Myogenic progenitor cells used in an optimized manner for the prevention and treatment of anal incontinence - Patent Application 20070123333

Abstract

The present invention relates to myogenic precursor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in a subject, said subject being at risk of developing anal incontinence or having suffered from anal incontinence for up to 20 years, more preferably from about 6 months to about 20 years or from about 6 months to about 10 years. The present invention also refers to a pharmaceutical composition comprising MPCs and a pharma- ceutical acceptable excipient and / or carrier for use in a method for the prevention and / or treatment of anal incontinence according to the present invention.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to myogenic precursor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in a subject, the subject being at risk of developing anal incontinence or having suffered from anal incontinence for up to 20 years, more preferably from about 6 months to about 20 years, or from about 6 months to about 10 years. The present invention also relates to a pharmaceutical composition comprising MPCs and a pharma- ceutical acceptable excipient and / or carrier for use in a method for the prevention and / or treatment of anal incontinence according to the present invention. [Background technology]

[0002] 2. Background of the Invention Anal incontinence and its epidemiology The ability to maintain self-control is fundamental to our well-being as social beings. Loss of anal continence results in physical, physiological and social handicaps. Although anal incontinence is generally thought to affect mainly the elderly and disabled, these conditions can occur in any age group. The spectrum of anal incontinence, i.e. the inability to control bowel contents, ranges from small fecal marks (traces) left on underwear to lost flatus and major episodes of uncontrollable defecation of loose or solid stool. The reasons can be multi-layered and complex. Regardless of the extreme impairment of the quality of life of affected individuals, impaired anal continence represents a cost factor that cannot be underestimated for the public health system. In the United States, more than $400 million is spent annually on anal incontinence support programs. Moreover, anal incontinence is the second most common reason for nursing home admission (even more than dementia). One-third of older adults in nursing homes and hospitals have fecal incontinence.

[0003] Anatomy and Physiology of Anal Incontinence The anatomical structures necessary for anal continence have been studied in more detail in the past decade, thanks to the availability of endanonal imaging techniques. This has also led to a better understanding of the mechanisms of anal continence and the factors involved in maintaining it. Anal continence requires the coordination of different anatomical structures with different physiological functions. Intact sensation ensures awareness of rectal fullness and awareness of stool quality. Functional innervation allows specialized ring-shaped muscles (sphincters) to respond in an appropriate manner (voluntarily and involuntarily) to the increasing anal "demand for closure". Finally, an intact sphincter completely obstructs the anal canal until defecation is appropriate. Dysfunction of one of these structures results in impaired anal continence. The function of the sphincter tissue is based on the involuntary rest pressure of the anal sphincter inside the anus (internal sphincter), which is made of smooth muscle tissue, and the involuntary rest pressure and voluntary squeeze pressure arising from the anal sphincter outside the anus (external sphincter). The constant baseline tone of the puborectalis muscle results in a "distortion" of the anorectal junction towards a junction that forms a 90° angle between the anal canal and the rectum. This anorectal angle also contributes to the maintenance of anal continence. However, the puborectalis muscle alone is not able to maintain continence. Anal continence is further provided by the interaction of the internal and external anal sphincters. The rectal cushion of the anal canal mucosa, tightened by the sphincter, eventually results in an air-tight occlusion. In the resting state, the anal canal is occluded by the constant tonic activity of the external anal sphincter and the baseline resting pressure of the internal anal sphincter. The internal anal sphincter is an extension and dilation of the circular smooth muscle layer of the colon, providing about 75-85% of the baseline pressure of a closed anal canal. The activity of this smooth muscle component is completely inhibited by distension of the rectum, the so-called rectoanal inhibitory reflex. This relaxation is accompanied by a reflex contraction of the external anal sphincter and the puborectalis muscle, which prevents defecation in inappropriate cases. If it is convenient to have a bowel movement at that time, the external anal sphincter and puborectalis muscles relax, triggering involuntary motor activity of the colon and rectum that results in rectal pressure greater than anal pressure and subsequent expulsion of the stool. If it is not convenient to have a bowel movement when the urge to defecate arises, the puborectalis and external anal sphincter muscles can be voluntarily contracted, pushing the stool back into the rectum until it is convenient to have a bowel movement.

[0004] Causes of Anal Incontinence The importance of the described structures with respect to voluntary continence is emphasized, since dysfunction of these structures leads to incontinence. Most patients suffering from anal incontinence are diagnosed with anal sphincter abnormalities. These abnormalities may occur in the external anal sphincter, the internal anal sphincter, or both. Sphincter-related incontinence may be caused by (1) obstetric or accidental trauma, (2) iatrogenic trauma such as surgery or radiation therapy, (3) neurogenic diseases such as multiple sclerosis or diabetes, or (4) age-related muscle atrophy. Obstetric trauma is the leading cause of incontinence in women, since up to 9% of vaginal deliveries lead to anal sphincter rupture.

[0005] Treating anal incontinence Patients are referred for invasive treatments when conservative treatments such as dietary changes, biofeedback exercises, and antidiarrheal medications fail, are poorly successful, or are not feasible in the first place due to severe anatomical, physiological, or neurological dysfunction. Traditional invasive treatments include surgical approaches such as repair and recreation of the anal sphincter (sphincteroplasty, colostomy, laminaplasty) when the sphincter is defective, or colostomy or antegrade inhibition enemas when incontinence occurs due to a complete spinal cord injury. However, traditional surgical approaches for sphincter repair have a high failure rate of 50%, and surgical approaches to create a new sphincter have a high morbidity. One less invasive treatment for incontinence is perianal injections of bulking agents that are believed to increase pressure in the anal canal. A single randomized sham-controlled trial found that injections of NASHA Dx (dextranomer stabilized with hyaluronic acid) produced significantly better treatment outcomes than sham injections. In detail, 52% of patients were found to have at least a 50% reduction in incontinence episodes per week, compared with 32% of patients who received sham treatment. However, only 6% of patients were found to be fully regulated, and to date, available efficacy data only cover short-term effects of up to 6 months. It was also noted that bulking agents have a limited impact on patients' quality of life. Therefore, more effective treatments for anal incontinence are needed. In the past two decades, neuromodulation has gained attention as a treatment option for incontinence, among which sacral nerve stimulation (SNS) has become the most promising in terms of efficacy. SNS therapy is based on the implantation of a medical device (e.g., Interstim, Medtronic, USA) that uses chronic low-voltage electrical stimulation of the sacral nerves to improve neuromuscular function, but its mode of action (MoA) is still unclear. Success rates, defined as the proportion of patients who achieved at least a 50% reduction in incontinence episodes per week (IEF) after SNS, were aggregated from 61 studies and median short-term (<12 months), medium-term (12-36 months) and long-term (>36 months) success rates were found to be 63%, 58% and 54%, respectively.However, 13.1% of screened patients did not respond to test stimulation and therefore did not undergo permanent implantation, and approximately 25% of patients included in the long-term follow-up underwent interpretation of the stimulator due to complications (technical or infectious) or loss of efficacy.

[0006] Since incontinence is primarily caused by defects / weakness in rectal muscle tissue, animal models of muscle regeneration suggest that intramuscularly injected cells will engraft within the host muscle, and early clinical trials for the treatment of stress urinary incontinence by injection of muscle-derived cells seemed promising. The idea of ​​regenerating the anal sphincter by cell therapy was born (Frudinger et al., 2009). However, to allow market approval and thus widespread application of cell therapy for the treatment of incontinence, the efficacy of the therapy needs to be better than a placebo effect and be clinically relevant in that it provides sufficient benefit and / or complete remission in the subjects in need. An effect of at least a 50% reduction in incontinence episodes per week is considered clinically relevant. For the method to be useful, a significantly higher proportion of patients receiving the cell therapy treatment compared to those receiving the placebo treatment is required, and such a reduction is necessary for the method to be considered effective.

[0007] As outlined above, incontinence is a highly complex condition with many different causes, histories and severities, and therefore it is necessary to select the most appropriate patient population that will benefit from a particular method. Different treatment modalities may target different patient populations to achieve significant and relevant therapeutic effects.

[0008] EP210976B1 discloses a method of using myoblasts for the prevention and treatment of anal incontinence by injecting the myoblasts into the external anal sphincter of a subject suffering from a rectal injury. WO2014 / 044867A1 discloses a method of treating urinary and / or anal incontinence, or women and men at risk of developing urinary or anal incontinence, by administration of skeletal muscle derived cells. WO2019 / 115790 discloses skeletal muscle derived cells for use in treating incontinence. WO2020 / 193460 discloses induced smooth muscle cells for use in a method of treating a disease or disorder in a subject. Clinical studies have been conducted to test the potential efficacy of muscle cells in treating fecal incontinence, but no superior efficacy of these methods compared to a placebo effect has been demonstrated. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention has been made in view of the above-mentioned prior art. Therefore, one object of the present invention is to provide an effective method of using myogenic precursor cells for the prevention and / or treatment of anal incontinence.

[0010] The inventors have surprisingly discovered that while in some subjects injection of myogenic precursor cells has no or only minimal effect, in other subjects injection of myogenic precursor cells significantly improves the subject's condition.

[0011] None of the above disclosures selects a narrow patient population that will ultimately be eligible for administration of myogenic precursor cells for improved prevention / treatment of anal incontinence within the broad indications of incontinence.The objective underlying the present invention was therefore to identify a group of subjects that can be effectively treated by injection of myogenic precursor cells in the context of an individualized therapeutic approach.Another objective of the present invention is to provide an optimized method for the use of myogenic precursor cells for the prevention and / or treatment of anal incontinence, which results in a more effective treatment of anal incontinence.

[0012] None of the above disclosures select a patient population according to the subject's condition related to incontinence, duration of incontinence, and / or severity of incontinence. Therefore, another object of the present invention is to provide a method of using myogenic precursor cells for the prevention and / or treatment of anal incontinence, which selects a patient population according to the subject's condition related to incontinence, duration of incontinence, and / or severity of incontinence.

[0013] None of the above disclosures select a safe and effective number of cells for the prevention and / or treatment of anal incontinence. Therefore, one object of the present invention is to select a specific cell dose in the method of using myogenic precursor cells for the prevention and / or treatment of anal incontinence.

[0014] Furthermore, none of the above disclosures selects an optimized accompanying stimulus in the method of using myogenic precursor cells for the prevention and / or treatment of anal incontinence. Therefore, another object of the present invention is to select an optimized accompanying stimulus in the method of using myogenic precursor cells for the prevention and / or treatment of anal incontinence. [Means for solving the problem]

[0015] The object of the invention is solved by the subject matter defined in the claims. [Brief description of the drawings]

[0016] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] [Figure 1]FIG. 1 shows the change in incontinence episode frequency (IEF) per week (A, B) from baseline (A, B) and 50% response rate (C, D) over the study period of Example 5 in the treatment groups (low cell count-LCC, high cell count-HCC, placebo-PBO) in the intent-to-treat (ITT) population, where episodes classified as trace were either counted for analysis (A, C) or excluded (B, D). [Diagram 2] Figure 2 shows the change in IEF per week from baseline to 12 months after treatment (A, B) and the 50% response rate at 12 months after treatment (C, D) according to Example 5, which is revealed by counting each as an incontinent episode, calculated by including (A, C) or excluding (B, D), respectively. Data was visualized between treatment groups (PBO, LCC, HCC) in different patient populations according to the subject selection of Example 6. The different patient populations defined in Example 6, called TPP1, are patients suffering from fecal incontinence for 10 years or less. TPP2 are patients suffering from more than 2 episodes at baseline, classified as small or large. TPP3 are patients suffering from fecal incontinence for 10 years or less and with IEF more than 2 times per week at baseline, classified as small or large. NR1 are patients suffering from fecal incontinence (FI) for more than 10 years, and NR2 are patients suffering from 2 or less incontinence episodes (IE) classified as "small" or "large". p values ​​of two-tailed Wilcoxon tests (A, B) or Fisher's exact tests (C, D) comparing PBO with LCC or HCC are shown whenever they reached a significance threshold of p < 0.05. [Diagram 3] Figure 3 shows phase contrast microscopy images of myogenic progenitor cells isolated according to Example 1 and tested for differentiation potential according to Example 2. Myotubes are visible at 600x magnification and contain multiple nuclei (black arrows), thereby demonstrating the potential of mononuclear MPCs to fuse with each other. [Figure 4]Figure 4 shows an overview of the clinical study conducted according to Example 5. Study visits including parameters evaluated and a timeline of data collection are shown in A. Treatment assignments are indicated by numbers (N) in B. QoL = quality of life, V = visits, VAS = visual analog scale, WIE = incontinent episodes per week, CGI = bed global impression scale. [Diagram 5] FIG. 5 shows Tukey's box plot summarizing acetylcholinesterase (AChE) activity of LCC (A) and HCC (B) batches containing MPCs isolated according to Example 1, differentiated in vitro according to Example 2, and then measured for AChE activity according to Example 3. Activity is shown as relative mU per 2×105 cells. LCC batches (n=83) were found to possess a mean ± SD AChE activity of 241.90 ± 151.90, ranging from 36 to 568. HCC batches (n=75) were found to possess a mean ± SD AChE activity of 213 ± 137.40, ranging from 49 to 680. [Figure 6] FIG. 6 shows the percentage of MPCs positive for surface cell markers CD34, CD56 and CD90 for either low cell count (LCC, n=83) (A) and high cell count (HCC, n=75) formulations prepared according to Example 1 and tested for surface markers according to Example 4. Data are visualized as Tukey's bar graphs (outliers are visible as points). All LCC and HCC batches were found to be CD34 negative (%CD34 positive with mean±SD of 1.29±1.37 for LCC and 1.10±0.90 for HCC). All LCC and HCC batches were found to be CD56 positive (%CD56 positive with mean±SD of 92.06±6.73 for LCC and 89.86±7.06 for HCC). All LCC and HCC batches were found to be CD90 positive (mean ± SD %CD90 positivity of 94.00 ± 5.00 for LCC and 94.99 ± 4.52% for HCC). [Figure 7]Figure 7 shows the change in IEF from baseline to 6 months after treatment among treatment groups (PBO, LCC, HCC) in the ITT set patients further subgrouped according to increasing number of baseline IEFs (including traces). Data are visualized as mean ± SEM. The change in IEF from baseline to 6 months increased by successively excluding patients with low baseline IEF in all groups, but was more pronounced in LCC and HCC than in PBO patients. [Figure 8] Figure 8 shows the effect size (Cohen's d) of IEF from baseline to 12 months after treatment (A, B) and odds ratios for 50% responder rate at 12 months after treatment (C, D) calculated by comparing LCC or HCC with PBO treatment, either considering (A, C) or not considering (B, D) trace as incontinent episodes, in the ITT, TPP1, TPP2, TPP3, NR1 and NR2 patient populations, respectively. [Figure 9] Figure 9 shows the change in IEF from baseline to 12 months after treatment between treatment groups (PBO, LCC, HCC) (A, B) and 50% responder rate (C, D) in TPP3 set patients and TPP3 patients further subgrouped to select only those with fecal incontinence related to anal sphincter injury (TPP3_injury). Data are shown either with (A, C) or without (B, D) evidence of incontinent episodes. Data are shown as mean ± SEM. P values ​​of two-sided Wilcoxon rank sum tests comparing LCC or HCC with PBO with alpha error of less than 0.05 are shown for the change in IEF. P values ​​of Fisher's exact tests comparing response rates of LCC or HCC with PBO treatment with alpha error of less than 0.05 are shown. [Figure 10]Figure 10 shows the effect size (Cohen's d) of IEF from baseline to 12 months after treatment (A, B) and odds ratios for 50% responder rate at 12 months after treatment (C, D) calculated by comparing LCC or HCC with PBO treatment, either considering (A, C) or not considering (B, D) traces as incontinent episodes in the TPP3 and TPP3_injured patient populations, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] [Terms and definitions] The term "anal incontinence" as used herein refers to the unwanted loss of bowel contents through the anus, such as flatus, liquid or solid feces. The term is comprised of all three severity grades: Grade 1 is gas only, Grade 2 is liquid and loose stool, and Grade 3 is solid formed stool.

[0019] As used herein, the term "anal sphincter" or "anal sphincter tissue" preferably refers to the levator ani and puborectalis muscles as part of the anal sphincter, but may also include the pubococcygeus, coccygeus, iliococcygeus, and pudendal nerves.

[0020] The term "fecal incontinence" as used herein refers to the unwanted loss of bowel contents through the anus, preferably limited to liquid or solid feces. The term may include severity grades of "anal incontinence": Grade 2 = liquid and loose stool, Grade 3 = solid formed stool.

[0021] The term "myogenic progenitor cells" or "MPC" for short as used herein preferably refers to cells with myogenic potential, which may be primary cells and / or in vitro cultured cells derived from muscle tissue, or may relate to other cells derived from myogenic potential, such as, but not limited to, from adipose tissue or other stem cell containing tissues, such as bone marrow. The term also includes cells that are pluripotent stem cells, or cells derived from pluripotent stem cells that can become muscle cells upon isolation and culture, or can become muscle cells after administration within or adjacent to muscle tissue. Since myogenic progenitor cells are preferably obtained by isolation of muscle tissue, the term "myogenic progenitor cells" may also refer to a suspension of several different cell types, at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% of said cells are "myogenic progenitor cells" as defined herein. Preferably, the suspension is a suspension of single cells, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% of the single cells are homogenous of the same cell type, i.e., myogenic precursor cells. In addition to myogenic precursor cells as defined herein, the suspension may also contain other cell types, such as cells of adipose, chondrogenic, osteogenic, and / or fibrogenic tissue origin. Such cells may be mesenchymal cells, adipocytes, chondrocytes, osteocytes, and / or process-related impurities, and / or any other cell type that may be isolated together with the myogenic precursor cells. Such cells may comprise up to 1%, 2%, 5%, 10%, 20%, 30%, 40%, or 50% of the myogenic precursor cell suspension.

[0022] The term "myogenic potential" preferably refers to the potential of a cell, cells, or cell population to develop, regenerate, and / or enhance muscle tissue. As used herein, myogenic potential may further relate to the potential of a cell, cells, or cell population to differentiate in vitro into multinucleated myotubes and / or to express enzymatically active acetylcholinesterase.

[0023] The term "penetration" as used herein preferably refers to the process of introducing an injection device, e.g., a needle, into body tissue without still affecting the injection process.

[0024] The term "injection" as used herein preferably refers to the ejection of an injection solution from an injection device to a specific site in the human body, in particular in or adjacent to the muscle tissue that results in anal incontinence (e.g., anal sphincter tissue), which releases the above-mentioned cells. The injection process may be, but is not limited to, a static injection. That is, the injection device remains in the position reached. The static injection process preferably refers to an injection process in which the injection device is stationary, i.e. does not move relative to the ejected injection solution, during the ejection of the injection solution. Alternatively, the injection process may be dynamic. The dynamic injection process preferably refers to an injection process in which the injection device is dynamic, i.e. moves relative to the ejected injection solution, during the ejection of the injection solution. The dynamic injection process may be performed, for example, by moving a hollow needle, which passes in the opposite direction through the needle trajectory in the external anal sphincter, thereby simultaneously ejecting the injection solution. Alternatively, and preferably, the term "injection" as used herein refers to any administration route suitable for placing the cells adjacent to the anal sphincter tissue. Such administration routes preferably include oral, topical, intravenous, or intra-arterial administration.

[0025] The term "injection site" as used herein preferably refers to a site in the human body where the injection process may begin, such as, but not limited to, a site close to the anal incontinence or a site close to muscle tissue. The injection site may or may not be the same as the site where the injection process ends.

[0026] The term "injection device" as used herein includes any device suitable for penetrating human tissue to reach an injection site in a subject and deliver a solution, particularly a solution containing myogenic precursor cells, to said injection site in the subject.

[0027] As used herein, the term "passive incontinence" preferably refers to the lack of sensory awareness of fecal loss, including low baseline anal pressure and lack of sensory capacity of the anal and rectal mucosa.

[0028] As used herein, the term "urge incontinence" or "urgency" preferably refers to the inability to delay defecation for more than 5 minutes after the recognition of the urge to defecate. Such patients have an immediate need to go to the toilet and / or are unable to go to the toilet quickly enough to empty the bowel contents into the toilet, resulting in an involuntary defecation.

[0029] The term "CD56+" or "CD56 positive" as used herein preferably refers to cells expressing the cell marker CD56. The term "CD56+" or "CD56 positive" may also be used for cell populations containing different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the CD56 cell marker.

[0030] The term "CD56-" or "CD56 negative" as used herein preferably refers to cells that do not express the cell marker CD56. The term "CD56-" or "CD56 negative" may also be used for cell populations containing different cell types, preferably where at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD56.

[0031] As used herein, the term "A2B5+" or "A2B5 positive" preferably refers to cells that express the cell marker A2B5. The term "A2B5+" or "A2B5 positive" may also be used for cell populations containing different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker A2B5.

[0032] The term "A2B5-" or "A2B5 negative" as used herein preferably refers to cells that do not express the cell marker A2B5. The term "A2B5-" or "A2B5 negative" may also be used for a cell population containing different cell types, preferably where at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker A2B5.

[0033] The term "desmin positive" as used herein preferably refers to cells that express the cell marker desmin. The term "desmin positive" may also be used in reference to a cell population comprising different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population express the cell marker desmin.

[0034] The term "desmin negative" as used herein preferably refers to cells that do not express the cell marker desmin. The term "desmin negative" may also be used in reference to a cell population comprising different cell types, preferably where at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker desmin.

[0035] The term "CD105+" or "CD105 positive" as used herein preferably refers to cells expressing the cell marker CD105. The term "CD105+" or "CD105 positive" may also be used for cell populations containing different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker CD105.

[0036] The term "CD105-" or "CD105 negative" as used herein preferably refers to cells that do not express the cell marker CD105. The term "CD105-" or "CD105 negative" may also be used for cell populations containing different cell types, preferably where at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker CD105.

[0037] As used herein, the term "CD34+" or "CD34 positive" preferably refers to cells expressing the cell marker CD34. The term "CD34+" or "CD34 positive" may also be used for cell populations containing different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population express the cell marker CD34.

[0038] The term "CD34-" or "CD34 negative" as used herein preferably refers to cells that do not express the cell marker CD34. The term "CD34-" or "CD34 negative" can also be used for cell populations that include different cell types, preferably if less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent express the cell marker CD34. In a particularly preferred embodiment, the term "CD34-" or "CD34 negative" can also be used for cell populations that include different cells, preferably if at most 19, 10, 5, 4, 3, 2, 1 or 0 percent express the cell marker CD34.

[0039] The term "CD90+" or "CD90 positive" as used herein preferably refers to cells expressing the cell marker CD90. The term "CD90+" or "CD90 positive" can also be used in reference to a cell population comprising different cell types, preferably where at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population express the cell marker CD90.

[0040] The term "CD90-" or "CD90 negative" as used herein preferably refers to cells that do not express the cell marker CD90. The term "CD90-" or "CD90 negative" can also be used for cell populations containing different cell types, preferably when less than 50%, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population express the cell marker CD90.

[0041] The term "Tuj1+" or "Tuj1 positive" as used herein preferably refers to cells expressing the cell marker Tuj1. The term "Tujl+" or "Tujl positive" can also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker Tuj1.

[0042] The term "Tuj1-" or "Tuj1 negative" as used herein preferably refers to cells that do not express the cell marker Tuj1. The term "Tujl-" or "Tujl negative" can also be used for cell populations containing different cell types, preferably when less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population expresses the cell marker Tuj1.

[0043] As used herein, the term "nestin+" or "nestin positive" preferably refers to cells that express the cell marker nestin. The term "nestin+" or "nestin positive" may also be used in reference to a cell population comprising different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker nestin.

[0044] The term "nestin-" or "nestin negative" as used herein preferably refers to cells that do not express the cell marker nestin. The term "nestin-" or "nestin negative" can also be used in reference to a cell population comprising different cell types, preferably when less than 50%, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent of the cell population express the cell marker nestin.

[0045] As used herein, the term "Sca-1+" or "Sca-1 positive" preferably refers to cells that express the cell marker Sca-1. The term "Sca-1+" or "Sca-1 positive" may also be used in reference to a cell population comprising different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population express the cell marker Sca-1.

[0046] The term "Sca-1-" or "Sca-1 negative" as used herein preferably refers to cells that do not express the cell marker Sca-1. The term "Sca-1-" or "Sca-1 negative" can also be used for cell populations containing different cell types, preferably when less than 50% or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population express the cell marker Sca-1.

[0047] As used herein, the term "MyoD+" or "MyoD positive" preferably refers to cells that express the cell marker MyoD. The term "MyoD+" or "MyoD positive" can also be used in reference to a cell population comprising different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98 or 99 percent of the cell population expresses the cell marker MyoD.

[0048] The term "MyoD-" or "MyoD negative" as used herein preferably refers to cells that do not express the cell marker MyoD. The term "MyoD-" or "MyoD negative" can also be used for cell populations containing different cell types, preferably when less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1 or 0 percent, expresses MyoD.

[0049] The term "differentiation medium" as used herein preferably refers to a cell culture medium that induces fusion in multinuclear fusion-competent cells or myogenic precursor cells, such as myoblasts. However, the term may also refer to a cell culture medium that does not contain substances necessary for induction of fusion, if the multinuclear fusion-competent cells or myogenic cells are capable of fusing without the respective induction.

[0050] The term "cell growth medium" as used herein refers to any medium suitable for the incubation of mammalian cells, such as myogenic progenitor cells, preferably capable of attaching said mammalian cells onto the surface of the incubation vessel (flask or dish).

[0051] The term "incontinent episode" or "IE" as used herein preferably refers to an event in which there is an uncontrolled, unexpected, and / or involuntary loss of liquid or solid feces through the rectum. Incontinent episodes can be classified as "trace," "small amount," or "larger amount." This classification can be done by having the patient keep a diary, which allows them to track the date and time when the incontinent event occurred, and preferably classify the size and / or amount of incontinent events. The diary is preferably kept for at least one week, more preferably for about one to about four weeks, about one to about three weeks, or about one to about two weeks.

[0052] As used herein, "frequency of incontinent episodes per week", also referred to as "IE frequency per week" or "IEF per week", preferably refers to the number of incontinent episodes occurring during a 28-day period normalized to a 7-day period and calculated as follows: IEF per week = (number of incontinent episodes reported during the period / number of completed days during the period) x 7

[0053] An incontinent episode classified herein as "trace" preferably corresponds to anal incontinent episodes in which liquid or solid feces leak unexpectedly from the subject's rectum, resulting in a quantity of feces that is so small that it appears as a stain or mark on the linen. Such "trace" may not require the patient to change the undergarment, as the quantity is absorbed by the undergarment linen.

[0054] The term "responder" as used herein preferably refers to a patient who has at least a 50% reduction in IEF per week calculated by comparing two incontinence diaries, one diary filled out during a specific period before a specific intervention (e.g., a method for the use of myogenic precursor cells according to the present invention) and one diary filled out during a specific period after the specific intervention. Such period before and / or after the intervention is preferably 1 week, more preferably 2 weeks, even more preferably 3 weeks, even more preferably 4 weeks.

[0055] An incontinent episode classified as "small amount" herein preferably refers to anal incontinent episodes in which liquid or solid stool unexpectedly escapes from the subject's rectum, with the amount of stool being enough to stain or mark the subject's linens, but is only a small amount compared to the results of normal bowel movements. In such episodes, the patient may need to change undergarments because the amount is not efficiently absorbed by the linens and causes discomfort.

[0056] Incontinent episodes classified herein as "heavier" preferably refer to episodes of anal incontinence in which liquid or solid feces unexpectedly leak from the subject's rectum, such that the amount of feces may be greater than that which appears as stains or marks on the subject's linens. The amount may be comparable to that produced by normal bowel movements. In such episodes, both undergarments and secondary garments are affected, leading to severe discomfort and preferably requiring the patient to change both.

[0057] As used herein, the term "incremental squeeze pressure" preferably corresponds to the maximum anal squeeze pressure minus the anal rest pressure.

[0058] The term "conditions related to incontinence" as used herein preferably corresponds to the results obtained by a physician when examining a subject at risk of developing anal incontinence or a subject who has developed anal incontinence. Such a subject may be examined for anal sphincter tissue, for example by ultrasound and / or manometric examination, to define the state of muscle damage and / or muscle atrophy. In addition, the examiner may consider whether conditions such as pelvic floor dysfunction, nerve damage, or loss of storage capacity are associated with the risk of developing anal incontinence or associated with developed anal incontinence. Also classified as symptoms related to incontinence are diarrhea and constipation.

[0059] The term "pelvic floor dysfunction" as used herein preferably corresponds to a condition in which a subject is unable to properly relax and regulate the muscles of the pelvic floor for urination or defecation. Pelvic floor organs affected by pelvic floor dysfunction include the bladder, uterus, vagina, prostate, rectum, etc. Painful urination, incontinence, or a consistent urgency to go to the toilet may be signs of pelvic floor dysfunction.

[0060] The term "muscle damage" as used herein preferably corresponds to muscle loss in anal sphincter tissue. Such muscle damage may be detected by a physician when performing ultrasound or other useful examination techniques to visualize the anal sphincter. Such muscle damage may affect, for example, the external anal sphincter and / or the internal anal sphincter. A typical example of muscle damage visible on ultrasound is scar formation after sphincter rupture during childbirth.

[0061] The term "muscle atrophy" or alternatively "atrophy" as used herein preferably corresponds to a decrease in muscle mass of the anal sphincter. Such a decrease in muscle mass may be accompanied by the deposition of connective tissue and / or fatty tissue between muscle cells. Muscle atrophy may occur with age or when muscles are left unused. A physician may be able to detect muscle atrophy of the anal sphincter tissue, for example, by ultrasound.

[0062] The term "nerve damage" as used herein preferably corresponds to a lack of function of nerve tissue that normally functionally innervates and is part of the anal sphincter tissue. Such nerves may be the pudendal nerve and / or nerves leading to the pudendal nerve. The term may further correspond to a deficit in nerve tissue that may be related to incontinence.

[0063] The term "loss of storage capacity" as used herein preferably corresponds to an abnormality in the rectal capacity. Loss of storage capacity can occur when surgery, inflammation, or radiation therapy causes the rectum to become stiff and lose normal elasticity. Loss of rectal storage capacity leads to a lack of ability of the rectum to stretch (i.e., elongate), resulting in excess stool leakage through the rectum.

[0064] The term "duration of incontinence" as used herein preferably corresponds to the time elapsed from the date of onset or diagnosis of incontinence. The date of onset of incontinence preferably corresponds to the date of onset of symptoms, i.e. the date of the first occurrence of an incontinence episode. The date of diagnosis of incontinence preferably corresponds to the date on which the doctor diagnosed the patient with incontinence. The date of diagnosis of incontinence is preferably the same as the date of onset of symptoms, or alternatively later than the date of onset of symptoms. In the case of fecal incontinence, the term "duration of incontinence" may also be referred to as "duration of FI" or "duration of incontinence". If the duration of incontinence is measured from the diagnosis of incontinence, the term "duration of incontinence" may also refer to "time from the first diagnosis of FI".

[0065] The term "severity of incontinence" as used herein preferably corresponds to the amount of burden on a subject with anal incontinence. Such severity may be expressed in terms of the number of incontinence episodes per week, the impact of incontinence on the patient's life, a fecal incontinence quality of life score and / or a Wexner score. Preferably, the Wexner score corresponds to the continence scale disclosed in Table 3 of Jorge et al., 1993.

[0066] The term "stimulate" as used herein preferably corresponds to any action performed on the anal sphincter tissue that results in the contraction of the muscle. Stimulation as used herein may include spontaneous contraction of the anal sphincter or may be performed by electrical stimulation. The latter may be performed by stimulating the nerves that innervate the anal sphincter or by directly stimulating the anal sphincter, such as the external and / or internal anal sphincter.

[0067] The term "comprising" as used herein should not be construed as being limited to the meaning of "consisting of" (i.e., excluding the presence of additional other materials). Rather, "comprising" means that additional materials may optionally be present. The term "comprising" encompasses as specifically contemplated embodiments within its scope "consisting of" (i.e., excluding the presence of additional other materials) and "including but not consisting of (i.e., requiring the presence of additional other materials)," with the former being more preferred.

[0068] As used herein, the term "AChE positive" or "AChE+" preferably refers to a cell or cell population that exhibits (i.e., positive) acetylcholinesterase enzyme (AChE) activity. In particular, such AChE activity is greater than or equal to 2×10 5 Each cell is about 20mU rel ~about 1000mU rel , more preferably about 30 mU rel ~about 800mU rel , and even more preferably about 50 mU rel ~about 700mU rel The positive result is when the AChE activity of the above 2×10 5 The cells are cultured in a skeletal muscle differentiation medium, for example, as described in the Examples of this specification. rel " was measured 60 minutes after the addition of acetylthiocholine iodide (ATI) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 2×10 5 "mU / ml" refers to the relative "mU / ml" of cells relative to the linear equation obtained by a dilution series of AChE stock solution ranging from 4 to 500 mU / ml under the same conditions, except that the OD of the dilutions of the stock solution has already been measured 6-8 minutes, preferably 6, 7 or 8 minutes after the addition of ATI and DTNB. "mU rel " refers to the 2×10 5 It can also refer to the relative "mU / ml" of an individual cell.

[0069] As used herein, the term "AChE negative" or "AChE-" preferably refers to a cell or cell population that does not have AChE activity (i.e., is negative). 5 Each cell is approximately 0mU rel ~about 19mU rel If the cells exhibit an AChE activity of 0.1 to 100%, then such AChE activity is negative and the cells are preferably cultured in, for example, a skeletal muscle differentiation medium, as described in Example 2 herein.

[0070] The term "multipotency" as used herein refers to the differentiation potential of mesenchymal cells, preferably characterized by in vitro differentiation potential towards at least the adipogenic, chondrogenic and osteogenic lineages.

[0071] The term "oligopotent" as used herein refers to the differentiation potential of cells characterized by an in vitro differentiation potential that is preferably restricted to the myogenic lineage, such as smooth, striated, and / or cardiac muscle.

[0072] The term "myogenic differentiation potential" as used herein preferably refers to the ability of a cell to express detectable amounts of proteins known to be expressed by myogenic cells in vivo, such as, but not limited to, one or more of the following markers: desmin and myosin. Tests for the expression of such markers in in vitro cultured cells are well known to those skilled in the art. Such tests preferably include flow cytometry and / or other immunocytochemical assays, including fluorescent immunostaining and Western blotting. Alternatively, the use of the term myogenic differentiation potential, which refers to the ability of a cell (e.g., a myogenic progenitor cell) to form multinucleated myotubes, is preferred. Such myotubes are known to those skilled in the art as muscle cells that preferably contain at least three separate nuclei. The formation of such myotubes by originally mononucleated cells occurs, for example, in cells. Thus, the differentiation medium can be used as a test of myogenic differentiation potential.

[0073] As used herein, the term "myotube" refers to a muscle cell that preferably contains at least three distinct nuclei. Preferably, such myotubes are formed by the fusion of mononuclear cells (e.g., myogenic progenitor cells).

[0074] The term "neurogenic potential" or "neurogenic potential" as used herein preferably refers to a cell population expressing a neural marker, such as, but not limited to, one or more of the markers A2B5, TUJ1, NCAM, nestin or equivalent markers. Preferably, the term refers to a cell population in which at least 60% of the cells express A2B5, more preferably at least 60% of the cells express A2B5 and TUJ1, or at least 60% of the cells express A2B5 and NCAM, or at least 60% of the cells express A2B5 and nestin, or at least 60% of the cells express TUJ1 and NCAM, or at least 60% of the cells express TUJ1 and nestin, or at least 60% of the cells express NCAM and nestin. More preferably, the term refers to a cell population in which at least 60% of cells express A2B5, TUJ1 and NCAM, or at least 60% of cells express TUJ1, NCAM and nestin, or at least 60% of cells express A2B5, NCAM and nestin. Even more preferably, the term refers to a cell population in which at least 60% of cells express A2B5, TUJ1, NCAM and nestin. Tests for the expression of such markers in in vitro cultured cells are well known to those skilled in the art. Such tests preferably include flow cytometry and / or other immunocytochemical assays, including immunofluorescence staining and Western blotting.

[0075] The term "adjacent" as used herein preferably refers to the distance between a pre-specified location and the location actually reached when injecting a pharmaceutical component into a patient. Preferably, the "adjacent" location is one directly adjacent to the pre-specified location. Preferably, the term "adjacent" refers to any type of tissue that is directly adjacent to another type of tissue, i.e., by contacting each other directly, but is indirectly, more preferably directly, attached to the muscle of the anal sphincter tissue, including but not limited to the mucosa, submucosa, muscularis mucosa, or anal canal epithelium. Alternatively, preferably, the term "adjacent" refers to the distance, preferably 0.3 to 15 mm, that cells, preferably myogenic precursor cells, may travel from the injection site to the desired site in the subject. Alternatively, "adjacent" refers to the relative distance between the actual injection site and the desired location, preferably no more than 5 cm, more preferably no more than 1 cm, even more preferably no more than 0.5 cm apart from each other.

[0076] [Description of the embodiment] The present invention is directed to myogenic precursor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in a subject, the subject being characterized as having suffered from anal incontinence for up to 20 years, more preferably from 6 months to about 20 years, or from about 6 months to about 10 years.

[0077] Selecting a target The inventors selected a broad patient population (ITT) in Example 5 and found that when patients in this patient population were treated with MPC according to the method of the present invention, they showed a significantly higher reduction in incontinence symptoms than the equivalent patient population in the ITT population treated with placebo (Figure 1). However, statistical significance only indicates that the effect is not random (e.g., not due to a placebo effect or an effect unrelated to MPC). Secondly, the observed effect must be clinically relevant. The higher the weekly IEF reduction rate and / or the higher the proportion of responders, the more clinically relevant the outcome of the treatment in a particular patient population. The applicants found that the treatment effect changes by selecting different subpopulations of patients from the ITT, as described in Example 7.

[0078] From the ITT population (TPP1 population) of Example 5, defined as having IE for 10 years or less, a subgroup of patients was selected according to Example 7, and it was observed that in these patients, LCC and / or HCC treatment resulted in a higher reduction in IEF per week and a higher responder rate than LCC or HCC treatment did in ITT patients (Figure 2). By introducing such a narrow target population, the inventors found that the method of using MPC was more effective in terms of effect size and odds ratio compared to a broader patient population (Figure 8), and thus the method of using MPC was advantageous compared to already known methods.

[0079] Thus, in a preferred embodiment of the present invention, the subject suffering from anal incontinence, more preferably fecal incontinence, suffers from said incontinence for a limited time. More preferably, such period is limited to about 6 months to about 20 years, more preferably limited to about 6 months to about 10 years, more preferably limited to about 6 months to about 8 years, more preferably limited to about 6 months to about 6 years, more preferably limited to about 6 months to about 4 years, even more preferably limited to about 6 months to about 2 years. The preferred starting point of the period is either the onset of incontinence or the diagnosis of incontinence.

[0080] In a preferred embodiment, the present invention provides an MPC for use in a method for preventing anal incontinence, preferably fecal incontinence, particularly when the subject is a healthy subject in terms of anal incontinence, preferably fecal incontinence, but is at risk of developing said incontinence, preferably due to histological and / or anatomical abnormalities of the sphincter tissue. The abnormalities are caused by muscle damage (e.g., due to obstetric anal sphincter injury or anal intercourse), genetic diseases (e.g., Hirschsprung's disease, Duchenne muscular dystrophy), perianal fistula, surgical procedures (e.g., Grasilplasty, sphincter overlap repair, cancer resection). Preferably, the abnormalities are associated with a tear or rupture of the anal sphincter, such as the external anal sphincter and / or the internal anal sphincter. More preferably, the tear or rupture is at an angle of less than 180° relative to the total angle of 360° at which the anal sphincter is visible in an ultrasound image.

[0081] Prevention should desirably begin from the time when there is a risk of developing anal incontinence, such as when a muscle is injured or when a person is diagnosed as being at risk of developing anal incontinence.

[0082] Another preferred embodiment of the invention provides the MPC for use in a method for treatment, wherein the subject to be treated suffers from anal incontinence, in particular faecal incontinence, more preferably urge faecal incontinence and / or passive faecal incontinence, more preferably urge faecal incontinence.

[0083] In a preferred embodiment of the present invention, the subject's anal incontinence, preferably fecal incontinence, or risk of developing said incontinence, is caused by muscle damage, for example as described above. Preferably, said muscle damage is present as scarring in the external and / or internal anal sphincter, which is visualized by endoanal ultrasound. The muscle damage may occur in the external anal sphincter, the internal anal sphincter, or both. Causes include (1) obstetric trauma or accidental trauma, (2) heterotopic trauma, such as surgery or radiation, (3) neurogenic diseases, such as multiple sclerosis or diabetes, and (4) age-related muscle atrophy. Obstetric trauma is the leading cause of incontinence in women, as up to 9% of vaginal deliveries lead to anal sphincter rupture.

[0084] Preferably, said subject suffers from fecal incontinence with a well-defined severity.Preferably, such severity is defined by suffering from a certain amount of incontinence episode frequency.More preferably, such anal incontinence or fecal incontinence patient suffers from more than 2, more preferably more than 3, even more preferably more than 4, even more preferably more than 5, even more preferably more than 6, even more preferably more than 7, even more preferably more than 8, even more preferably more than 9, even more preferably more than 10, even more preferably more than 11, even more preferably more than 10 incontinence episodes per week, where all types of episodes classified as "trace", "small amount" and / or "heavier amount" are considered.

[0085] We found that patients with increasing severity (i.e., increasing baseline IEF) were more responsive to MPC treatment (Figure 7). By selecting such a narrow subgroup of patients defined by having a weekly IEF of more than 6 before treatment (TPP2) in addition to the definition of TPP1 according to Example 7, we found that in said patient population after administration of MPC (LCC or HCC), the reduction in weekly IEF was increased, the response rate was increased, and the effect size and odds ratio were increased compared to those observed in the broader patient population (ITT, TPP1) (Figure 2). This is further highlighted by the increase in the overall effect size and odds ratio (Figure 8).

[0086] Furthermore, the inventors found that the treatment of anal or fecal incontinence according to the invention is most effective for episode types defined as "larger" and "smaller" compared to "trace" (Table 3). Thus, preferably, the severity is defined by suffering from a certain amount of weekly incontinence episode frequency. In a preferred embodiment, the episode frequency for assessing severity is limited to incontinence episodes defined as "small" and "larger", thereby excluding "trace". The inventors found that a subpopulation of TPP1 (TPP3) defined by pre-treatment incontinence severity as having more than two "small" or "larger" incontinence episodes per week was more responsive to treatment with LCC or HCC than the other patient populations (ITT, TPP1 and TPP2), as illustrated in Example 7 by the increased effect size and odds ratio (Figure 8).

[0087] In an alternative preferred embodiment, the MPC is for use in a method for the treatment of anal incontinence in a subject, wherein the subject has a severity of incontinence defined as more than 2, more preferably more than 3, even more preferably more than 4 incontinence episodes per week classified as "light" or "heavier" before treatment, with the incontinence episodes classified as "light" or "heavier" before treatment being defined. Pre-treatment preferably refers to treatment according to the present invention. Thus, preferably, the weekly incontinence episodes are classified before administration of the MPC described herein. Preferably, "pre-treatment" does not include any attempt at treatment other than the treatment according to the present invention, such as, for example, conservative treatment with loperamide or surgical treatment with graciloplasty or sphincteroplasty.

[0088] The inventors found that, in addition to the definition of TPP1, a subgroup of patients (TPP2) defined by having an IEF of more than 6 weeks before treatment was selected according to Example 7, which resulted in an increased reduction in weekly IEF and an increased responder rate in this patient population after administration of MPC (LCC or HCC) compared to that observed in the broader patient population (ITT, TPP1) (Figure 2). This is further highlighted by the increase in the overall effect size and odds ratio (Figure 8). Thus, the selection of this narrow patient population is advantageous compared to the selection of the broader patient population already known in the art.

[0089] Thus, in a highly preferred embodiment of the invention the MPCs are for use in a method of treatment of anal incontinence, more preferably fecal incontinence, in a subject, wherein said subject comprises (i) have suffered from anal incontinence, more preferably fecal incontinence, for 20 years or less, more preferably from about 6 months to about 10 years; and (ii) >6 incontinent episodes per week prior to treatment; and / or (iii) having an incontinence severity of more than 2, more than 3, or more than 4 incontinence episodes per week classified as light or heavy prior to treatment;

[0090] The inventors have found that the combination of a limited incontinence period and an increase in the severity of FI in terms of weekly incontinence episode frequency, excluding episodes classified as "trace", is a suitable selection process of Example 7 for selecting patients who will benefit much better from the method of treating fecal incontinence using MPC according to the present invention. The inventors have found that by selecting such a narrow subgroup of patients according to Example 7, defined by having more than 2 IEFs per week before treatment (TPP3) where traces are not counted as incontinence episodes, in addition to the definition of TPP1, the reduction of weekly IEFs and the responder rate are increased in said patient population after administration of MPC (LCC and / or HCC) compared to those observed in the broader patient population (ITT, TPP1) (Figure 2). This is further highlighted by the increase in the overall effect size and odds ratio (Figure 8). Therefore, the selection of this narrow range of patient population is advantageous compared to the selection of a broad range of patient populations already known in the art.

[0091] The inventors have found that a select subgroup of patients suffering from fecal incontinence with muscle damage, having been diagnosed with fecal incontinence for 10 years or less and having IEF more than twice a week before treatment, are particularly responsive to treatment of the condition with the methods of the invention (Example 7, Figures 9 and 10).

[0092] In a preferred embodiment, the subject is 18 years of age or older. In a further preferred embodiment, the subject suffers from fecal incontinence for 6 months or more, which is confirmed at screening by relevant medical history and anal examination. In a further preferred embodiment, the subject has a Wexner score of more than 9 and at least 3 episodes of fecal incontinence per week measured by bowel diary for a period of time, such as 2, 3 or 4 weeks. In a further preferred embodiment, the subject has at least 3 episodes of non-air incontinence per week measured by diary as described above.

[0093] In a preferred embodiment, the subject has not undergone anal surgery within the past 6 months prior to the date of administration of the MPC. In a further preferred embodiment, the subject has not undergone one or more previous overlap repair surgeries. The term "overlap repair" as used herein preferably refers to a primary overlap repair as is commonly performed in the art to correct acute injury in cases of birth clefts. Preferably, the term also refers to an equivalent treatment performed with a primary overlap repair but at a later time (without acute injury), which may also be called "sphincteroplasty". In a further preferred embodiment, the subject has not undergone more than two total anal surgeries (e.g., a primary repair after delivery and one overlap repair performed later, or the insertion and removal of a permanent neurostimulation system). In a further preferred embodiment, the subject does not have an overlap repair and associated early atrophy of the external anal sphincter. In a further preferred embodiment, the subject has no history of artificial anal sphincter (AAS) surgery. In a preferred embodiment, the subject has not undergone any transanal or perianal injection of bulking products. In a preferred embodiment, the subject has not undergone bowel and pelvic radiation therapy. In a preferred embodiment, the subject has not received chemotherapy within the past 5 years prior to the date of administration of the MPC. In a preferred embodiment, the subject does not have chemotherapy-associated neuropathy of the intestine and pelvis. In a preferred embodiment, the subject is not receiving immunosuppressive therapy. In a preferred embodiment, the subject has not been diagnosed with chronic inflammatory bowel disease. In a preferred embodiment, the subject has no current or diagnosed anal fistula disease. In a preferred embodiment, the subject has no chronic diarrhea. In a preferred embodiment, the subject has no acute anal sphincter injury, including obstetric and other trauma, acute disc loss, or neurological disease (spinal cord injury, multiple sclerosis, Parkinson's disease, stroke, etc.). In a preferred embodiment, the subject does not have uncontrolled type I or type II diabetes or suffer from diabetic peripheral neuropathic pain.In a preferred embodiment, the subject has not been diagnosed with human immunodeficiency virus (HIV), acute or chronic viral hepatitis HCV, acute or chronic viral hepatitis HBV, active syphilis, HTLV (e.g., as determined by a risk assessment by the investigator). In a preferred embodiment, the subject has no metal parts embedded in the electrical stimulation treatment area. In a preferred embodiment, the subject does not have chronic constipation and / or overflow urinary incontinence.

[0094] In a preferred embodiment, the subject has a condition associated with incontinence due to muscle damage, pelvic floor dysfunction, nerve damage, loss of storage capacity, or atrophic incontinence. In a preferred embodiment, the subject's fecal incontinence is passive incontinence, urge incontinence, or fecal wetting. In a highly preferred embodiment, the subject has a condition associated with incontinence due to muscle damage, atrophy, and / or urge incontinence.

[0095] The MPCs for use according to the invention are preferably administered to or adjacent to the anal sphincter tissue of said subject, preferably to the external anal sphincter, the internal anal sphincter and / or the pubic muscle, as further described herein. The administration is preferably carried out by injection. The injection step may be, but is not limited to, a static injection, i.e. an injection where the injection device remains in the position where it is reached. Alternatively, the injection step may be dynamic. Alternatively, the administration of the MPCs may include any administration route suitable for placing the cells adjacent to the anal sphincter tissue. Such administration routes may consist, for example, of oral administration, topical administration, intravenous administration or intra-arterial administration.

[0096] In any of the above preferred embodiments of this embodiment, the method further comprises stimulating the anal sphincter tissue before and / or after administration of MPC. Preferably, the stimulation comprises anal sphincter tissue stimulation for at least 2 weeks, more preferably at least 4 weeks, after administration of MPC, more preferably comprising anal sphincter tissue stimulation before administration of MPC and for at least 2 weeks, more preferably at least 4 weeks, after administration of MPC.

[0097] Preferably, the stimulation is performed by Kegel exercises and / or electrical stimulation, more preferably by Kegel exercises and / or transcutaneous electrical stimulation. The transcutaneous electrical stimulation is preferably performed every day, more preferably twice a day, even more preferably three times a day. Preferably, any of the stimulations is performed for at least 10 minutes, more preferably at least 20 minutes. Also, preferably, the stimulation is performed multiple times a day, more preferably 1 to 10 times, even more preferably 1 to 5 times, even more preferably 3 times.

[0098] The electrical stimulation is preferably performed for a specific time per day, for example at least 5 minutes per session, preferably at least 15 minutes, more preferably at least 20 minutes. Preferably, the patient performs the stimulation three times per day, each for about 1 to 60 minutes, preferably about 10 to 30 minutes, more preferably about 20 minutes. Preferably, the electrical stimulation includes the use of an anal rectal probe attached to a stimulator. Preferably, the anal rectal probe is inserted into the subject's rectum during stimulation. Preferably, the stimulator is programmed to transmit electrical pulses to the probe, the pulses being preferably biphasic or monophasic. Preferably, the pulses are implanted in a sequence of pause, increase, balance, decrease, pause. Preferably, the biphasic or monophasic pulses have a frequency of about 10 Hz to about 100 Hz, more preferably about 25 Hz to about 75 Hz, more preferably about 50 Hz, at the balance. Preferably, each balance phase has the same duration as each increase or decrease phase. Preferably, each rest, increase, decrease or equilibrium phase lasts from about 1 second to about 15 seconds, more preferably from about 2 seconds to about 8 seconds, more preferably about 4 seconds. Preferably, the current intensity in the equilibrium phase is set to about 1 mA to about 1000 mA, more preferably from about 10 mA to about 500 mA, more preferably from about 50 mA to about 250 mA, more preferably from about 75 mA to about 150 mA, more preferably about 100 mA. Preferably, the biphasic or monophasic pulse has a width of about 50 μs to about 500 μs, more preferably from about 150 μs to about 300 μs, more preferably from about 200 μs to about 300 μs, more preferably about 250 μs.

[0099] Kegel exercises are preferably performed by voluntary contraction of the pelvic floor muscles by the subject. Preferably, the contraction is held for about 1 to about 10 seconds, more preferably about 2 to about 5 seconds, more preferably about 3 seconds. After the contraction, the muscles are preferably relaxed for about 1 to about 10 seconds, more preferably about 2 to about 5 seconds, more preferably about 3 seconds. Preferably, the sequence of contraction and relaxation is repeated 1 to 20 times, more preferably 5 to 15 times, more preferably about 10 times. Preferably, each set of the sequence of times is repeated 1 to 10 times, more preferably 3 to 5 times, more preferably 3 times, preferably every day.

[0100] The inventors found that the combination of electrical stimulation and cell injection (e.g., HCC or LCC treatment according to Example 1) was better than electrical stimulation and sham injection alone (e.g., PBO treatment according to Example 1) (Figure 1), suggesting that the combination of MPC and electrical stimulation is an effective treatment for incontinence.

[0101] Cell dose selection Preferably, the MPCs are for use in a method for the prevention and / or treatment of anal incontinence in a subject, the method comprising administration of an effective amount of MPCs, preferably from about 1 million to about 200 million MPCs, preferably from about 4 million to about 60 million MPCs, more preferably from about 4 million to about 6 million MPCs or from about 40 million to about 60 million MPCs.

[0102] The inventors found that in a broad patient population (ITT), after injection of 40-60 million MPCs (HCC), 6 months after treatment, when traces were counted as incontinent episodes (Fig. 1A), and 12 months after treatment, when traces were excluded as incontinent episodes (Fig. IB), a significantly higher reduction in weekly incontinent episode frequency was observed compared to placebo injection (PBO). A trend was observed for a higher reduction in weekly incontinent frequency with HCC than with PBO during the entire test period of Example 5, regardless of whether traces were included as incontinent episodes (Fig. 1). Furthermore, when traces were included in the ITT patient population (Fig. 1C), a consistently higher number of responders was observed in the LCC and HCC groups compared to the PBO group (Fig. I). Also preferably, according to this embodiment, the amount is 4-6 million cells per patient. The applicants found that both a higher trend for a reduction in weekly IEF and an increase in responder rate was observed after treatment of ITT patients with 4-6 million cells (LCC) compared to PBO treatment (Figure 1). This trend was more pronounced when 4-6 million cells were used for fecal incontinence patients who had not experienced fecal incontinence for more than 10 years. The inventors found that for such patients, LCC transplantation significantly reduced IEF from baseline to 12 months after treatment (Figures 2A and B) and was clinically significant by reducing weekly IEF (excluding trace) by at least 50% in most patients (Figure 2D). The inventors further found that patients injected with 40-60 million cells showed a higher trend for a reduction in fecal incontinence symptoms (weekly IEF) compared to patients injected with 4-6 million cells.

[0103] Injection procedure In a preferred embodiment of the present invention, MPCs are injected into or adjacent to the anal sphincter tissue of a subject. Preferably, the cells are injected into or adjacent to the external anal sphincter, internal anal sphincter, and / or pubic muscle. More preferably, the cells are injected into the external anal sphincter, internal anal sphincter, or pubic muscle. Most preferably, the cells are injected into the external anal sphincter. The inventors found that injection of MPCs into the external anal sphincter is effective compared to placebo treatment (Figure 1). The inventors found that cells isolated according to Example 1 can be successfully injected into a subject in need according to Example 5. The injection site was the external anal sphincter, which belongs to the anal sphincter tissue.

[0104] In one preferred embodiment of the invention, the MPCs are injected into a given tissue or injury site, thereby providing a therapeutically effective number of cells in solution or suspension, e.g., about 1×10 6 ~Approx. 2×10 8 The number of cells for injection is preferably suspended in about 0.1 mL to about 100 mL, more preferably about 1 mL to about 10 mL, more preferably about 3 mL to about 6 mL, and even more preferably 6 mL of solution. The injection solution is a physiologically acceptable medium, with or without serum. The physiologically acceptable medium may be, as non-limiting examples, physiological saline or phosphate buffer solution.

[0105] In a preferred embodiment of the invention, MPC injection into anal sphincter tissue is performed as a treatment for anal incontinence to enhance, improve, and / or repair the external and / or internal anal sphincter. Preferably, MPC are injected into or adjacent to the external and / or internal anal sphincter and survive and differentiate into mature muscle cells to enhance the sphincter and / or improve sphincter function. The feasibility and long-term survival of MPC according to this embodiment has been shown previously (Messner et al., 2021; Thumer et al., 2020).

[0106] In another preferred embodiment of the present invention, MPC injections are performed into the anal sphincter tissue to prevent anal incontinence by augmenting and / or strengthening existing incontinent tissue. The feasibility of MPC injections into healthy muscle tissue has already been demonstrated (Frudinger et al., 2015).

[0107] In a preferred embodiment of the invention, the effective number of cells is administered in one or more aliquots of cell suspension. Preferably, multiple aliquots are injected per patient, more preferably 2-50 aliquots, more preferably 2-20 aliquots, even more preferably 5-15 aliquots, preferably a total of 0.1-100 ml, more preferably 1-10 ml, even more preferably 3-6 ml per patient. The inventors have found that such aliquots and volumes are efficacious (i.e., superior to placebo application) and clinically relevant (i.e., most patients reduce IEF per week by at least 50%) (Figures 1 and 2).

[0108] In a preferred embodiment of the invention, MPC is administered by one or more injections into and / or adjacent to the anal sphincter tissue of said subject, preferably by one or more injections into the external anal sphincter, internal anal sphincter and / or pubic muscle. Preferably, MPC for use according to the invention is injected into the internal anal sphincter of a subject, wherein the subject is further characterized as suffering from passive fecal incontinence or passive fecal incontinence and urge fecal incontinence. Preferably, MPC for use according to the invention is injected into the external anal sphincter and / or pubic muscle, more preferably the external anal sphincter, of a subject, wherein the subject is further characterized as suffering from urge fecal incontinence or urge and passive fecal incontinence. Preferably, injection of MPC is performed at multiple locations throughout the anal sphincter tissue.

[0109] Preferably, the MPC is injected into one or more sites, more preferably about 2 to about 20 sites, more preferably about 6 to about 18 sites, more preferably about 10 to about 15 sites, and even more preferably about 12 sites. The inventors have found that injections of the MPC of Example 1 distributed in patients according to Example 5 is effective and significantly more effective than placebo treatment (Figure 1).

[0110] MPC used in the present invention The present invention provides MPCs for use according to the invention. In one embodiment of the invention, the MPCs are characterized as "muscle-derived cells" as already disclosed in EP2120976B1. Non-limiting examples of such cells include myoblasts, fibroblasts, and muscle-derived stem cells present in muscle tissue. The present invention also contemplates the use of cells with myogenic potential (e.g., from liposuctioned tissue15, or other stem cell-bearing tissue (bone marrow), or adipose-derived cells), particularly for use in repairing anal sphincter tissue. In particular, the cells used in the present invention are capable of fusing (forming a syncytium of at least three cells) in vitro and / or in vivo and establishing an oriented contractile cytoskeleton (actin-myosin arrangement). In accordance with the present invention, the MPCs, including myoblasts, may be primary or cultured cells. They may be histocompatible (autologous) or non-histocompatible (allogeneic) with respect to the recipient, including humans. Particular embodiments of the present invention are myoblasts and muscle-derived stem cells, including autologous myoblasts and muscle-derived stem cells that are not recognized as foreign by the recipient. In this regard, the myoblasts can be matched against major histocompatibility centers (MHC or HLA in humans). Such MHC or HLA matched cells can be autologous cells, or they can be cells from a human with the same or similar MHC or HLA antigen profile. The patient can also be tolerant to allogeneic MHC antigens, or the cells can be engineered to lack MHC proteins, thereby rendering them immune tolerant to the originally HLA-mismatched recipient.

[0111] In another embodiment of the invention, the MPCs lack MHC class I and / or II antigens, as described in US Pat. No. 5,538,722.

[0112] In a highly preferred embodiment, MPCs are a cell population of skeletal muscle derived cells (SMDCs), in which at least 60% of the cells are CD56 positive, at least 80% of the cells are CD90 positive and at most 10% of the cells are CD34 positive. Preferably, at least 60% of the cells are also positive for A2B5 and CD105. Preferably, said cell population of SMDCs is Sca-1 negative, in particular at most 10% or 0% of the cells express Sca-1. Preferably, also, at least 60% of the cells are positive for desmin. Preferably, said cell population of SMDCs is MyoD negative, in particular at most 5% or 10% of the cells express MyoD.

[0113] In a further preferred embodiment, the MPCs are a cell population of SMDCs, in which about 64% to about 99.9% of the cells are CD56 positive, about 80% to about 99.9% of the cells are CD90 positive, and about 0% to about 9% of the cells are CD34 positive. Preferably, at least 60% of the cells are A2B5 and CD105 positive. Preferably, said cell population of SMDCs is Sca-1 negative, in particular at most 10% or 0% of the cells express Sca-1. Preferably, also, at least 60% of the cells are desmin positive. Preferably, said cell population of SMDCs is MyoD negative, in particular at most 5% or 10% of the cells express MyoD.

[0114] In another embodiment of the invention, the MPCs are characterized as "skeletal muscle derived cells" (SMDCs) as disclosed in WO2019 / 115790 (Thumer et al., 2019, p. 115790). The SMDCs preferably exhibit a characteristic expression pattern. Preferably, about 60% or more, 70% or more, 80% or more, 90% or more, 95% or more or 98% or more of the SMDCs express CD56 and A2B5. Preferably, the SMDCs do not express CD34, Sca-1 and MyoD. Hereby, the term "does not express" preferably means that less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 2% of the SMDCs express said markers. In a preferred embodiment, 0% of the SMDCs express Sca-1.

[0115] In a further preferred embodiment, the MPCs are a cell population of SMDCs, which consists of at least 90% CD56 positive, at least 90% A2B2 positive, at least 90% CD105 positive, and at least 90% desmin positive cells, with less than 10% of the population expressing CD34, Sca-1 and MyoD. In a preferred embodiment, 0% of said SMDCs express Sca-1.

[0116] The expression pattern of SMDCs as described above can be used to determine the myogenic index of cell cultures without the need for differentiation, and thus can be used to determine whether skeletal muscle-derived cells can be used to treat muscle dysfunction, particularly incontinence, such as urinary incontinence and / or anal incontinence.

[0117] In another embodiment of the invention, the MPCs described herein are characterized as "myogenic progenitor cells" as disclosed in WO2020 / 193460. Preferably, these cells are characterized by positive expression of CD56 and CD90, and negative expression of CD34. In a further preferred embodiment of the invention, the MPCs are oligogenic. The inventors have found that such cells are useful in the methods of the invention (Figure 1).

[0118] In another embodiment of the invention, the MPCs described herein are characterized as "mesenchymal stromal cells" or "MSCs" as disclosed in WO2020 / 193460 and are characterized by positive expression of CD105, CD73, and negative expression of CD34, and CD56. In a further preferred embodiment of the invention, the MPCs are MSCs characterized by negative expression of desmin and / or positive expression of CD90. In a further preferred embodiment of the invention, the MPCs are multipotent MSCs.

[0119] In another embodiment of the invention, the MPCs described herein are characterized by positive expression of aSMA, CD49a, desmin, CD56, and CD146, and negative expression of CD34.

[0120] In another embodiment of the invention, the MPCs described herein are characterized by positive expression of aSMA, CD49a and CD146, and negative expression of CD56.

[0121] In a highly preferred embodiment, the MPCs for use in the method according to the invention are characterized by positive expression of the markers CD56 and CD90, in particular at least 60%, more preferably at least 80%, more preferably at least 95% of the MPCs express CD56 and at least 60%, more preferably at least 80%, more preferably at least 95% of the myogenic precursor cells express CD90. Preferably, about 60 to about 99.9% of said MPCs express CD56 and about 80 to about 99.9% of said MPCs express CD90. Preferably, said MPCs are further characterized by negative expression of the markers CD34 and / or Sca-1, in particular at most 10%, more preferably at most 5%, even more preferably at most 1% of the MPCs express CD34 and / or Sca-1. Even more preferably, the MPCs are further characterized by positive expression of the marker desmin, in particular at least 60%, more preferably at least 80%, even more preferably at least 95% of the cells are desmin positive.

[0122] In a preferred embodiment, MPCs for use in the methods according to the invention are composed of a cell population having the following marker expression characteristics: About 60 to about 99.9% of the cell population (i.e., MPCs) express the cell marker CD56, about 60 to about 99.9% of the cell population express the marker CD90, and about 0 to about 15% of the cell population express the marker CD34. More preferably, about 60 to about 99.9% of the cell population express the cell marker CD56, about 70 to about 99.9% of the cell population express the cell marker CD90, and about 0 to about 10% of the cell population express the cell marker CD34. More preferably, about 80 to about 99% of the cell population express the cell marker CD56, about 80 to about 99.9% of the cell population express the marker CD90, and about 0 to about 5% of the cell population express the marker CD34.

[0123] MPCs for use according to the invention may further have the following marker expression characteristics: About 60 to about 100% of the cell population express the cell marker desmin and / or about 60 to about 100% of the cell population express the marker CD105. More preferably, about 60 to about 99.9% of the cell population express the cell marker desmin and about 90 to about 100% of the cells express the cell marker CD105. Preferably, about 60 to about 100% of the cell population further express the cell marker A2B5.

[0124] In a further particularly preferred embodiment, the MPCs have the following marker expression profile: About 60% to about 100% of the cell population express the cell marker CD56, about 60% to about 100% of the cell population express the cell marker CD90, about 0% to about 10% of the cell population express the cell marker CD34, about 60% to about 100% of the cell population express the cell marker desmin, about 60% to about 100% of the cell population express the cell marker A2B5, and about 60% to about 100% of the cell population express the cell marker CD105. Preferably, about 0% to about 10% of the cell population shows expression of the cell marker Sca-1.

[0125] In further preferred embodiments of all of the above embodiments relating to MPCs, about 0 to about 10% of the MPCs express MyoD and / or about 0 to about 10% of the MPCs express Sca-1.

[0126] In certain preferred embodiments, MPCs for use according to the invention have the following marker expression profile: About 60% to about 100% of the cell population express the cell marker CD56, about 80% to about 99.9% of the cell population express the cell marker CD90, about 0% to about 10% of the cell population express the cell marker CD34, about 60% to about 100% of the cell population express the cell marker desmin, about 60% to about 100% of the cell population express the cell marker A2B5, about 60% to about 100% of the cell population express the cell marker CD105, about 0% to about 10% of the cell population express the cell marker MyoD, and / or about 0% to about 10% of the cell population express the cell marker Sca-1.

[0127] In another preferred embodiment of the invention, the MPCs for use in the methods of the invention are characterized by their differentiation potential into cell lineages. Preferably, the MPCs are characterized as comprising myogenic differentiation potential in vitro and / or in vivo, whereby differentiation potential is defined as the ability to expand, form or become mature muscle tissue, i.e. by the formation of multinucleated muscle fibers or by electrophysiological coupling of multiple mononucleated cells. Preferably, the MPCs are capable of expanding, forming or becoming mature muscle tissue in vitro and / or in vivo. Preferably, the MPCs are capable of expanding, forming or becoming mature skeletal and / or smooth muscle tissue in vitro and / or in vivo. The MPCs are further characterized by their ability to self-renew by mitosis in culture and to exit mitosis after administration to a subject.

[0128] In another embodiment of the invention, MPCs are characterized by having myogenic and neurogenic differentiation potential. Furthermore, MPCs can have further differentiation potential, i.e. oligo-, multi- or multi-potential. Preferably, MPCs include differentiation potential for all tissues adjacent to said injection site of an anal incontinent subject in need. More preferably, such differentiation potential includes differentiation into skeletal, smooth and / or neuronal tissue. Preferably, said cells are multipotent to allow augmentation and regeneration of skeletal and smooth muscle tissue of anal sphincter tissue. More preferably, said cells are multipotent to allow augmentation and regeneration of skeletal and smooth muscle tissue of anal sphincter tissue, as well as neuronal tissue. Cells containing myogenic and / or neurogenic potential can be isolated from muscle tissue, preferably by performing a muscle biopsy. Preferably, cells with myogenic potential are isolated from muscle tissue by obtaining skeletal muscle derived cells (SMDCs).

[0129] Such cells can be tested for myogenic potential by methods known to those of skill in the art. To determine the myogenic and / or neurogenic potential of MPCs according to the invention, the cells test positive for their AChE activity according to the examples of the invention or other methods known to those of skill in the art (e.g., Thumer et al., 2018).

[0130] The MPC for use according to the present invention is preferably about 20 mU rel ~about 1000mU rel , more preferably about 30 mU rel ~about 800mU rel , and more preferably about 50 to about 700 mU rel Each AChE activity is preferably 2 x 10 5 Measured per cell.

[0131] The selected cell dose administered to a subject as described above is preferably about 1x10 2 mU rel _ total ~about lx10 6 mU rel _total and more preferably exhibits a total AChE activity of about 1x10 3 mU rel _ total ~about 5x10 5 mU rel _ total , and more preferably about 7x10 4 mU rel _ total ~about 2x10 5 mU rel _ total To determine the total AChE activity, 2x10 5 AChE activity determined for a batch of cells can be extrapolated. For example, if a batch of cells is 2x10 5 50mU per cell rel of acetylcholinesterase enzyme activity, a cell suspension containing 50 million such cells will yield a total of 1.25 x 10 4 mU rel Therefore, it can be extrapolated that the total acetylcholinesterase enzyme activity is 1000 mg / kg. rel _ total " preferably refers to the extrapolated AChE activity of a certain number of cells, where the AChE activity is in mU rel In a further preferred embodiment, the relative AChE activity is linearly extrapolated from the measured AChE activity in mU. rel _ total The total AChE activity given in mU is the total AChE activity of a cell population or cell dose of MPC relative to an AChE standard as described herein. rel _ total " may also refer to the relative "mU / ml" of the cell dose of said cell population or MPC measured 60 minutes after addition of ATI and DTNB, with respect to the linear equation obtained by a dilution series of AChE stock solution in the range of 4-500 mU / ml under the same conditions, except that the OD of the dilutions of the stock solution is already measured 6-8 minutes, preferably 6, 7 or 8 minutes, after addition of ATI and DTNB.

[0132] The inventors have found that the MPC obtained according to the method disclosed in the present invention has a 36 mUrel ~568mU rel The AChE activities were found to be in the range of 241.90±SD AChE activity, respectively, of 2×10 5 The HCC batch (n=75) had 49mU rel ~680mU rel Mean ± SD AChE activity 213 ± 137.40 mU rel It was found to possess.

[0133] Methods for obtaining myogenic precursor cells The MPCs used in the method according to the invention are preferably isolated cells of muscle tissue. They can be obtained by methods well known to those skilled in the art, for example, isolation from muscle biopsy. Preferably, the cells can be isolated from muscle tissue, preferably by obtaining a muscle biopsy. Preferably, the muscle biopsy is obtained from a subject by surgery. Preferably, the MPCs are isolated from a subject, more preferably from a human subject, even more preferably from a human subject in need, such as a subject suffering from incontinence. Preferably, the MPCs are isolated from a muscle biopsy of a subject, or alternatively, from another tissue source, such as, but not limited to, adipose tissue or connective tissue. Preferably, the muscle biopsy is obtained from skeletal muscle, more preferably from the pectoralis major, biceps brachii, or latissimus dorsi.

[0134] MPCs are preferably obtained from the obtained muscle biopsy by a method comprising the following steps: (a) cooling the obtained muscle biopsy; (b) processing and cooling the sample; (c) resuspending the sample of step (b) in a serum-containing medium containing at least one enzyme and heating to 38° C. for 1 to 20 hours; pelleting the sample; and (d) resuspending the pellet of the sample of step (c) to provide a single cell suspension from the sample of step (c), thereby obtaining MPCs. Step (a) comprises performing a muscle biopsy. Such a muscle biopsy, which is the source of MPCs, can be obtained from the muscle at the site of injury or from another site that is more easily accessible to the clinical surgeon. In a further preferred embodiment, step (a) is performed at a temperature below 16° C., preferably in the temperature range of 1 to 16° C., preferably 4 to 10° C., particularly preferably 7° C.; and for a time in the range of up to 96 hours. Thus, step (a) can be carried out at a temperature range of 1-16°C, or any temperature intermediate this range, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C, or any intermediate temperature within this range, such as a temperature range of 6-8°C. Alternatively, the temperature range is 4°C or less, preferably in the range of 1-3°C. Step (a) is preferably carried out for a time period ranging up to 96 hours, or any time period within this range, such as 12-96 hours, 12-72 hours, 12-48 hours, 24-96 hours, 24-72 hours, 24-48 hours, or any other intermediate range. Preferably, the treatment of step (b) includes the use of scissors, scalpels, tweezers, filters, ball mills, centrifuges. Said treatment refers in particular to mechanical disruption of the tissue sample. The treatment of the sample is preferably carried out at room temperature. The cooling in step (b) is preferably carried out after the treatment of the sample. The cooling in step (b) can be carried out at a temperature in the range of 1-16° C., or any temperature within this range, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C., or any intermediate temperature within this range, such as a temperature in the range of 4-8° C., or in the range of 1-3° C. Step (b), in particular the cooling, can be carried out for a definite time range, such as 2-48 hours, 2-36 hours, or 2-24 hours.Step (c) preferably comprises enzymatic treatment using a solution containing one or more selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase, deoxyribonuclease and DNAse. In a preferred embodiment, step (c) comprises the use of collagenase. Resuspension in step (c) preferably comprises centrifuging the sample of step (b), discarding the supernatant and resuspending the cell pellet in serum-containing medium containing at least one enzyme such as trypsin. Step (c) may further comprise one or more washing steps, including vortexing the cells in a suitable solution such as a buffer. The sample of step (b) is preferably centrifuged after the incubation period to pellet the cells of the sample and discard the enzyme-containing supernatant. Step (c) may be carried out at a temperature in the range of 25-38°C, preferably 36-38°C. Preferably, step (d) comprises a method selected from at least one of FACS sorting, centrifugation, electrokinetic sorting, acoustophoretic sorting, bead-based cell sorting, and optical sorting. A suitable enrichment method for obtaining a single cell suspension is magnetically activated cell sorting (MACS®). In the MACS method, cells can be separated by incubating them with particles coated with antibodies against a specific surface antigen. The incubated cells are then transferred to a column placed in a magnetic field. In this step, cells that express the antigen and are attached to the nanoparticles remain on the column, while other cells that do not express the antigen pass through the column. This method separates cells positively and / or negatively for a specific antigen. Another example of a suitable enrichment method is fluorescence activated cell sorting (FACS®), as described for example in Webster et al. (Exp Cell Res. 1988 Jan; 174(l):252-65). After step (d), which comprises incubation of the single cell suspension obtained in step (d), a further optional step (e) can be carried out, the incubation of step (e) being preferably carried out at a temperature in the range of 25-38°C, preferably 36-38°C, particularly preferably 37°C, thereby obtaining adherent MPCs.This further optional incubation step allows the cells to expand and obtain larger amounts of MPC. Step (e) may optionally be followed by a further step (f) comprising discarding the non-adherent cells of step (e), which step (f) is preferably carried out after at least 6 hours to 4 days. Step (f) may optionally be followed by a further step (g) of expanding the adherent cells of step (e), the expansion step (h) consisting of culturing the adherent cells for 1 to 5 passages until 70 to 80% confluent.

[0135] In another embodiment of the invention, MPCs are obtained from any donor and from any somatic cells that can be cultured and reprogrammed in vitro. Preferably, these somatic cells are first reprogrammed into induced pluripotent stem cells by ectopic expression of reprogramming factors consisting of Klf-4, Sox-2, Oct4 and Myc (Takahashi et al., 2007; Takahashii and Yamanaka, 2006; Yamanaka, 2008), followed by directed differentiation into MPCs as already described in the prior art (Bajpai et al., 2012; Incitti et al., 2020; Miyagoe-Suzuki & Takeda, 2017; Xuan et al., 2021). More preferably, myogenic progenitor-derived cells are derived from somatic cells by direct reprogramming without pluripotent intermediates, according to methods known in the art (Hirai et al., 2018; Ito et al., 2017).

[0136] The invention also provides a method of preventing or treating anal incontinence in a subject comprising: (a) selecting a subject at risk of developing or suffering from anal incontinence, more preferably fecal incontinence, according to a condition of the subject related to incontinence; (b) administering, preferably injecting, an effective amount of MPC into or adjacent to the anal sphincter tissue; and (c) optionally stimulating the anal sphincter tissue before and / or after step (b).

[0137] Preferably, said method is the same as the method described above for the prevention and / or treatment of anal incontinence in a subject to which the MPC for use is provided. Thus, all the embodiments described above, such as those relating to the selection of the subject in terms of duration, severity and causality of the incontinence, those relating to the characteristics of the MPC, those relating to the injection of an effective amount of MPC in terms of cell dose, target tissue and distribution, and / or those relating to stimulation in terms of electrical stimulation and Kegel exercises, also represent embodiments of the method according to the invention.

[0138] Step (a) of the method according to the invention may comprise selecting a subject with muscle damage as an incontinence-related condition. Alternatively or additionally, step (a) may comprise selecting a subject with an anal incontinence duration of 20 years or less, preferably 10 years or less, or any of the further respective time ranges disclosed above. Alternatively or additionally, step (a) may comprise selecting a subject with a severity of incontinence defined as more than 6, preferably more than 7, preferably more than 8, more than 9, or more than 10 incontinence episodes per week before treatment. Alternatively or additionally, step (a) may comprise selecting a subject with a severity of incontinence defined as more than 2 incontinence episodes per week classified as light or heavy before treatment. Step (c) comprises anal sphincter tissue stimulation, preferably for at least 2 weeks, after injecting MPC, or before and after injecting MPC.

[0139] The invention also provides a pharmaceutical composition comprising MPC and a pharma- ceutically acceptable excipient and / or carrier for use in a method for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in a subject, wherein the subject is at risk of developing or has been suffering from anal incontinence, preferably fecal incontinence, for up to 20 years, more preferably from about 6 months to about 20 years, or from about 6 months to about 10 years. In particular, the invention provides a pharmaceutical composition comprising (i) MPC for use in a method according to any embodiment of the invention, and (ii) a pharma- ceutically acceptable excipient and / or carrier.

[0140] The pharmaceutical compositions according to the invention may further comprise one or more conventional additives, examples of which include physiologically acceptable buffers, albumin, collagen, laminin, and dimethylsulfoxide.

[0141] The present invention is further directed to a process for preparing a pharmaceutical composition for use in a method according to the invention, preferably comprising the step of combining MPC with a pharma- ceutically acceptable diluent, excipient or carrier.

[0142] The present invention is further directed to a pharmaceutical pack comprising one or more compartments, wherein at least one compartment contains an MPC for use in a method according to the invention.

[0143] The present invention is further directed to the use of MPC as a medicament for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in a subject as described herein.

[0144] The present invention is further directed to the use of MPC in the manufacture of a medicament for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in a subject as described herein.

[0145] The subject in whom a disease is to be treated or prevented from occurring according to any of the above-described embodiments is preferably a human or an animal, particularly a mammal, and most preferably a human.

[0146] The following examples are intended to illustrate the invention but are not to be construed as limiting it. EXAMPLES

[0147] Example 1 - Isolation of skeletal muscle derived myoblasts (MPCs) In a clinical trial conducted by the applicant (EudraCT number: 2010-021463-32), MPCs were isolated from human fecal incontinence patients, as already disclosed in WO2019115790. WO2019115790 performed the isolation of myogenic skeletal muscle progenitor cells from a small number of patient samples, whereas the above clinical trial performed the isolation of skeletal muscle-derived myogenic progenitor cells from approximately 170 separate patient samples. Therefore, the quality of the samples used in the study may have been significantly different in part from the samples used in WO2019115790, depending on the patient and the quality of the biopsy obtained. In detail, a skeletal muscle biopsy was taken from the pectoralis major or biceps brachii muscle of each incontinent patient treated according to Example 5. To perform the biopsy, first, the skin was opened by making an incision over the muscle with a length of approximately 1 cm until the fascia of the pectoralis major was reached. After opening the fascia, a 1 cm incision was made. 3 100 pieces of muscle tissue (biopsies) were taken. The biopsies were transferred directly into biopsy transport medium, consisting of Ham's F10 basal medium supplemented with gentamicin (final concentration 1-5 μg / ml), pre-cooled to approximately 4°C. The biopsies were stored in the biopsy transport medium at 1-11°C for approximately 26 hours. The biopsies were then transferred into a petri dish filled with 1x PBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. The muscle tissue was then transferred to another petri dish filled with 1x PBS and cut into 2-3 mm pieces using a scalpel. 2After the additional transfer step as described above, the tissue pieces were further cut into 1 mm cubes. The pieces were finally transferred to a centrifuge tube filled with lx PBS and centrifuged at 1300 rpm for 10 min. After centrifugation, the supernatant was removed and the muscle tissue was suspended in lx PBS supplemented with 8 μg / ml gentamicin. The muscle tissue suspension was then cooled to 2-8 °C for 48 h. After cooling, the muscle tissue suspension was centrifuged at 1300 rpm for 10 min, the supernatant was removed, and 2.5 ml of digestion solution containing 1-5 mg / ml collagenase, 2-4% v / v Hepes buffer, 0.1-10% v / v fetal calf serum, and 5-10 μg / ml gentamicin dissolved in Ham's F10 was added. The muscle tissue suspension was incubated at 37 °C, 5% CO 2 The suspension was then centrifuged at 1300 rpm for 10 min, the supernatant was removed, and the pellet was resuspended in Ham's F10 medium containing 10-20% v / v FCS, 1-3 ng / ml bFGF, and 3-10 μg / ml gentamicin, and plated in cell culture flasks. MPCs attached to the bottom of the culture flask were further maintained by changing the medium every 3-4 days, detaching after reaching confluence, and then subcultured. Subcultures were performed at 1x10 6 ~8x10 7 The MPCs isolated according to this example were found to form multinucleated myotubes when cultured under differentiation conditions (Examples 2 and 3) and to be highly positive for AChE activity (Figures 3 and 5). Furthermore, when the cells were analyzed according to Example 4, they were positive for CD56 and CD90 and negative for CD34 (Figure 6). Furthermore, when cultured under differentiation conditions according to Example 2, the cells were found to form multinucleated myotubes (Figure 3).

[0148] Example 2 - Differentiation Potential of MPCs 2x10 obtained according to Example 1 6MPCs were seeded into 24-well Nunclon® Delta Surface plastic plates. Approximately 2-4 days after seeding, the proliferation medium was replaced with skeletal muscle cell differentiation medium (500 mL, PromoCell GmbH, Germany) and skeletal muscle differentiation of the cells was initiated by adding 10 mL of skeletal muscle cell differentiation medium supplement pack (PromoCell GmbH, Germany) and 240 μL of gentamicin (8 mg / mL, Sandoz GmbH, Austria). Cells were cultured for approximately 5-7 days and analyzed by phase contrast microscopy.

[0149] The MPCs obtained according to Example 1 were tested for in vitro differentiation into skeletal myogenic lineage under appropriate culture conditions. Successful differentiation was observed in all batches of MPCs used for treatment according to Example 5. Successful myogenic differentiation was determined by microscopic observation of at least one myotube, i.e., a muscle cell with at least three separate nuclei formed by fusion of multiple single-nucleated MPCs (Figure 3).

[0150] Example 3 - AChE enzyme activity measurement Cells isolated according to Example 1 were cultured under differentiation conditions according to Example 2 for 5-7 days and then analyzed for AChE expression.

[0151] Reagent preparation American Public Health Association (APHA) phosphate buffer, pH 7.2 (Sigma-Aldrich Co. LLC, Germany) was prepared according to the manufacturer's instructions. Briefly, 17 g of the powder mixture (monopotassium phosphate 22.66 g / L and sodium carbonate 7.78 g / L) was added to 400 mL of distilled water. After adding 0.5 mL of Triton X-100, the mixture was dissolved on a magnetic stirrer at room temperature for 30 min. The final volume was made up to 500 mL with a graduated cylinder and used without further dilution. The buffer was stored at 4 °C until use. Ellman's reagent (5,5'-dithiobis-2-nitrobenzoic acid, DTNB, 0.5 mM) was freshly prepared for each AChE assay by weighing 2 mg into a 1.5 mL Eppendorf tube. This was dissolved in 1 mL of phosphate buffer (pH 7.2, 0.1% triton X-100) by vortexing for 1–2 min. The final volume was made up to 10 mL in a 15 mL Falcon tube with phosphate buffer (pH 7.2, 0.1% triton X-100) and stored at 4 °C until use. Acetylthiocholine iodide (ATI, 5.76 mM) was freshly prepared for each AChE assay by weighing 2 mg into a 1.5 mL Eppendorf tube. It was dissolved in 1.2 mL of distilled water by vortexing for 1-2 min and stored at 4 °C until use.

[0152] Preparation and measurement of AChE standard enzyme: AChE standard dilutions were prepared in phosphate buffer (pH 7.2, 0.1% triton X-100) and used immediately. A ready-to-use 50 U / mL AChE stock (from Electrophorus electricus) was purchased from AAT Bioquest® Inc. Sunnyvale, CA, USA. This AChE was diluted to prepare 1000 mU / mL AChE according to the manufacturer's instructions and further diluted in a 1:2 ratio to obtain eight dilutions ranging from 4 to 500 mU / mL. 200 μL of each AChE standard enzyme dilution was mixed with 300 μL of 0.5 mM DTNB and 50 μL of 5.76 mM ATI. Additionally, at least one blank reaction mix was prepared by mixing 0 mU / mL phosphate buffer with the above DTNB and ATI. Standards and blanks were incubated at 30°C in the dark for 6, 7, or 8 min, followed by OD measurements at 412 nm using an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK). The OD values ​​of the blank reactions were subtracted from the OD values ​​of the standard enzyme reactions. The correlation between the blank-corrected OD values ​​and the AChE concentrations (mU / mL) of the standard dilutions was visualized using GraphPad Prism software. The linear equation of AChE concentration versus the corrected OD values ​​was calculated.

[0153] Cell measurements To measure the AChE activity of cells, cells obtained by culturing in skeletal muscle differentiation medium according to Example 2 were treated as follows: the differentiation medium was carefully removed from the 24-well plate, and 300 μL of 0.5 mM DTNB solution (prepared in phosphate buffer, pH 7.2, 0.1% triton X-100) was immediately added. After 2 minutes of incubation at room temperature in the dark, 50 μL of 5.76 mM ATI (prepared in distilled water) was added. In addition, at least one blank reaction mix was made by mixing phosphate buffer with DTNB and ATI as above. All reaction mixes were incubated at 30° C. in the dark for 60 minutes, and then OD measurements were performed at 412 nm on an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK).

[0154] mU rel Calculation of AChE in: After colorimetric measurement of cells for 60 minutes, the OD412nm values ​​obtained as above were corrected by subtraction of the OD412nm value from the blank reaction. The corrected OD412nm values ​​of cells after 60 minutes were entered into the linear equation generated from the AChE standard enzymatic reaction (outlined above where the OD at 412nm was measured 6, 7 or 8 minutes after addition of ATI and DTNB) and a 2x104 correlation with the AChE standard was obtained. 5 The AChE activity per cell was determined. The determined AChE activity unit was therefore 2 x 10 5 mU, which represents the relative AChE activity of a cell rel is given by:

[0155] MPCs produced according to Example 1 and used in treatment according to Example 5 had a cell count of 2x10 as measured according to this Example 3. 5 At least 36 and up to 680 mU per cell rel It was found that the AChE was present in the 14.

[0156] Example 4 - Surface marker expression Cells isolated according to Example 1 and derived from approximately 170 different subjects were tested for the expression of surface markers CD34, CD56, CD90. To determine surface marker expression, flow cytometry was performed on a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells obtained according to Example 1 were harvested by overlaying with IX trypsin for 5 min at 37° C. and 400 μl of trypsin were added to the 100 μl plate. *The plates were centrifuged at 1000 x g and resuspended in Ix PBS supplemented with 1% FCS. 40,000 cells were suspended in 195 μl 1x PBS and incubated in 1.5 mL Eppendorf tubes for 20 min at 4 °C in the dark after adding 5 μL of CD34-PE, CD56-PE, CD90-PE (Beckman Coulter). Then, 5 μL of the viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was added to each reaction and the plate was incubated for 10 min at room temperature in the dark. Finally, cell events were acquired using Guava InCyte™ v.2.3 software. Histograms and dot plots were generated with a minimum of 5000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison to isotype controls set as at least 95% negative or by comparison to control (negative) cells.

[0157] Furthermore, individual random and illustratively selected batches of cells obtained according to Example 1 were tested for the expression of Sca-1, A2B5 and CD105 by flow cytometry. Thus, flow cytometric analysis was performed using a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells were harvested with trypsin for 5 min at 37°C, centrifuged at 400rcf and resuspended in lx PBS supplemented with 1% FCS. Cells at a concentration of 40000 / reaction were incubated with 5 μL of IgGl-PE (Beckman Coulter), Isotype Alexa488 (Sigma), anti-CD105-PE (Beckman Coulter, France) or A2B5-Alexa488 antibody (Millipore) in 1.5 mL Eppendorf tubes in the dark for 15 min at 4°C. Cells were washed with 1 mL of PBS, centrifuged at 400 rcf, and resuspended in 200 μL of 1xPBS for FACS analysis in 96-well round-bottom plates. After washing and resuspension, 5 μL of the viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was added to each reaction and plates were incubated for 10 min at 4°C. Cell events were acquired using Guava InCyte® v2.3 software. Histograms and dot plots were generated with a minimum of 3000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with isotype controls set as at least 99% negative.

[0158] MPC batches produced according to Example 1 and used for treatment according to Example 5 were found to be CD56 positive in the range of 64.10%-99.83%, CD90 positive in the range of 80.05%-99.88%, and CD34 positive in the range of 0.04%-8.33%. This expression profile was determined for MPC batches isolated from biopsies of approximately 170 different patients. Furthermore, individual batches, illustratively randomly selected from said MPC batches, were found to be Sca-1 negative with at least 60% of cells per batch positively expressing A2B5 and CD105 for each marker, and at most 10% of cells positively expressing Sca-1.

[0159] Immunocytochemistry was performed for intracellular detection of desmin and MyoD expression in cells isolated according to Example 1. First, the supernatant of the cell culture dish was discarded and the cells were washed three times with PBS. Permeabilization was performed by covering the cells with a 4% formaldehyde solution (v / v; diluted in PBS) for 20 min at room temperature. After each incubation step, the cells were washed three times with PBS. Then, the cells were covered with 500 μl of hydrogen peroxide block (Thermo Fisher Scientific) and incubated for 5 min at room temperature. A final concentration of 40 μg pro ml (w / v) of primary antibody (desmin or MyoD) was pipetted onto the cells and incubated for at least 90 min (37°C, 5% CO 2 ). Cells were then covered with 500 μl of biotin-labeled secondary antibody (goat anti-rabbit, polyclonal, Thermo Fisher Scientific) and incubated under the same conditions as the primary antibody, but for at least 60 min. To visualize antibody binding, 500 μl of horseradish peroxidase (Vectorlabs) was added to a final concentration of 2–5 μg / ml (diluted in PBS) and incubated at 37 °C, 5% CO 2 The cells were incubated for 20 min at RT. A final wash with PBS was performed before observing the results. Cells that stained positive for desmin by immunocytochemistry are visualized in dark red.

[0160] Randomly and illustratively selected individual batches of MPCs isolated according to Example 1 as described above were found to be desmin positive, where at least 60% of the cells were found to be positive. Additionally, randomly and illustratively selected individual batches of MPCs isolated according to Example 1 were found to be MyoD negative, where at most 10% of the cells were found to be MyoD positive.

[0161] Example 5 - Treating Patients with Fecal Incontinence with MPC The applicant conducted a multinational, multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical Phase IIb study as a proof-of-concept study to investigate the efficacy of myogenic progenitor cell-based therapy in a patient population that reflects the diversity actually encountered in clinical practice. Patient inclusion and exclusion criteria are shown below (Table 1). An overview of the consultations and tests performed and the patient flow are shown in Figure 4.

[0162] Table 1: Inclusion and exclusion criteria for patients to be included in the ITT population of the clinical trial according to Example 5.

[0163] [Table 1-1]

[0164] [Table 1-2]

[0165] [Table 1-3]

[0166] Eligible patients were randomized into one of three treatment arms (see Supplementary Methods for randomization procedure) and received a low dose of infused cells (LCC, 5±1x10) isolated according to Example 1. 6 ) or high dose (HCC, 50±10x10 6) combined with electrical stimulation for 4 weeks or a control treatment of infusion of cell-free medium combined with electrical stimulation. Transplantation of MPCs was performed as previously described in anesthetized patients by trained physicians. Patients were placed in supine position and cells or placebo were injected under direct ultrasound guidance using a specially designed injection device. Each patient received an aliquot of frozen cells diluted with lactated Ringer's to the appropriate cell concentration. As a result, a total volume of 6 ml was injected in 12 depots (12x0.5 ml), each depot arranged in a circular fashion directly in the external anal sphincter (EAS). Cell injection into the longitudinal muscle, internal anal sphincter and subepithelium was avoided. For this treatment, all patients were hospitalized for 1 day. Cells isolated according to Example 1 and used for transplantation were analyzed for skeletal muscle differentiation potential (Example 2), AChE enzyme activity (Example 3) and surface marker expression (Example 4). All cell batches produced during the study were found to be positive for the potential to form multinucleated myotubes (Figure 3). Furthermore, the cell populations used to treat both LCC and HCC were found to be 64.10%-99.83% positive for CD56, 80.05%-89.99% positive for CD90, and 0.04%-8.33% positive for CD34 (Figure 6). Pelvic floor muscle electrical stimulation therapy is considered the gold standard for conservative treatment of fecal incontinence, as it is known to stimulate muscle growth and growth-related signaling. An overview of the study is shown in Figure 4A. Patients selected for treatment in this study (intent-to-treat population, ITT) had basic population demographic parameters shown in Table 2.

[0167] Table 2: Demographics, time since first FI visit, and associated sphincter abnormalities in the ITT patient population of the clinical trial

[0168] [Table 2-1]

[0169] [Table 2-2]

[0170] [Table 2-3]

[0171] Example 6 - Efficacy Analysis After the first screening visit in Example 5, patients were asked to keep an incontinence diary for 2 weeks before taking a muscle biopsy sample from the pectoralis major muscle at the next visit. Four weeks of electrical stimulation treatment were then started. After another four weeks of keeping the diary, patients received a cell (LCC or HCC) or control (PBO) injection, followed by a post-transplant control the next day. Patients received another four weeks of electrical stimulation treatment and kept the diary. Patients were surveyed at 3, 6, and 12 months after the injection, and interviewed with the control group before keeping the four-week diary. In the diary, patients were asked to score daily on a visual analog scale (VAS) how badly fecal incontinence affected them, record fecal incontinence episodes, and classify them into three categories: "trace", "small amount", and "larger amount". Further parameters consisted of response rate assessment, anal manometry, ultrasound measurements, and the FI-QoL questionnaire, as outlined in the scheme. The primary outcome was the change in 4-week incontinence episode frequency (IEF) up to 6 months after injection (V4 = 6 months after treatment in Figure 1) compared to the baseline value (VO) 4 weeks before injection. Secondary outcomes were the change in VAS calculated from the mean value of the 4-week diary period and quality of life. In addition, the proportion of patients with a reduction in IEF of ≥ 25%, ≥ 50%, ≥ 75%, or ≥ 90% compared to baseline, as well as the change over time in anal manometry and ultrasound data were evaluated. Further exploratory endpoints were the change in IEF and other parameters from baseline to 12 months after injection, the change in different types of incontinence episodes (IE), and the number of incontinence-free days.

[0172] Anal endoscopy, ultrasound, and anal manometry were performed according to standards established by each participating center. Evaluation items included anal canal length, resting pressure, and maximum squeezing pressure. Balloon expulsion tests were used to measure the amount of filling required to reach the first sensation, the amount of defecation desire, the amount of defecation urgency, and the maximum tolerated capacity.

[0173] During the study period, the occurrence of adverse events (AEs) and serious AEs were recorded, standard physical examinations, hematology, blood chemistry, and urinalysis were performed, and concomitant medications were registered. The study was overseen by an independent Data Safety Monitoring Board. According to this evaluation, a total of 252 patients were randomly assigned to the three treatment groups in a 1:1:1 ratio. For the primary endpoint analysis, a one-sided Wilcoxon rank sum test was applied, with a p value of <0.025 considered significant. Secondary endpoints of continuous variables were compared between study groups by unpaired t-test (if the compared study groups were normally distributed) or Wilcoxon rank sum test (if the compared study groups were not normally distributed), with a p value of <0.05 considered significant. Non-continuous variables were compared between study groups by chi-square test or Fisher's test.

[0174] Analysis of treatment effects in terms of 50% responder rates at the fifth visit (12 months after treatment) for PBO, LCC, and HCC treatments for different types of incontinence episodes revealed that responder rates were consistently lowest for episodes classified as “trace” (Table 3). Therefore, for further analysis of IEF changes and responder rates, calculations were made both for counting all episode types and for counting only “low volume” and “higher volume” episode types.

[0175] Table 3: Proportion of patients with at least a 50% reduction in weekly incontinence episodes from baseline to 12 months post-treatment by treatment group (LCC, HCC, PBO) and type of episode (trace, light, heavier).

[0176] [Table 3]

[0177] Analysis of the change in IEF between treatment groups in the ITT patient population showed a consistent decrease across post-transplant visits in all treatment groups, whether all episode types were counted or traces were excluded (Figure 1A, Figure IB, Table 4, Table 5). The decline in IEF was highest in the HCC group at all post-treatment visits, followed by the LCC group, and finally the PBO group. In the ITT set, a two-sided Wilcoxon Mann-Whitney test with an alpha level of 0.05 was performed between HCC vs. PBO and LCC vs. PBO, and it was found that the change in IEF from baseline to the post-transplant visit was significantly increased in HCC compared to PBO at 6 months post-treatment including traces (p=0.035) and at 12 months excluding traces (p=0.034) (Figure 1A and B), suggesting that high-cell-number transplantation of MPC is superior to placebo treatment.

[0178] Table 4: Absolute change in weekly IEF (including trace) from baseline (VO) during the study period of Example 5 by treatment group in the ITT patient population. PBO, placebo. LCC, low cell count. HCC, high cell count. SD, standard deviation. N, number of patients.

[0179] [Table 4]

[0180] Table 5: Absolute change in weekly IEF (excluding trace) from baseline (VO) during the study period of Example 5 by treatment group in the ITT patient population. PBO, placebo. LCC, low cell count. HCC, high cell count. SD, standard deviation. N, number of patients.

[0181] [Table 5]

[0182] Responder Rate Consistent with other researchers (Rao, 2016), we considered a reduction in IEF of 50% or more as a clinically meaningful improvement and classified patients with this degree of reduction as responders, distinguishing them from non-responders with a smaller reduction. Therefore, we evaluated the data on the proportion of responders in each treatment group, including data at 1 and 3 months after injection, and reconsidered the above subgroups. We detected that in all defined groups and subpopulations, the proportion of responders increased continuously up to the 6-month follow-up, with the cell group exceeding the control group in all cases, and HCC exceeding LCC in most cases (Percentage of patients with at least a 50% reduction in IEF (including trace) per week from baseline to post-treatment visit according to Example 5 by treatment group in the ITT patient population). PBO, placebo. LCC, low cell count. HCC, high cell count (Table 6). Between 6 and 12 months, the responder rates remained stable or declined in the control and LCC groups, but continued to increase in the HCC group to over 50% when traces were included (Table 6) and over 60% when traces were excluded (Table 7). Fisher's exact test was performed for the 50% responder rates between LCC or HCC and PBO treatment in ITT patients, with significance at p = 0.05, and the superiority of HCC treatment over PBO treatment was observed at 1 month (p = 0.037) and 12 months (p = 0.006) after treatment when traces were excluded from the analysis (Figure 1D, Table 7). This suggests that the application of high cell numbers of myogenic progenitor cells is an effective and clinically relevant treatment for incontinence.

[0183] Table 6: Proportion of patients with at least a 50% reduction in weekly IEF (including trace) from baseline to post-treatment visit according to Example 5 by treatment group in the ITT patient population. PBO, placebo. LCC, low cell count. HCC, high cell count.

[0184] [Table 6]

[0185] Table 7: Proportion of patients with at least a 50% reduction in IEF (excluding trace) per week from baseline to post-treatment visit according to Example 5 by treatment group in the ITT patient population. PBO, placebo. LCC, low cell count. HCC, high cell count.

[0186] [Table 7]

[0187] Example 7 - Relationship between patient characteristics and treatment outcome In the clinical trial outlined in Example 5, 288 patients were screened and 251 were randomized, of which 244 received the study drug. 218 women and 19 men completed the study to at least 6 months of follow-up (96%) (Figure 4). Participants had a median age of 63 years (interquartile range, IQR, 53.8-70) and had had FI for a median duration of 5.3 years (IQR, 2.7-9.9). Baseline demographic and clinical characteristics of ITT patients available for analysis of the primary endpoint are summarized in Table 2.

[0188] Subgroup-specific effects To establish patient groups that respond specifically to cell therapy over placebo, we performed an exploratory post-hoc analysis. To this end, we applied the following hypothesis-driven approach. We hypothesized that for the injected cells to restore the function of the EAS, it is effective to inject them into or close to existing muscle tissue. This may be due to the limited migratory capacity of MPCs after transplantation. Thus, muscle regeneration may be impaired in patients with sparse muscle tissue, either because of persistent scar formation in the damaged EAS or because of time-dependent sarcopenia. Both conditions correlate with the duration of FI before the study. When we reanalyzed the treatment effect only in patients suffering from FI for less than 10 years (73% of the ITT set; subject population 1, TPP1), we found that the reduction in IEF in the LCC and HCC groups eventually significantly exceeded that in the control group, resulting in a much higher change in IEF from baseline to 12 months, when traces were excluded, compared to the respective changes in the ITT set (Figure 2B). Responder Rates and TPP1Within the patient subgroup, a significantly higher 50% responder rate was observed with HCC than with PBO at 12 months after treatment when traces were excluded (Figure 2B).

[0189] Another factor that reduces the clear impact of cell therapy is that the IEF also decreased in the control group. Therefore, to clearly capture the effect of cell infusion, we hypothesized that those with a higher baseline IEF would have a greater decrease in IEF after treatment, and that a clear difference would be observed between the PBO group and the cell group (LCC, HCC). This hypothesis was tested by subgrouping the ITT set patients according to their baseline IEF (including traces) and comparing the change in IEF from baseline to 6 months between the treatment groups and subgroups. As a result, it was found that when patients with low baseline IEF were subsequently excluded, the change in IEF increased in all groups. However, the difference between the PBO group and the cell group became increasingly pronounced as the baseline IEF became higher (Figure 7, Table 8). This suggests that patients with high baseline IEF would benefit more from MPC-based therapy than patients with low baseline IEF. Because the difference between the treatment groups rapidly increased when the baseline IEF exceeded 6, we combined this feature with TPP1 to create TPP2 (<10 years since first FI visit, baseline IEF >6). When the treatment effect was reanalyzed only for these patients, the decline in IEF in the LCC and HCC groups was significantly greater than that in the control group, resulting in a much larger change in IEF from baseline to 12 months compared with the respective changes in the ITT and TPP1 groups (Figure 2A). Regarding the responder rate in the TPP2 patient subgroup, a significantly higher 50% responder rate was observed in HCC than in PBO at 12 months after treatment, finally resulting in a higher responder rate in the HCC group in TPP2 compared with the HCC group in ITT patients (Figure 2C). This suggests that TPP2 cases are more responsive to MPC-based treatment compared with ITT cases.

[0190] As demonstrated above that incontinent episodes classified as trace are least responsive to treatment with Example 5, and patients with high baseline IEF respond better to MPC-based treatment, we analyzed TPP1 patients with baseline IEF >2, who may not be classified as "trace". This patient population was called TPP3. When the treatment effect was reanalyzed only for TPP3 patients, the reduction in IEF in the LCC and HCC groups was significantly greater than that of the control group in which traces were not counted (Figure 2B), and finally, the change in IEF from baseline to 12 months was much larger when compared with the changes in ITT, TPP1, and TPP2, respectively (Figures 2A and 2B). Regarding the response rate of the TPP3 patient subgroup, when traces were included, a significantly higher 50% response rate was observed in HCC than in PBO at 12 months after treatment. When traces were not included in the analysis, a significantly higher responder rate was observed in both HCC and LCC compared with PBO. Finally, the TPP3 responder rate in HCC-treated patients was the highest compared to all other patient groups, regardless of whether traces were counted as episodes (Figure 2C, Figure 2D) (Table 11, Table 12). This suggests that TPP3 patients respond best to MPC-based therapy compared to ITT, TPP1, and TPP2 patients. Conversely, patients excluded by TPP3 subgrouping (i.e., "NR1" with FI duration >10 years or "NR2" with baseline IEF <2 (excluding traces)) had lower IEF change and response rate than TPP1, TPP2, and TPP3 patients. The selection of patient groups may affect not only how cell therapy with LCC or HCC affects IEF decline and response rate, but also how placebo (PBO) treatment affects it. Therefore, we further calculated effect sizes and odds ratios for IEF change rate and responder rate, respectively. In detail, the comparison of LCC or HCC with PBO treatment in patient subgroups was performed and Cohen's d and odd ratios were calculated according to Table 13. Regardless of whether traces were counted as episodes or not, TPP3 patients had the highest treatment benefit with LCC or HCC treatment (compared with PBO treatment, respectively) in terms of effect size and odd ratio (Figure 8).

[0191] In addition, we analyzed IEF change from baseline to 12 months after treatment and responder rate in all TPP3 patients compared with TPP3 patients with external anal sphincter injury (TPP3_injury) associated with pretreatment fecal incontinence, and associated effect sizes and odds ratios (Tables 9, 10, 11, and 12). Comparing these patient groups, we found that TPP3 patients suffering from FI due to muscle injury had a higher IEF reduction after HCC treatment compared with TPP3 patients with no identified cause of FI (Figure 9 A and B). This effect was observed regardless of whether traces were counted as FI. Also, in the TPP3_injury population, IEF reduction after HCC treatment was significantly higher compared with PBO treatment. Similarly, we found that the 50% responder rate was higher in the TPP3_injury population, regardless of whether traces were counted or not (Figure 9 C and D). In TPP3_injured patients, a significantly higher responder rate was observed in HCC treatment than in PBO treatment, but when the traces were excluded from the analysis, the responder rate in HCC-treated patients was finally 87.5%, the highest in the entire period (Table 12). When the effect size and odds ratio of IEF change and responder rate due to LCC or HCC compared with PBO treatment were analyzed in TPP3_injured patients, the TPP3_injured patient population showed a higher effect size in HCC treatment than in the TPP3 patient population, regardless of whether the traces were considered as incontinent episodes (Figure 10 A, B). The odds ratio of the 50% responder rate was also found to be higher in the TPP3_injured patient population compared to the TPP3 patient population for both LCC and HCC treatment, independent of counting or excluding traces, respectively (Figure 10 C, D). These results suggest that patients who suffered from EAS damage prior to treatment experienced a greater reduction in incontinence episodes and a greater change in response when treated with different numbers of MPCs compared to a broader patient population with multiple possible FI-related conditions (e.g., EAS atrophy, pelvic floor dysfunction, etc.).

[0192] Table 8: Change in IEF from baseline to 6 months after treatment in the ITT set further grouped according to baseline IEF between treatment groups (PBO, LCC, HCC). Means, standard deviations "SD" and number of patients "N" are shown.

[0193] [Table 8]

[0194] Table 9: Changes from baseline to 12 months post-treatment in IEF (including trace) treatment groups (PBO, LCC, HCC) and patient populations shown with mean, standard deviation (SD) and number of patients (N).

[0195] [Table 9]

[0196] Table 10: Changes in IEF (excluding traces) from baseline to 12 months after treatment by treatment group (PBO, LCC, HCC) and patient population shown as mean, standard deviation (SD) and number of patients (N).

[0197] [Table 10]

[0198] Table 11: Proportion of patients with at least a 50% reduction in IEF (including trace) from baseline to 12 months post-treatment by treatment group (PBO, LCC, HCC) and patient population.

[0199] [Table 11]

[0200] Table 12: Proportion of patients with at least a 50% reduction in IEF (excluding trace) from baseline to 12 months post-treatment by treatment group (PBO, LCC, HCC) and patient population. [Table 12]

[0201] Table 13: Overview of the patient population analyzed according to Example 7, by number of patients (N), severity of fecal incontinence, time since diagnosis of fecal incontinence, and conditions related to FL. MD=muscle injury, PFD=pelvic floor dysfunction, ND=nerve injury, LOSC=loss of storage capacity, AT=atrophy.

[0202] [Table 13]

[0203] References Bajpai, VK, Mistriotis, P., Loh, Y.-H., Daley, GQ, & Andreadis, ST (2012). Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates. Cardiovascular Research, 96(3), 391-400. Frudinger, A., Kolle, D., Schwaiger, W., Pfeifer, J., Paede, J., & Halligan, S. (2009). Muscle-derived cell injection to treat anal incontinence due to obstetric trauma. Gut, 59(01), 55- Frudinger, A., Pfeifer, J., Paede, J., Kolovetsiou-Kreiner, V., Marksteiner, R., & Halligan, S. (2015). Autologous skeletal-muscle-derived cell injection for anal incontinence due to obstetric trauma: a 5-year follow-up of an initial study of 10 patients. Colorectal Disease: The Official Journal of the Association of Coloproctology of Great Britain and Ireland, 17(9), 794-801. Hirai, H., Yang, B., Garcia-Barrio, M. T., Rom, O., Ma, P. X., Zhang, J., & Chen, Y. E. (2018). Direct Reprogramming of Fibroblasts Into Smooth Muscle-Like Cells With Defined Transcription Factors-Brief Report. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(9), 2191-2197. Incitti, T., Magli, A., Jenkins, A., Lin, K., Yamamoto, A., & Perlingeiro, R. C. R. (2020). Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy chain isoforms associated with slow and oxidative muscles. Skeletal Muscle, 10(f), 17. Ito, N., Kii, I., Shimizu, N., Tanaka, H., & Takeda, S. (2017). Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells. Scientific Reports, 7(1), 8097. Jorge, J. Marcio N., Wexner, Steven D. (1993). Etiology and Management of Fecal Incontinence. Dis Colon Rectum, Vol. 36, No.l, pp. 77-96. Messner, F., Thumer, M., Muller, J., Blumer, M., Hofmann, J., Marksteiner, R., Couillard- Despres, S., Troppmair, J., Ofiier, D., Schneeberger, S., & Hautz, T. (2021). Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion. Stem Cell Research & Therapy, 12(V), 146. Miyagoe-Suzuki, Y., & Takeda, S. (2017). Skeletal muscle generated from induced pluripotent stem cells - induction and application. World Journal of Stem Cells, 9(6), 89-97. Rando, T. A., & Blau, H. M. (1994). Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. The Journal of Cell Biology, 125(6), 1275-12M. Rao, S. S. C. (2016). Endpoints for therapeutic interventions in faecal incontinence: small step or game changer. In Neurogastroenterology and Motility (Vol. 28, Issue 8, pp. 1123-1133). Blackwell Publishing Ltd. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861-872. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663-676. Thumer, M., Deutsch, M., Janke, K., Messner, F., Kreutzer, C., Beyl, S., Couillard-Despres, S., Hering, S., Troppmair, J., & Marksteiner, R. (2020). Generation of myogenic progenitor cell-derived smooth muscle cells for sphincter regeneration. Stem Cell Research & Therapy, 11(1), 233. Xuan, W., Khan, M., & Ashraf, M. (2021). Pluripotent stem cell-induced skeletal muscle progenitor cells with givinostat promote myoangiogenesis and restore dystrophin in injured Duchenne dystrophic muscle. Stem Cell Research & Therapy, 72(1), 131. Yamanaka, S. (2008). Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors. Cell Proliferation, 41 Suppl 1, 51-56.

Claims

1. 1. Myoblasts for use in a method for the treatment of anal incontinence in a subject, wherein the myoblasts are administered by one or more injections into and / or near the anal sphincter of the subject, and wherein the subject has suffered from anal incontinence for between 6 months and 10 years, with a severity of incontinence defined as more than two incontinence episodes per week prior to treatment, the incontinence episodes resulting in the unexpected leakage of liquid or solid stool from the subject's rectum, and excluding incontinence episodes that only manifest as a trace in the form of stains or marks on linens.

2. Myoblasts for use as described in claim 1, wherein the incontinent episode results in an amount of stool that exceeds the amount that appears as a stain or mark on the subject's linen, and which amount is comparable to the amount that would result from a normal bowel movement.

3. The myoblasts for use according to claim 1, characterized in that the subject's anal incontinence is caused by muscle damage.

4. 2. The myoblasts for use according to claim 1, characterized in that the subject has a severity of incontinence defined as more than 6 incontinence episodes per week prior to treatment.

5. 2. The myoblasts for use according to claim 1, wherein the method comprises stimulating the anal sphincter tissue before and / or after administration of the myoblasts.

6. 6. The myoblasts for use according to claim 5, characterized in that the stimulation comprises stimulation of the anal sphincter tissue for at least two weeks after administration of the myoblasts.

7. Myoblasts for use as described in claim 5, characterized in that the stimulation includes stimulation of anal sphincter tissue for at least two weeks before and after administration of the myoblasts.

8. 6. Myoblasts for use according to claim 5, characterized in that the stimulation is carried out by Kegel exercises and / or transcutaneous electrical stimulation.

9. 9. The myoblasts for use according to claim 8, wherein the transcutaneous electrical stimulation is performed daily, at least twice or at least three times per day.

10. 6. Myoblasts for use according to claim 5, characterized in that each stimulation lasts for at least 10 minutes.

11. Myoblasts for use as described in claim 5, characterized in that each stimulation lasts for at least 20 minutes.

12. (i) by positive expression of the markers CD56 and CD90, whereby at least 60% of the myoblasts express CD56 and at least 60% of the myoblasts express CD90; and / or (ii) 20 mU rel Measured by AChE activity of ∼1000 mUrel, the AChE activity is 2 × 10 5 Measured per cell 2. A myoblast for use according to claim 1, characterized in that 13. The myoblast for use according to claim 12, characterized in that (ii) it has an AChE activity of 30 mU rel to 800 mU rel measured per 2×10 5 cells.

14. The myoblast for use according to claim 12, characterized in that (ii) it has an AChE activity of 50 to 700 mU rel measured per 2×10 5 cells.

15. Myoblasts for use as described in claim 1, characterized in that 60 to 99.9% of the myoblasts express the cell marker CD56, 80 to 99.9% of the myoblasts express the cell marker CD90, and 0 to 10% of the myoblasts express the cell marker CD34.

16. The method comprises: (i) administration of 1 million to 200 million myoblasts; and / or (ii) 1 × 10 2 mU rel total ~1 x 10 6 mU rel Administration of an amount of myoblasts having a total AChE activity of 2. The method of claim 1, comprising administering an effective amount of:

17. The method comprising: (ii) administration of an amount of myoblasts having a total AChE activity of 1×10 3 mU rel total to 5×10 5 mU rel total ; 17. The method of claim 16, comprising administering an effective amount of myoblasts.

18. The method comprising: (ii) administration of an amount of myoblasts having a total AChE activity of 7×10 4 mU rel total to 2×10 5 mU rel total ; 17. The myoblasts for use according to claim 16, characterized in that it comprises administering an effective amount of:

19. 2. The myoblasts for use according to claim 1, characterized in that the myoblasts are administered by one or more injections into the external anal sphincter, the internal anal sphincter and / or the puborectalis muscle.

20. Myoblasts for use as described in claim 1, the myoblasts being contained in a pharmaceutical composition comprising a pharmaceutically acceptable additive and / or carrier.

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