an artificial tissue comprising an in vitro selection step of cultured cells expressing the susd-2 gene. Another aspect of the invention relates to a method of obtaining a cell or an isolated cell population useful for the preparation of a cell implant and / or an artificial tissue, hereinafter method of the invention, which comprises determining the expression of the biomarker of the invention.

Another aspect of the invention relates to an isolated cell or an isolated cell population, hereinafter cell or isolated cell population of the invention, obtainable by the method of the invention.

Another aspect of the invention relates to a composition, hereinafter composition of the invention, comprising an isolated cell or an isolated cell population of the invention. The cells of the invention, the cell population of the invention, as well as the composition of the invention, can be part of a pharmaceutical composition for administration to a subject. Therefore, another aspect of the invention relates to a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising an isolated cell of the invention or a cell population of the invention. In a preferred embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier. In another preferred embodiment, the pharmaceutical composition of the invention further comprises another active ingredient.

The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government regulatory agency or a state government or listed in the State Pharmacopoeia in the European Pharmacopoeia, or other pharmacopoeia generally recognized for its use in animals, and more specifically in humans.

The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the cells or the cell population of the invention or of said composition comprising cells of the invention obtainable according to the method of the invention should be administered; obviously, said vehicle must be compatible with said cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (eg, 0.9% sterile saline NaCI, phosphate buffered saline (PBS), Ringer-lactate solution, etc. ), optionally supplemented with serum, preferably with autologous serum; cell culture media (eg, DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polyiornitine, hydrogels, agarose, dextran sulfate silicone. Also, if desired, the support medium may, in specific embodiments, contain growth factors or other agents. If the support is solid, semi-solid, or gelatinous, the cells can be introduced into a liquid phase of the vehicle that is subsequently treated in such a way that it becomes a more solid phase.

The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the cells, which comprise said pharmaceutical composition, and / or auxiliary substances or Pharmaceutically acceptable substances, such as buffering agents, surfactants, cosolvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, and so on. Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions. Examples of suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that they promote a chemical change in the preparation of the drug and are present therein in a planned modified manner that provides the specific activity or effect. Another aspect of the invention relates to the use of a cell or a population of cells of the invention, or of a pharmaceutical composition of the invention, in the manufacture of a medicament. Alternatively, it refers to a cell or a population of cells of the invention for use in the manufacture of a medicament.

Another aspect of the invention relates to the use of a cell or a population of cells of the invention, or of a pharmaceutical composition of the invention, in the preparation of a medicament for tissue repair and regeneration. Alternatively, it refers to a cell or a population of cells of the invention for use in the manufacture of a medicament for tissue repair and / or regeneration.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.

The isolated adult cell of the invention, or the cell population of the invention, may be of autologous, allogeneic or xenogenic origin. Preferably, they are of autologous origin. The administration can also be carried out with the help of a "scaffold" or bioactive scaffolding, that is, of a support structure that serves to transport cells and / or growth or differentiation factors, and which usually consist of a structure quite rigid, covered or impregnated with a drug that helps cells to establish themselves and build a tissue similar to the one you want to replace. Different scaffold are known in the state of the art. If that is the case, the cell may be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in its genome that modify the expression of genes that are important for their basic properties (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest such as, for example, proteins with therapeutic properties. Therefore, in another preferred embodiment, the cell of the invention has been genetically modified. In the present invention, "marker" or "biomarker" means a protein or a fragment thereof, or the sequence encoding said protein or fragment, which distinguishes a cell (or group of cells) from another cell (or cell group). The cell population of the invention is positive for a given marker if at least 20% of the cells in the population show a detectable expression of the marker, preferably 70%, 80%, 90%, 95%, and much more. preferably, 98%. Sometimes, 99% or 100% of the cells of the invention show a detectable expression of the marker.

The term "isolated" indicates that the cell or cell population of the invention to which it refers, are not in their natural environment. That is, the cell or cell population has been separated from its surrounding tissue. Particularly it means that said cell or the cell population is substantially free (free) of other cells normally present in its environment. In general, a cell is essentially free of other cells normally present in its environment when it is separated from at least 60%, preferably at least 80%, preferably from at least 90%, more preferably from, at least 95%, even more preferably of at least 96%, 97%, 98% or even 99%, of said cells normally present in the environment of it. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Quantification of the mRNA of the susd-2 gene in different types of cartilage as well as in different cell types. The values obtained are expressed in fluorescence units. These data demonstrate that the expression of the susd-2 gene is significantly higher in all types of cartilage compared to cells from epithelial tissue and mesenchymal cells.

EXAMPLES The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1. Quantification of the expression levels of the susd-2 gene in chondrocytes in culture. The analysis was performed by quantifying the expression of SUSD2 at the RNA level using commercial RNA microarrays from the Affymetrix® commercial house (Human-Genome U133 plus 2.0 system). The results show that the average expression of this gene in human cartilage is 957.9 UF (fluorescent units of the Affymetrix ^® trading house), being 1 612.7 U .F. in human hyaline cartilage and 303, 1 U .F. in fibrous cartilage (fibrocartilage). These values are significantly higher (p <0.001) than those found in other types of mesenchymal cells, including fibroblasts of the oral and skin mucosa and mesenchymal stem cells of the Wharton's jelly of the umbilical cord (mean 85.6 UF) and in epithelial cells of the cornea and oral mucosa (mean 29.6 UF) (Figure 1 and Table 1).

These results demonstrate that chondrocytes maintained in culture express significantly higher amounts of the SUSD2 gene at the RNA level than other cell types of both mesenchymal and epithelial type, so that their quantification could be used as a specific marker of human chondrocytes for clinical use.

Table 1. Expression in fluorescent units

Claims

Biomarker of cartilage precursor cells characterized in that it comprises at least one expression product of the susd-2 gene or some fragment of at least one of them.

Biomarker according to claim 1 characterized in that it comprises SEQ ID NO: 1, or some fragment thereof.

Biomarker according to claim 2 characterized in that the fragment is the sequence between position 2106 and position 2640 of SEQ ID NO: 1.

Biomarker according to claim 2 characterized in that the fragment is the sequence between position 2765 and position 3143 of SEQ ID NO: 1.

Biomarker according to claim 1 characterized in that it comprises SEQ ID NO: 2, or some fragment thereof.

Use of a biomarker according to any of claims 1 to 5, in the selection of in vitro cells intended for transplantation.

7. Use of a biomarker according to claim 6, in the selection of cells in vitro for the preparation of a cellular implant.

8. Use of a biomarker according to claim 6, in the selection of cells in vitro for the preparation of an artificial tissue. 9. Method of obtaining a cell or an isolated cell population useful for the preparation of a cell implant and / or an artificial tissue, which comprises determining the expression of the biomarker according to any one of claims 1 to 5.

10. Cell or isolated cell population obtainable according to the method of claim 9.

eleven . Use of a cell or a cell population according to claim 10, in the manufacture of a medicament. 12. Use of a cell according to claim 10, in the preparation of a medicament for tissue repair and / or regeneration.