JP7610865B2 - Methods for protecting dopamine neurons in vivo - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act 1. Disclosure by publishing a paper in an online journal Brain Sciences, Volume 8, Issue 9 (September 2018) (URL: https://www.mdpi.com/2076-3425/8/9/167/htm) "The Healing Effect of Human Milk Fat Globule-EGF Factor 8 Protein (MFG-E8) in A Rat Model of Parkinson's Disease" Publication date: August 31, 2018

Application of Article 30, Paragraph 2 of the Patent Act Publication by distribution of research report Name of research report: GSK Japan Research Grant 2016 Research Report Publisher: GlaxoSmithKline K.K. Date of issue: February 1, 2019

The present invention relates to a therapeutic agent for Parkinson's disease. More specifically, the present invention relates to dopamine neurons in the substantia nigra of the midbrain, a drug for protecting therapeutic dopamine neurons, and therapeutic cells.

Parkinson's disease was first reported by James Parkinson in 1817 (Non-Patent Document 1).
Parkinson's disease is a neurodegenerative disease that often develops in people in their 50s and 60s, and causes the degeneration of nerve cells in the brain and peripheral autonomic nervous system. Symptoms of Parkinson's disease include motor symptoms such as bradykinesia, tremors, muscle rigidity, and impaired posture, as well as various non-motor symptoms such as olfactory disorders, autonomic symptoms, psychiatric symptoms, cognitive impairment, and sleep disorders. The cause of Parkinson's disease has not yet been fully elucidated, but it is thought that in Parkinson's disease, a protein called α-synuclein aggregates and accumulates in nerve cell bodies and nerve fibers, and that the process of this aggregation may damage nerve cells. The spherical structures formed by the accumulation of α-synuclein are called Lewy bodies.

The number of Parkinson's disease patients increases with age, and in Japan, the number of patients is thought to be around 150 per 100,000 population. In Parkinson's disease, motor symptoms occur due to a decrease in dopamine nerve terminals in the striatum, which is accompanied by the degeneration and loss of dopamine neurons in the substantia nigra, and a decrease in the amount of dopamine in the striatum. It is said that motor symptoms appear when the number of dopamine neurons in the substantia nigra decreases by around 50% and the amount of dopamine in the dopamine nerve terminals falls to 20% or less (Non-Patent Document 2).

The main treatment for Parkinson's disease is drug therapy. Other options include brain surgery such as deep brain stimulation, rehabilitation, and cell transplantation therapy. As an example of therapeutic dopamine neurons, a group led by Professor Jun Takahashi of Kyoto University conducted basic research using monkeys, and in 2018 began clinical trials in humans using therapeutic dopamine neurons produced from human induced pluripotent stem cells (iPS cells) (https://www.cira.kyoto-u.ac.jp/j/pressrelease/news/180730-170000.html).
The most common drug treatment is dopamine replacement therapy, which administers levodopa, a precursor of dopamine.Other drugs that may be used include dopamine agonists that have the same effect as dopamine, drugs that promote the release of dopamine (amantadine), inhibitors of substances that cause the breakdown of dopamine (selegiline, entacapone), non-dopamine zonisamide, adenosine receptor antagonist istradefylline, and drugs that reduce acetylcholine (anticholinergics).

The following new drugs have been approved in Japan in the past six years (January 2011 to January 2017).
- Dopamine agonists (Mirapex LA [Boehringer Ingelheim Japan Co., Ltd.], Requip CR [GlaxoSmithKline K.K.], Apokyn [Kyowa Hakko Kirin Co., Ltd.], Neupro (patch [Otsuka Pharmaceutical Co., Ltd.])
- Adenosine A2A receptor inhibitor with a new mechanism that acts on the non-dopamine system (Nouriast [Kyowa Hakko Kirin Co., Ltd.])
- Levodopa-carbidopa hydrate combination for jejunal administration (Duodopa [AbbVie])
- A combination of the COMT inhibitor entacapone and a combination drug of levodopa and carbidopa (Stalevo [Novartis Pharma])

Milk fat globule-EGF factor 8 (MFG-E8) is a human lactadherin homologue and is a membrane-bound glycoprotein found in breast milk and breast epithelial cells (see non-patent document 3).
Non-Patent Document 4 describes that administration of MFG-E8 can be effective in treating sepsis and ischemia-reperfusion injury.
Non-Patent Document 5 describes that MFG-E8 secreted from activated macrophages binds to apoptotic cells and induces phagocytosis by phagocytes.
Patent Document 1 describes a method for culturing cells using MFG-E8, and shows a technique that enables human iPS cells to be cultured without using a scaffold material.
Although various reports have been published on MFG-E8, there have been no reports to date of administering MFG-E8 as a treatment for Parkinson's disease or of using MFG-E8-producing cells as therapeutic cells.

WO2015-182140

J Neuropsychiatry Clin Neurosci. 2002 Spring;14(2):223-236 Tomoyuki Orimo, New developments in the diagnosis and treatment of Parkinson's disease, Clinical Neurology 2017;57:259-273 Pediatric Research. 1996 April 39:66. Mol Med. 2011 Jan-Feb;17(1-2):126-133. Nature. 2002 May 9;417(6885):182-187. Brain Sci. 2018 Aug 31;8(9).

The main objective of the present invention is to provide a technology capable of protecting dopamine neurons in the substantia nigra of the midbrain and therapeutic dopamine neurons.

The inventors have discovered that a fragment of MFG-E8 promotes the protection of dopamine neurons in the body, and have completed the invention of a composition for protecting dopamine neurons that contains this as an active ingredient.

That is, in order to solve the above problems, the present invention provides the following [1] to [3].
[1] A composition containing SEQ ID NO: 2 (24-387 fragment of human MFG-E8) as an active ingredient is added,
A culture medium for protecting dopamine neurons in the substantia nigra from inflammation-induced loss.
[2] A composition containing a cell expressing a polypeptide or protein represented by SEQ ID NO: 2 (24-387 fragment of human MFG-E8) as an active ingredient is added,
A culture medium for protecting dopamine neurons in the substantia nigra from inflammation-induced loss.
[3] The medium according to [1] or [2],
By administering it directly into the human brain,
A culture medium for protecting dopamine neurons in the substantia nigra from inflammation-induced loss.

The present invention makes it possible to provide technology that can protect dopamine neurons in the substantia nigra of the midbrain and therapeutic dopamine neurons.

This is a microscopic image showing the effect of MFG-E8 fragments when lipopolysaccharide (LPS), a component of the cell wall outer membrane of gram-negative bacteria, was administered to the substantia nigra of the midbrain in rats (Example 1). The arrows indicate TH staining-positive cells in the immunohistochemical section image of the rat substantia nigra (scale bar 200 μm). This figure shows the results of evaluating the effect of MFG-E8 fragments when LPS was administered to the substantia nigra of the midbrain using rats (Example 1). LPS was administered to the substantia nigra of the midbrain, and the rats were kept for two weeks in the presence or absence of MFG-E8. The brain tissue sections were then immunostained using a tyrosine hydroxylase (TH) antibody, and the area ratio of the stained positive site to the area in the microscopic image was calculated. This figure shows the results of evaluating the effect of MFG-E8 fragments when LPS was administered to the substantia nigra of the midbrain using rats (Example 1). LPS was administered to the substantia nigra of the midbrain, and the rats were kept for two weeks in the presence or absence of MFG-E8. After removal, brain tissue sections were immunostained with tyrosine hydroxylase (TH) antibody, and the number of stained dopamine neurons per area in the microscopic image was measured.

The following describes a preferred embodiment of the present invention. Note that the embodiment described below is an example of a typical embodiment of the present invention, and is not intended to narrow the scope of the present invention.

<<Principle of the invention and active ingredients>>
The present invention was discovered based on the fact that a fragment of human MFG-E8 shown in SEQ ID NO:2 has a protective effect on dopamine neurons in the body.
Human MFG-E8 is a protein consisting of 387 amino acid residues as shown in SEQ ID NO: 1 (GenBank Accession No. Q08431), and is also called lactadherin, breast epithelial antigen BA46, HMFG, MFGM, and secreted EGF repeat and discoidin domains-containing protein 1 (SED1).

In SEQ ID NO:1, the amino acid sequence from 1 to 23 is a secretory signal sequence, and also has functional sites as shown in the sequence below.
[SEQ ID NO: 3] Shows the EGF-like domain site (amino acid residues 24-67 of the amino acid sequence of SEQ ID NO: 1).
[SEQ ID NO: 4] Shows the F5/8 type C1 domain site (amino acid sequence 70-225 of SEQ ID NO: 1).
[SEQ ID NO: 5] Shows the F5/8 type C2 domain site (amino acid sequence 230-387 of SEQ ID NO: 1).
[SEQ ID NO: 6] Lactadherin Chain site (amino acid sequence 24-387 of SEQ ID NO: 1).
[SEQ ID NO: 7] Lactadherin short form chain site (amino acid sequence 202-387 of SEQ ID NO: 1).
[SEQ ID NO: 8] Medin Chain site (amino acid sequence 268-317 of SEQ ID NO: 1).
[SEQ ID NO: 9] A portion of the sequence of MFG-E8 secreted into the culture supernatant of human adipose-derived mesenchymal stem cells (amino acid sequence 70-107 of SEQ ID NO: 1).
[SEQ ID NO: 10] A portion of the MFG-E8 sequence secreted into the culture supernatant of human adipose-derived mesenchymal stem cells (amino acid sequence 207-219 of SEQ ID NO: 1).
[SEQ ID NO: 11] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in a clinical medium (amino acid sequence 93-107 of SEQ ID NO: 1).
[SEQ ID NO: 12] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in clinical medium (amino acid sequence 111-134 of SEQ ID NO: 1).
[SEQ ID NO: 13] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in a research medium (amino acid sequence 111-135 of SEQ ID NO: 1).
[SEQ ID NO: 14] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in research medium (amino acid sequence 220-237 of SEQ ID NO: 1).
[SEQ ID NO: 15] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in research medium (amino acid sequence 254-268 of SEQ ID NO: 1).
[SEQ ID NO: 16] A portion of the MFG-E8 sequence expressed by human adipose-derived mesenchymal stem cells cultured in research medium (amino acid sequence 272-308 of SEQ ID NO: 1).
In addition, SEQ ID NO:1 has the following modifications or functional sites:
[Disulfide bond area]
Amino acids represented by combinations of positions 27-38, 32-55, 57-66, 70-225, 212-216, and 230-387 in SEQ ID NO:1.
[Glycosylation site]
In sequence number 1, amino acids at positions 228, 238, 325, 329, and 350.
[Phosphoserin modification site]
The 42nd amino acid in sequence number 1.

The MFG-E8 fragment used in the present invention consists of the amino acid sequence from the 24th to the 387th amino acid residues in the amino acid sequence of human MFG-E8, and is a fragment consisting of approximately 94% of human MFG-E8 (387 amino acid residues).
That is, the MFG-E8 fragment is composed of an amino acid sequence from which the secretory signal sequence portion in human MFG-E8 has been removed, and its functions, except for its secretory ability, are equivalent to those of human MFG-E8. In addition, the MFG-E8 fragment contains various functional sites shown in SEQ ID NOs: 3 to 16. Thus, a composition that contains any one or more of the functional sites of the MFG-E8 fragment, or that can be evaluated as having a function functionally equivalent to or including the MFG-E8 fragment, can be expected to have a similar effect on protecting dopamine neurons.
Compositions that are expected to have such a protective effect on dopamine neurons include the following polypeptides and proteins.
(1) Human MFG-E8 (GenBank Accession No. Q08431, sequence number 1).
(2) Human MFG-E8 fragment (sequence number 2).
(3) An amino acid sequence representing a functional site of a human MFG-E8 fragment (an amino acid sequence represented by any one of SEQ ID NOs: 3 to 16).
(4) An amino acid sequence containing multiple functional sites of a human MFG-E8 fragment (an amino acid sequence containing two or more of SEQ ID NOs: 3 to 16).
(5) An amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added among the amino acid sequences shown in (1) to (4) above.
(6) An amino acid sequence substantially comprising any one or more of the disulfide bond site, sugar modification site, and phosphoserin modification site contained in MFG-E8, among the amino acid sequences represented by (1) to (5) above.
(7) An amino acid sequence in which the amino acid sequences shown in (1) to (6) above are dimerized.
(8) Human MFG-E8 isoforms (GenBank Accession Nos. Q08431-1, Q08431-2, Q08431-3, etc.) and MFG-E8 homologues, as well as amino acid sequences corresponding to (1) to (6) above.
In other words, compounds that are expected to have a protective effect on dopamine neurons are compounds having the amino acid sequences shown below.
(A) A protein represented by the amino acid sequence of SEQ ID NO: 1 or 2.
(B) A polypeptide or protein comprising any one or more of the amino acid sequences set forth in SEQ ID NOs: 3 to 16.
(C) A polypeptide or protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the polypeptide or protein shown in (A) or (B).
(D) A polypeptide or protein represented by any one of (A) to (C), which has a modification or functional site equivalent to that shown in SEQ ID NO:1 below.
[Disulfide bond area]
Amino acids represented by combinations of positions 27-38, 32-55, 57-66, 70-225, 212-216, and 230-387 in SEQ ID NO:1.
[Glycosylation site]
In sequence number 1, amino acids at positions 228, 238, 325, 329, and 350.
[Phosphoserin modification site]
The 42nd amino acid in sequence number 1.
(E) A polypeptide or protein in which any of the amino acid sequences described in (A) to (D) is dimerized.

Polypeptides and proteins comprising a part of the amino acid sequence of MFG-E8 or an MFG-E8 fragment are also compositions produced by cells, and examples of such compositions are shown in the results of analysis performed by the inventors using liquid chromatography/mass spectrometry (LC/MS/MS) (Non-Patent Document 6). By changing the cell culture conditions, the amino acid sequence of the MFG-E8 fragment expressed and secreted by human adipose-derived mesenchymal stem cells was identified, and the functional sites shown in SEQ ID NOs: 11 to 16 were clarified.
Human adipose-derived mesenchymal stem cells (hMSC-AT-c) were obtained from PromoCell (Heidelberg, Germany). Clinical medium (MSCGM-CD Mesenchymal Stem Cell Bullet Kit) was obtained from Lonza (Basel, Schweiz). DMEM containing 10% FBS was used as the research medium.

The MFG-E8 fragment or MFG-E8 (hereinafter collectively referred to as "MFG-E8, etc.") used in the present invention can be expected to have a dopamine neuroprotective effect as long as its structure or function is not impaired, so partial shortening or elongation of the amino acid sequence can also be performed.

In the case of truncation of the MFG-E8 fragment, it is composed of 10 or more amino acid sequences, and includes the amino acid sequences of SEQ ID NO: 2 to 16. For example, in SEQ ID NO: 2, at least 90% to 99% (amino acid residues 327-363) of the MFG-E8 fragment is included, preferably 91 to 99% (amino acid residues 331-363), more preferably 92 to 99% (amino acid residues 334-363), particularly preferably 93 to 99% (amino acid residues 338-363), and most preferably 94 to 99% (amino acid residues 342-363).
In the case of extension of the MFG-E8 fragment, the fragment is composed of 10 or more amino acid sequences and includes the amino acid sequences of SEQ ID NOs: 2 to 16. For example, in the case of SEQ ID NO: 2, the fragment can be at least 100% to 120% (365-436 amino acid residues), preferably 101% to 118% (365-429 amino acid residues), more preferably 101% to 116% (365-422 amino acid residues), particularly preferably 101% to 114% (365-414 amino acid residues), and most preferably 101% to 110% (365-400 amino acid residues) of the MFG-E8 fragment.

In the case of truncations of MFG-E8, the truncation comprises the amino acid sequence of SEQ ID NO: 1. In SEQ ID NO: 1, at least 90% to 99% (amino acid residues 348-386) of MFG-E8 is included, preferably 91 to 99% (amino acid residues 352-386), more preferably 92 to 99% (amino acid residues 356-386), particularly preferably 93 to 99% (amino acid residues 359-386), and most preferably 94 to 99% (amino acid residues 363-386).
Furthermore, in the case of extension of MFG-E8, it contains the amino acid sequence of SEQ ID NO: 1. In the case of SEQ ID NO: 1, it can be at least 100% to 120% (amino acid residues 388-464) of MFG-E8, preferably 101% to 118% (amino acid residues 388-456), more preferably 101% to 116% (amino acid residues 388-448), particularly preferably 101% to 114% (amino acid residues 388-441), and most preferably 101% to 110% (amino acid residues 388-425).

As described above, MFG-E8, MFG-E8 fragments, and polypeptides and proteins which have either MFG-E8 or MFG-E8 fragments as part of their sequence used as active ingredients in the present invention can undergo partial amino acid substitutions, deletions, insertions, or chemical modifications.
In other words, compounds that have undergone partial amino acid substitution, deletion, or insertion or chemical modification while maintaining structural and functional equivalence to these active ingredients are considered to be equivalent active ingredient compounds.

In the present invention, one embodiment of the composition includes an embodiment in which the active ingredient itself listed above is contained as a composition component. Another embodiment includes an embodiment in which the active ingredient is produced by therapeutic cells such as in a regenerative medicine product described below.
In addition, "treatment" with regard to therapeutic cells includes not only curative or symptomatic treatment of dopamine neuron disorders and Parkinson's disease, but also prevention or delay of these disorders.

<<Methods of producing therapeutic cells and cells for pharmaceutical production>>
[MFG-E8]
The cells for treating Parkinson's disease of the present invention are characterized by producing MFG-E8 (Milk fat globule-EGF factor 8), an MFG-E8 fragment, and a polypeptide or protein containing either MFG-E8 or an MFG-E8 fragment, or any one or more of these.
Furthermore, the cells for treating Parkinson's disease of the present invention can be prepared by the following steps.
(a) a step of designing or creating MFG-E8, an MFG-E8 fragment, and a polypeptide or protein containing either MFG-E8 or an MFG-E8 fragment, or a vector expressing one or more of these; (b) a step of cells taking up the vector; and (c) a step of selecting and culturing cells that produce MFG-E8, an MFG-E8 fragment, and a polypeptide or protein containing either MFG-E8 or an MFG-E8 fragment. The cells for treating Parkinson's disease produced by this series of steps can be stored and used for treatment, or the MFG-E8, an MFG-E8 fragment, and the polypeptide or protein containing either MFG-E8 or an MFG-E8 fragment produced by the cells for treating Parkinson's disease can be used by concentrating, extracting, or purifying them.

The genetic information of MFG-E8 used to produce therapeutic cells of the present invention is not limited to human MFG-E8 or isoforms shown in SEQ ID NO: 1, but may be genetic information of non-human MFG-E8 (MFG-E8 homologue) as long as the product has the activity of protecting dopamine neurons in the body.
The genetic information of the MFG-E8 homolog may be that of any animal, for example, the genetic information of a homolog from rodents such as rats, mice, hamsters, guinea pigs, etc.; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, sheep, etc.; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc.
As an example, the genetic information of mouse-derived MFG-E8 has been described in academic journals (PLoS One. 2012;7(4):e36368. and PLoS One. 2011;6(11):e27685.).
The genetic information of the MFG-E8 homolog used in the preparation of therapeutic cells according to the present invention can be appropriately selected depending on the origin of the therapeutic cells to be cultured for regenerative medicine products, etc., and preferably, the genetic information of the MFG-E8 homolog derived from the same animal as the animal from which the cells to be cultured are derived is used. For example, when preparing therapeutic cells derived from humans, it is preferable to use the genetic information of MFG-E8 derived from humans.
On the other hand, in producing cells for pharmaceutical production according to the present invention, it is also envisaged that the genetic information of human-derived MFG-E8 will be used in highly protein-expressing cell lines derived from non-human mammals, such as CHO (Chinese Hamster Ovary) cells, as is the case in the production of antibody pharmaceuticals.

Furthermore, the genetic information of MFG-E8 used to prepare therapeutic cells or cells for pharmaceutical production according to the present invention is capable of producing the amino acid sequence of human MFG-E8 and MFG-E8 homologues, and also capable of producing polypeptides of any length (fragments of MFG-E8), so long as they have the activity of promoting the protection of dopamine neurons in vivo. The length of the polypeptide, in terms of the number of amino acid residues, is, for example, 21 to 100, preferably 101 to 200, and more preferably 201 to 364. Specific examples of preferred fragments include polypeptides (10 to 364 amino acid residues) consisting of the amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 16.
As used herein, the term "polypeptide" is used interchangeably with the terms "peptide" and "protein."

Furthermore, the genetic information of MFG-E8 used to produce therapeutic cells or cells for pharmaceutical production according to the present invention is not particularly limited as long as it has activity to promote the protection of dopamine neurons in the body. In other words, it may be a nucleotide sequence that can translate an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of human MFG-E8 and MFG-E8 homologues.

"An amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added" means an amino acid sequence in which a number of amino acids (preferably 10 or less, more preferably 7 or less, and even more preferably 5 or less) that can be deleted, substituted, inserted, or added by a known method for producing mutant polypeptides, such as site-directed mutagenesis, have been deleted, substituted, inserted, or added. It is well known in the art that some amino acids in the amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein.

Furthermore, it is also well known that there are mutants of natural proteins that do not significantly change the structure or function of the protein. Therefore, "an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added" is not limited to a nucleotide sequence that corresponds to a sequence in which a mutation has been artificially introduced by a known method for producing mutant polypeptides, but also includes a nucleotide sequence that corresponds to the amino acid sequence of a naturally occurring mutant.

The vector used to generate therapeutic cells or cells for pharmaceutical production according to the present invention may be a nucleotide sequence of MFG-E8 protein isolated and purified from nature, a recombinant protein, or a synthetic protein, and may be one that can be chemically modified after translation. Amino acid analysis of natural proteins, expression of recombinant proteins, synthesis of proteins, or genetic manipulation of these can be performed by conventionally known methods.

The above nucleotide sequence may further include a nucleotide sequence encoding a signal sequence. To secrete the target protein extracellularly, a base sequence encoding a signal sequence may be introduced into the 5' end of the target protein gene. The base sequence encoding the signal sequence is not particularly limited as long as it encodes a signal sequence that can be secreted and expressed by animal cells, but when the host is an animal cell, the MFG-E8 signal sequence, insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used.

Nucleic acid homologs encoding the above proteins can be obtained from cDNA libraries prepared from cells or tissues of mammals other than humans that are known to express the gene by well-known techniques that use primers and probes made based on nucleotides synthesized from mRNA encoding the human-derived MFG-E8 gene. Such techniques include PCR, hybridization (Southern blotting, Northern blotting, etc.), etc.

The PCR method is a polymerase chain reaction, which is performed for about 25 to 40 cycles, each cycle consisting of a denaturing step (about 94 to 96°C, about 30 seconds to 1 minute) for dissociating double-stranded DNA into single strands, an annealing step (about 55 to 68°C, about 30 seconds to 1 minute) for binding a primer to the single-stranded DNA of a template, and an extension step (about 72°C, about 30 seconds to 1 minute) for extending the DNA strand. In addition, before the denaturing step, a pre-heating treatment is performed at about 94 to 95°C for about 5 to 12 minutes, and after the final cycle of the extension step, an extension reaction can be performed at 72°C for about 7 to 15 minutes. PCR is carried out in a commercially available thermal cycler in the presence of sense and antisense primers (size: about 17-30 b, preferably 20-25 b) and template DNA in a PCR buffer containing a heat-resistant DNA polymerase [e.g., AmpliTaq Gold (registered trademark) (Applied Biosystems)], MgCl ₂ , dNTPs (dATP, dGTP, dCTP, dTTP), etc. The amplified DNA can be separated and purified (ethidium bromide stained) by agarose gel electrophoresis.

Hybridization is a technique for detecting a target nucleic acid by forming a double strand with a labeled probe of about 20 to 100 b or more in length. For example, hybridization is performed in about 1 to 5× Standard Saline Citrate (SSC) at room temperature to about 40°C, followed by washing in about 0.1 to 1× SSC, 0.1% sodium dodecyl sulfate (SDS), at about 45 to 65°C. Here, 1× SSC refers to a solution of 150 mM NaCl, 15 mM Na-citrate, pH 7.0. Such conditions are known as a method that enables the detection of nucleic acids with sequence identity of about 80% or more, preferably 85% or more.

The above nucleic acid is inserted into a vector and used to produce polypeptides or proteins that are active ingredients of the therapeutic cells or pharmaceutical manufacturing cells of the present invention.

Vectors include, for example, plasmids, phages, viruses, etc. Examples of plasmids include, but are not limited to, E. coli-derived plasmids (e.g., pRSET, pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.), Ti plasmids, etc. Examples of phages include λ phages, etc. Examples of viral vectors include animal virus vectors such as retroviruses, vaccinia viruses, lentiviruses, adenoviruses, adeno-associated viruses, and Sendai viruses, and insect virus vectors such as baculoviruses, etc. Furthermore, vectors for human gene therapy may also be used. Furthermore, genome editing techniques using CRISPR/Cas9 proteins may also be used instead of gene introduction methods using vectors.

A vector may contain a polylinker or multiple cloning site for incorporating a target DNA, and may also contain several control elements for expressing the DNA. Control elements include, for example, a promoter, an enhancer, a polyA addition signal, a replication origin, a selection marker, a ribosome binding sequence, a terminator, etc.

Examples of selection markers include drug resistance genes (e.g., neomycin resistance gene, ampicillin resistance gene, kanamycin resistance gene, puromycin resistance gene, etc.), auxotrophy complementation genes (e.g., dihydrofolate reductase (DHFR) gene, HIS3 gene, LEU2 gene, URA3 gene, etc.), etc.

When an animal cell is used as the host, examples of promoters include the SRα promoter, SV40 promoter, LTR promoter, CMV promoter, human CMV early gene promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, metallothionein promoter, polyhedrin promoter, etc.

Examples of transformation or transfection include electroporation, spheroplast, lithium acetate, calcium phosphate, Agrobacterium, viral infection, liposome, microinjection, gene gun, and lipofection.

[Basal medium]
The composition of the medium used for culturing and transporting the therapeutic cells according to the present invention may be the same as that of a medium conventionally used for culturing therapeutic cells or cells for the production of pharmaceuticals, for example, the following media used for culturing cells derived from animal tissues:

RPMI-1640 medium, Eagle's MEM medium, Dulbecco's modified MEM medium, Glasgow's MEM medium, α-MEM medium, 199 medium, IMDM medium, DMEM medium, Hybridoma serum free medium, Chemically Defined Hybridoma serum free medium, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1, CMRL-1066, Williams' medium E, Brinster's BMOC-3 Medium, E8 Medium (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL, Inc.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 Medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMedTM iPSC Medium (ReproCELL, Inc.), Cellartis MSC Xeno-Free Culture Medium (Takara Bio Inc.), TeSR-E8 (Veritas Corporation), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Inc.), and mixtures thereof.

MFG-E8 can also be added in advance to the culture medium used for culturing, transporting, and storing the therapeutic cells of the present invention. The concentration of MFG-E8 added to the culture medium is not particularly limited, but can be, for example, 1 μg/mL to 5 μg/mL.

The therapeutic cells of the present invention may contain culture medium components when administered. MFG-E8 may also be added to the culture medium in advance. The concentration of MFG-E8 added to the administration solution when administering the therapeutic cells is not particularly limited, but may be, for example, 0.1 mg/mL to 1 mg/mL.

In addition, physiologically active substances and nutritional factors necessary for cell survival or proliferation can be added to the medium as necessary. These additives may be added to the medium in advance or may be added during cell culture.

Examples of physiologically active substances include insulin, IGF-1, transferrin, albumin, and coenzyme Q10.
The nutritional factors include sugars, amino acids, vitamins, hydrolysates, lipids, and the like.
Examples of the sugar include glucose, mannose, fructose, and the like, and one or more of them may be used in combination.
Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and these may be used alone or in combination of two or more.
Examples of vitamins include d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, niacinamide, pyrodoxal, riboflavin, thiamine, cyanocobalamin, and DL-α-tocopherol, and these may be used alone or in combination of two or more.
Hydrolysates include those derived from hydrolyzed soybeans, wheat, rice, peas, corn, cottonseed, yeast extracts, and the like.
The lipids include cholesterol, linoleic acid, and linolenic acid.

Furthermore, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin may be added to the medium as necessary. When an acidic substance such as sialic acid is added to the medium, it is desirable to adjust the pH of the medium to a neutral range suitable for cell growth, between pH 5 and 9, preferably between pH 6 and 8.

The medium for culturing and transporting therapeutic cells according to the present invention may be a medium that does not contain serum, i.e., a serum-free medium. The medium according to the present invention may contain serum, but it is preferable that it does not contain serum from the viewpoint of preventing contamination with components derived from different animals. Here, serum-free medium means a medium that does not contain unconditioned or unpurified serum. The serum-free medium may contain purified blood-derived components or animal tissue-derived components (e.g., growth factors).

The medium according to the present invention may or may not contain a serum substitute, as well as serum. Examples of serum substitutes include albumin substitutes such as albumin, lipid-rich albumin and recombinant albumin, vegetable starch, dextran, protein hydrolysates, transferrin or other iron transporters, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol or equivalents thereof. Specific examples of serum substitutes include those prepared by the method described in WO 98/30679, commercially available knockout serum replacement (KSR), Chemically-defined lipid concentrated (Life Technologies) and Glutamax (Life Technologies).

The "cells for producing pharmaceutical products or cells for regenerative medicine products" covered by the present invention include stem cells. Stem cells are immature cells that have the ability to differentiate and proliferate, and include multipotent stem cells. Multipotent stem cells refer to cells that have the ability to differentiate into multiple types of tissues and cells, although not all types.

Multipotent stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells. Multipotent stem cells are preferably mesenchymal stem cells. In addition, mesenchymal stem cells broadly refer to a population of stem cells or their precursor cells that can differentiate into all or some of the mesenchymal cells such as osteoblasts, chondroblasts, and adipblasts.

Here, the culture vessels used in the culture method according to the present invention are not particularly limited, and may include flasks, dishes, petri dishes, microwell plates, microslides, chamber slides, tubes, trays, culture bags, and stainless steel tanks. The substrates of these culture vessels are also not particularly limited, and may be glass or various plastics such as polypropylene and polystyrene. Conventionally, in order to induce adhesion of cells to the substrate surface of the culture vessel, a scaffold has been coated on the substrate surface. Such scaffolds include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, partial structures of laminin, fibronectin, and mixtures thereof (e.g., Matrigel), as well as dissolved cell membrane preparations (Lancet. 2005 May 7-13;365(9471):1636-1641.).

The culture density of therapeutic cells is not particularly limited as long as it is a density that can achieve the desired effect, such as improving cell viability, and is preferably about 1.0×10 ¹ to 1.0×10 ⁷ cells/mL, more preferably about 1.0×10 ² to 1.0×10 ⁷ cells/mL, even more preferably about 1.0×10 ³ to 1.0×10 ⁷ cells/mL, and most preferably about 3.0×10 ⁴ to 1.0×10 ⁷ cells/mL.

Culture conditions such as temperature, _CO2 concentration, dissolved oxygen concentration, and pH can be appropriately set based on conventional techniques. For example, the culture temperature is not particularly limited, but may be 30 to 40°C, preferably 37°C. The _CO2 concentration may be 1 to 10%, preferably 2 to 5%. The oxygen partial pressure may be 1 to 10%.

[Method of producing cells for producing pharmaceuticals or cells for regenerative medicine products]
The cultured cells according to the present invention, particularly stem cells, can be applied to a method for producing a cell composition containing stem cells or a cell composition containing differentiated cells from stem cells. By applying the method for producing therapeutic cells according to the present invention to a method for producing a cell composition containing stem cells or differentiated cells, continuous administration of MFG-E8 into the body becomes unnecessary.
The term "regenerative medical products" as used here refers to the products listed below and specified by government ordinance.
(1) Human or animal cells that have been processed by culture or other means,
A. Reconstruction, repair, and formation of the body's structure and functions
B. Those used for the purpose of treating or preventing a disease. (2) Those introduced into human cells for the purpose of gene therapy. See the website of the Pharmaceuticals and Medical Devices Agency (https://www.pmda.go.jp/review-services/drug-reviews/about-reviews/ctp/0007.html).

Specific steps of the method for producing a cell composition containing stem cells according to the present invention are, for example, as follows.
(a) Culturing stem cells.
(b) Introducing the vector into stem cells.
(c) Proliferating, maintaining, storing and transporting therapeutic cells that produce MFG-E8.

Step (a) is a step of proliferating and maintaining stem cells. Step (a) may be performed by applying a conventional method for culturing stem cells, and may involve culturing in the presence of a scaffold and/or serum (or a serum extract, etc.).

Step (b) is a step of introducing a vector into stem cells, and step (c) is a step of growing and maintaining the therapeutic cells produced by the genetic manipulation method of the present invention. The stem cells introduced with the vector in step (b) may be passaged again by step (a). In addition, a step of identifying the gene-introduced cells obtained in step (b) may be performed between steps (b) and (c). This additional step allows purification of only the MFG-E8-producing cells. Furthermore, when serum is used in step (a), a step of washing the cells obtained in step (b) with a serum-free medium or buffer solution may be performed between steps (b) and (c). In addition, since the inventors have clarified that human adipose-derived mesenchymal stem cells have the characteristic of expressing and secreting MFG-E8 (Non-Patent Document 6), the same interpretation can be made for human adipose-derived mesenchymal stem cells that are not subjected to genetic manipulation.

[Method of producing a cell composition containing differentiated cells]
In the method for producing a cell composition containing differentiated cells from stem cells according to the present invention, the stem cells proliferated in the above step (a) are induced to differentiate. The method for inducing differentiation of stem cells itself can be carried out by known techniques. For example, by adding a differentiation inducer such as retinoic acid to the medium, it is possible to differentiate the stem cells into neural cells or the like in the absence of a scaffold. In addition, differentiation induction may be carried out as a separate step after step (b), in which case differentiation induction may be carried out by known techniques.

[Cell composition]
The cell composition of the present invention comprising stem cells and/or differentiated cells that produce MFG-E8, etc. can be used for further culturing of stem cells or for cell production of regenerative medicine products, etc.

Here, the cell composition according to the present invention can be a composition obtained by the above-mentioned production method, in which dispersed stem cells such as small cell clusters are cultured and contain medium components.
When used for preservation such as cryopreservation, the cells of the regenerative medicine product according to the present invention may be mixed with the above-mentioned culture medium components, serum or its substitute, or an organic solvent such as DMSO, in addition to a known cell preservation solution. In this case, the concentration of the serum or its substitute is not particularly limited, but may be 1 to 50% (v/v), preferably 5 to 20% (v/v). The concentration of the organic solvent is not particularly limited, but may be 0 to 50% (v/v), preferably 5 to 20% (v/v).
The cells of the regenerative medicine product according to the present invention can be administered to a patient by means of transplantation, intravenous injection, intraventricular administration, and intracerebral parenchymal administration.

[Example 1: Protective effect of MFG-E8 on dopamine neurons in the substantia nigra of rats]
We created an LPS-administered Parkinson's disease model rat and examined the protective effect of MFG-E8 on dopamine neurons in the substantia nigra of the midbrain.

1. Experimental methods and procedures (1) Crl:CD (SD) rats (male, 6 weeks old) were used.
(2) LPS (Lipopolysaccharides from Escherichia coli 026:B6, SIGMA, L2762) and the MFG-E8 fragment shown in SEQ ID NO:2 (MFG-E8 protein Leu24-Cys387, product URL: https://www.rndsystems.com/products/recombinant-human-mfg-e8-protein-cf_2767-mf#product-details) were used.
(3) The drugs administered were test substance A (containing 0 μg LPS and 0 μg/1 μL MFG-E8 fragments), test substance B (containing 1 μg LPS and 0 μg/1 μL MFG-E8 fragments), and test substance C (containing 1 μg LPS and 200 ng/1 μL MFG-E8 fragments). Ultrapure water (Milli-Q water) was used as the solvent. Tissue sections were observed using a ZEISS Axioskop 2 plus microscope.

2. Injection of the administered sample The animal is anesthetized by intraperitoneally administering 40 mg/kg of sodium pentobarbital (Tokyo Chemical Industry Co., Ltd.) (volume of administration: 0.8 mL/kg). After anesthesia, 0. L-mL of levobupivacaine hydrochloride (Popscaine (registered trademark) 0.25% injection, Maruishi Pharmaceutical Co., Ltd.) is subcutaneously administered to the scalp. Next, the hair on the top of the animal's head is shaved and the head is fixed in a brain stereotaxic apparatus. The scalp is disinfected with iodine tincture and then incised to expose the skull. The connective tissue on the skull is removed with a cotton swab, and then dried with a blower to make the position of the bregma easier to see. A hole for inserting a stainless steel pipe is made in the skull 2.0 mm to the right and 5.3 mm behind the bregma using a dental drill. A stainless steel pipe connected to a 0.5 mm outer diameter silicon tube and a microsyringe is inserted vertically to a depth of 7.8 mm from the bone surface. 1 μL of the administered sample is injected into the substantia nigra over a period of 5 minutes using a microsyringe pump. After injection, the stainless steel pipe is left inserted for 5 minutes, slowly raised, and then removed. The skull hole is then sealed with a non-absorbable bone marrow hemostatic agent (Nestop, Alfresa Pharma Corporation) and the scalp is sutured. The animal is removed from the stereotaxic apparatus and returned to the breeding cage. The stainless steel pipe and silicone tube used were sterilized.

3. Extraction of the brain The brain is extracted two weeks after injection of the administered sample.
The animal is anesthetized with sodium pentobarbital (40 mg/kg, 1 mL/kg, intraperitoneal administration) and fixed in the supine position. The heart is exposed by opening the chest, and blood flow in the descending aorta is stopped with forceps. The left ventricle is cut open with scissors, and the tip of a disposable mouse catheter connected to a blood transfusion tube is introduced from the left ventricle to the aorta, and the tip of the catheter is fixed with a clamp. The atrial appendage is cut open, and 300 mL of physiological saline is perfused through the blood transfusion tube and the animal is euthanized by exsanguination. The animal is then perfused with 300 mL of 4% paraformaldehyde phosphate buffer solution and fixed. After fixation, the skull is removed and the whole brain is removed. The removed brain is immersed and fixed in a sample bottle containing 4% paraformaldehyde phosphate buffer solution under refrigeration.

4. Immunostaining in rat brain (Tyrosine Hydroxylase:HT staining)
The extracted brain is then cut into paraffin blocks in the usual manner. Sections are then prepared around the area where the administered sample was injected. The sections are then immunostained with TH antibody.

5. Immunostaining
1) Deparaffinization → Alcohol → DW
2) PBS Wash
3) Activation by autoclave: 120℃, 10 minutes
4) PBS Wash
5) 1% hydrogen peroxide/methanol
6) PBS Wash
7) Blocking non-specific reactions with skim milk
8) Primary antibody: Rabbit polyclonaL-to Tyrosine Hydroxylase
4℃/1h 100x dilution [sbcam: ab112]
9) PBS Wash
10) Histofine Simple Stain Rat MAX-PO (MULTI)
11) PBS wash
12) DAB starts coloring → DW stops
13) Nuclear staining (Mayer's hematoxylin) → water washing → alcohol dehydration → xylene clearing

6. Experimental Results (1) The results are shown in Figures 1 to 3.
(2) In the substantia nigra of the midbrain administered test substance A (containing 0 μg of LPS and 0 μg/1 μL of MFG-E8 fragment), 118.0 ± 28.6 cells positive for TH staining were observed in the microscopic photographs (Fig. 1A, Fig. 3).
(3) In the substantia nigra of the midbrain administered test substance B (containing 1 μg of LPS and 0 μg/1 μL of MFG-E8 fragment), 31.0 ± 13.2 cells positive for TH staining were observed in the microscopic photographs (Fig. 1B, Fig. 3).
(4) In the substantia nigra of the midbrain administered test substance C (containing 1 μg of LPS and 200 ng/1 μL of MFG-E8 fragment), 73.7 ± 38.6 cells positive for TH staining were observed in the microscopic photographs (Fig. 1C, Fig. 3).
(5) In this measurement, the cell number in the test substance B group was significantly decreased compared to that in the test substance A group (*, P<0.05, n=3) (Example 1, Figures 1 and 3).

(6) The area ratio of TH-positive staining sites in the images was measured using the image analysis software Image J 1.440 (http://imagej.nih.gov/ij).
(7) In the substantia nigra of the midbrain administered with test substance B, the number of positive staining sites in immunohistochemical staining was significantly decreased compared to that in the substantia nigra of the midbrain administered with test substance A (1.0±0.1 [relative value with the measured value of test substance A taken as 1]) (**, P<0.01, 0.56±0.1, n=3). On the other hand, in the substantia nigra of the midbrain administered with test substance B, no significant decrease was observed compared to that in the substantia nigra of the midbrain administered with test substance A (0.8±0.3, n=3) (Example 1, Figures 1 and 2).

7. Discussion
When LPS alone was administered without the MFG-E8 fragment, a significant decrease in TH-positive cells was observed in the substantia nigra of the midbrain, the injection site, two weeks after administration. On the other hand, when the MFG-E8 fragment and LPS were co-administered, no significant decrease in TH-positive cells was observed in tissue sections two weeks after administration, compared to administration of LPS alone.
These results demonstrated that the MFG-E8 fragment has a protective effect on dopamine neurons in vivo.

Claims (5)

Contains as an active ingredient the 24-387 fragment of human MFG-E8 consisting of the amino acid sequence shown in SEQ ID NO: 2,
A composition for protecting dopamine neurons in the substantia nigra. The composition contains, as an active ingredient, cells expressing the 24-387 fragment of human MFG-E8 consisting of the amino acid sequence shown in SEQ ID NO: 2.
A composition for protecting dopamine neurons in the substantia nigra. It is administered directly into the human cerebroventricular cavity;
The composition according to claim 1 or 2 . Contains as an active ingredient the 24-387 fragment of human MFG-E8 consisting of the amino acid sequence shown in SEQ ID NO: 2,
Culture medium for protecting dopamine neurons. The composition contains, as an active ingredient, cells expressing the 24-387 fragment of human MFG-E8 consisting of the amino acid sequence shown in SEQ ID NO: 2.
Culture medium for protecting dopamine neurons.