Use of glial cell line-derived neurotrophic factor (GDNF) to treat enteric neuropathy

Abstract

The present application relates to a method for inducing neurogenesis in aganglionic or hypoganglionic areas of the distal colon of a human subject suffering from an intestinal neuropathy, such as Hirschsprung's disease (HSCR) or enteric hypoganglionic syndrome, by administering an effective dose of recombinant glial cell line-derived neurotrophic factor (GDNF) polypeptide to the distal colon of the subject. The method also corrects an imbalance of nitrergic and cholinergic neuronal subtypes located upstream of the aganglionic or hypoganglionic areas, allowing for the restoration of distal colon function (e.g., motility and epithelial barrier) in the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 950,781, filed December 19, 2019, which is incorporated herein by reference.

[0002] The present invention relates generally to the treatment of intestinal neuropathies, such as Hirschsprung's disease (HSCR) and enteric oligoganglionosis. [Background technology]

[0003] The enteric nervous system (ENS) extends along the entire gastrointestinal tract and controls intestinal motility, blood flow, and epithelial activity in response to sensory stimuli (1). Interconnected enteric ganglia containing neurons and glia develop from neural crest-derived precursors that migrate through the intestinal tract during prenatal development. Incomplete colonization of the distal colon by ENS precursors leads to Hirschsprung's disease (HSCR), a condition that affects 1 in 5000 newborns (2, 3). In HSCR, the distal colon without neuronal ganglia (i.e., aganglionic colon) remains tonically contracted and does not propagate contractions, resulting in functional intestinal obstruction. Symptoms of HSCR include intractable constipation with stool and air retention, abdominal distension, growth failure, occasional vomiting, intestinal inflammation (enterocolitis), and risk of bacterial translocation into the blood causing sepsis and early death (2).

[0004] HSCR is clinically subdivided into short-range (S-HSCR) and long-range (L-HSCR) (4). S-HSCR, occurring in >80% of cases, refers to the absence of ENS in the rectum and sigmoid colon. L-HSCR refers to the aganglionic nature of longer regions of the distal intestine. The pathogenesis of HSCR is still not fully understood, and many genes influence HSCR risk (2). Furthermore, genetic risk variants may combine with nongenetic factors to inhibit complete intestinal colonization by ENS precursors (5). This non-Mendelian inheritance occurs because many proteins must function together for normal ENS development.

[0005] Since 1948, most children with HSCR have been saved by surgical removal of the distal aganglionic bowel (16, 17). However, this procedure is far from ideal. Postoperative complications are common and may last for a long time, affecting survival (e.g., enteritis) and / or quality of life (e.g., fecal incontinence or obstructive symptoms) (18-20). One promising approach would be “regenerative medicine” to reconstruct the ENS and reduce the need for surgery. This idea has inspired many groups to develop HSCR therapies based on cell transplantation (21). However, despite many promising results, several difficulties still exist (22). The optimal source of stem cells, the ideal expansion and / or differentiation strategy before transplantation, the method of cell delivery, as well as cell function and final outcome after transplantation are still poorly defined. In addition, non-autonomous cell transplantation may require immunosuppression.

[0006] Hypoganglionosis, also known as enteric hypoganglionosis, is a disorder that causes a reduction in the number of nerves in the intestinal wall. Enteric hypoganglionosis can mimic HSCR, and patients with both conditions can present with chronic constipation, intestinal obstruction, and enteritis (intestinal inflammation). Patients with hypoganglionosis may also suffer from severe complications, including fecaloma (hardening of stool in the colon), intestinal bleeding or perforation, and respiratory problems resulting from distension of the colon. The exact cause of hypoganglionosis is often unknown. In some cases, it is due to factors present at birth (congenital), while in other cases, it is thought to be an acquired condition. Management of isolated hypoganglionosis generally involves surgery to remove the affected bowel area.

[0007] Thus, there is clearly a need for alternative treatments for intestinal neuropathies such as HSCR and enteric hypoganglionosis, treatments aimed at inducing neurogenesis in the distal colon and, in particular, restoring distal colon motility in patients with HSCR and enteric hypoganglionosis.

[0008] This description makes reference to several documents, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention

[0009] The present disclosure relates to the use of GDNF to treat one or more pathological features of intestinal neuropathies, such as Hirschsprung's disease.

[0010] In aspects and embodiments, the present disclosure relates to the following clauses 1 to 90: 1. A method for inducing enteric neurogenesis in aganglionic or poorly ganglionated areas of the distal colon of a human subject suffering from enteric neuropathy, the method comprising administering to the distal colon of the subject a pharmaceutical composition comprising an effective dose of a recombinant glial cell line-derived neurotrophic factor (GDNF) polypeptide and a pharma- ceutical acceptable carrier. 2. The method of claim 1, wherein the GDNF polypeptide comprises an amino acid sequence having at least 70% identity to amino acids 78 to 211 of SEQ ID NO:1. 3. The method of claim 2, wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 78 to 211 of SEQ ID NO:1. 4. The method of claim 3, wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 78 to 211 of SEQ ID NO:1. 5. The method of claim 4, wherein the GDNF polypeptide comprises amino acids 78 to 211 of SEQ ID NO:1. 6. The method of any one of clauses 1 to 5, wherein the effective dose of recombinant GDNF polypeptide administered to a human subject corresponds to a dose of about 5 μg to about 20 μg in a mouse pup. 7. The method of any one of clauses 1-6, wherein the pharma- ceutically acceptable carrier comprises a saline solution or a gelling agent. 8. The method of any one of clauses 1 to 7, wherein the pharmaceutical composition is administered rectally via an enema. 9. The method of any one of clauses 1-7, wherein the pharmaceutical composition is administered by injection into the distal colon wall. 10. The method of any one of clauses 1 to 9, wherein the pharmaceutical composition is administered once a day up to four times a day. 11. The method of any one of clauses 1 to 10, wherein the pharmaceutical composition is administered on at least two consecutive days. 12. The method of any one of clauses 1 to 11, wherein the method is performed prior to surgical removal of an aganglionic or poorly ganglionated area in a subject. 13. The method of any one of clauses 1 to 11, wherein the method is performed after surgical removal of an aganglionic or poorly ganglionated area in the subject. 14. The method according to clause 12 or 13, wherein the surgical removal of the aganglionic or poorly ganglionic area is via pull-through surgery. 15. The method according to any one of clauses 1 to 14, wherein the intestinal neuropathy is enteric hypoganglionosis. 16. The method according to any one of clauses 1 to 14, wherein the intestinal neuropathy is Hirschsprung's disease (HSCR). 17. The method of clause 16, wherein the subject is suffering from short-zone HSCR. 18. The method according to clause 16 or 17, wherein the HSCR is sporadic HSCR. 19. The method according to any one of clauses 16 to 18, wherein HSCR is associated with reduced expression or activity of the RET receptor. 20. The method of clause 19, wherein the HSCR is associated with a mutation in the RET gene. 21. The method of any one of clauses 1 to 20, wherein enteric neurogenesis comprises the production of enteric neurons and / or enteric glial cells. 22. The method according to clause 21, wherein neurogenesis comprises the production of enteric neurons and enteric glial cells. 23. The method of clause 21 or 22, wherein the production of enteric neurons and / or enteric glial cells comprises proliferation of enteric neuronal and / or enteric glial precursors. 24. The method of any one of clauses 1 to 23, wherein the method corrects an imbalance of nitrergic and cholinergic neuronal subtypes located upstream of an aganglionic or ganglionic-poor area. 25. The method of any one of clauses 1 to 24, wherein the method restores distal colon motility in the subject. 26. The method of any one of clauses 1 to 25, wherein the method restores the proportion of lymphocytes and / or myeloid immune cells in the subject's distal colon. 27. The method of any one of clauses 1-26, wherein the human subject is under 5 years of age or under 1 year of age. 28. The method of clause 27, wherein the human subject is less than 6 months of age. 29. The method according to any one of clauses 1 to 28, wherein the pharmaceutical composition is administered to the rectum and / or sigmoid colon. 30. The method of clause 29, wherein the pharmaceutical composition is administered to the rectosigmoid region or is for administration to the rectosigmoid region. 31. A pharmaceutical composition for inducing enteric neurogenesis in aganglionic or poorly ganglionated areas of the distal colon of a human subject suffering from enteric neuropathy, comprising a recombinant glial cell line-derived neurotrophic factor (GDNF) polypeptide and a pharma- ceutical acceptable carrier, wherein the composition is for administration to the distal colon of the subject. 32. The pharmaceutical composition for use according to clause 31, wherein the GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78 to 211 of SEQ ID NO:1. 33. The pharmaceutical composition for use according to clause 32, wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78 to 211 of SEQ ID NO:1. 34. A pharmaceutical composition for use according to clause 33, wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78 to 211 of SEQ ID NO:1. 35. A pharmaceutical composition for use according to clause 34, wherein the GDNF polypeptide comprises amino acids 78 to 211 of SEQ ID NO:1. 36. A pharmaceutical composition for use according to any one of clauses 31 to 35, wherein the dose of recombinant GDNF polypeptide used corresponds to a dose of about 5 μg to about 20 μg in mouse pups. 37. A pharmaceutical composition for use according to any one of clauses 31 to 36, wherein the pharma- ceutically acceptable carrier is a saline solution or a gelling agent. 38. A pharmaceutical composition for use according to any one of clauses 31 to 37, wherein the pharmaceutical composition is for rectal administration via an enema. 39. A pharmaceutical composition for use according to any one of clauses 31 to 38, wherein the pharmaceutical composition is for administration by injection into the distal colon wall. 40. A pharmaceutical composition for use according to any one of clauses 31 to 39, wherein the pharmaceutical composition is for administration once a day up to four times a day. 41. A pharmaceutical composition for use according to any one of clauses 31 to 40, wherein the pharmaceutical composition is for administration on at least two consecutive days. 42. A pharmaceutical composition for use according to any one of clauses 31 to 41, wherein the pharmaceutical composition is for use prior to surgical removal of an aganglionic or poorly ganglionated area in a subject. 43. A pharmaceutical composition for use according to any one of clauses 31 to 41, wherein the pharmaceutical composition is for use after surgical removal of an aganglionic or poorly ganglionated area in a subject. 44. The pharmaceutical composition for use according to clause 42 or 43, wherein the surgical removal of the aganglionic or poorly ganglionic area is via pull-through surgery. 45. A pharmaceutical composition for use according to any one of clauses 31 to 44, wherein the intestinal neuropathy is enteric hypoganglionosis. 46. ​​A pharmaceutical composition for use according to any one of clauses 31 to 44, wherein the intestinal neuropathy is Hirschsprung's disease (HSCR). 47. The pharmaceutical composition for use according to clause 46, wherein the subject is suffering from short-spectrum HSCR. 48. The pharmaceutical composition for use according to clause 46 or 47, wherein the HSCR is sporadic HSCR. 49. A pharmaceutical composition for use according to any one of clauses 46 to 48, wherein HSCR is associated with a reduction in the expression or activity of the RET receptor. 50. The pharmaceutical composition for use according to clause 49, wherein the HSCR is associated with a mutation in the RET gene. 51. A pharmaceutical composition for use according to any one of clauses 31 to 50, wherein intestinal neurogenesis comprises the production of enteric neurons and / or enteric glial cells. 52. A pharmaceutical composition for use according to clause 51, wherein intestinal neurogenesis comprises the production of enteric neurons and enteric glial cells. 53. A pharmaceutical composition for use according to clause 51 or 52, wherein the production of enteric neuronal and / or glial cells comprises proliferation of enteric neuronal and / or enteric glial precursors. 54. A pharmaceutical composition for use according to any one of clauses 31 to 53, wherein the pharmaceutical composition corrects an imbalance of nitrergic and cholinergic neuronal subtypes located upstream of aganglionic or ganglionic-poor areas. 55. A pharmaceutical composition for use according to any one of clauses 31 to 54, wherein the pharmaceutical composition restores distal colon motility in a subject. 56. A pharmaceutical composition for use according to any one of clauses 31 to 55, wherein the pharmaceutical composition restores the proportion of lymphocytes and / or myeloid immune cells in the distal colon of the subject. 57. A pharmaceutical composition for use according to any one of clauses 31 to 56, wherein the human subject is under 5 years of age or under 1 year of age. 58. The pharmaceutical composition for use according to clause 57, wherein the human subject is less than 6 months of age. 59. A pharmaceutical composition for use according to any one of clauses 31 to 58, wherein the pharmaceutical composition is for administration to the rectum and / or sigmoid colon. 60. The pharmaceutical composition for use according to clause 59, wherein the pharmaceutical composition is for administration to the rectosigmoid region. 61. Use of a pharmaceutical composition comprising a recombinant glial cell line-derived neurotrophic factor (GDNF) polypeptide and a pharma- ceutical acceptable carrier for the manufacture of a medicament for inducing intestinal neurogenesis in aganglionic or poorly ganglionated areas of the distal colon of a human subject suffering from intestinal neuropathy, wherein the medicament is for administration to the distal colon of the subject. 62. The use according to clause 61, wherein the GDNF polypeptide comprises an amino acid sequence having at least 70% identity with amino acids 78 to 211 of SEQ ID NO:1. 63. The use according to clause 62, wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity with amino acids 78 to 211 of SEQ ID NO:1. 64. The use according to clause 63, wherein the GDNF polypeptide comprises an amino acid sequence having at least 95% identity with amino acids 78 to 211 of SEQ ID NO:1. 65. The use according to clause 64, wherein the GDNF polypeptide comprises amino acids 78 to 211 of SEQ ID NO:1. 66. The use according to any one of clauses 61 to 65, wherein the dose of recombinant GDNF polypeptide used corresponds to a dose of about 5 μg to about 20 μg in mouse pups. 67. The use according to any one of clauses 61 to 66, wherein the pharma- ceutically acceptable carrier is a saline solution or a gelling agent. 68. The use according to any one of clauses 61 to 67, wherein the pharmaceutical composition is for rectal administration via an enema. 69. The use according to any one of clauses 61 to 67, wherein the pharmaceutical composition is for administration by injection into the distal colon wall. 70. The use according to any one of clauses 61 to 69, wherein the pharmaceutical composition is for administration once a day up to four times a day. 71. The use according to any one of clauses 61 to 70, wherein the pharmaceutical composition is for administration on at least two consecutive days. 72. The use according to any one of clauses 61 to 71, wherein the pharmaceutical composition is used prior to surgical removal of an aganglionic or poorly ganglionated area in a subject. 73. The use according to any one of clauses 61 to 72, wherein the pharmaceutical composition is used following surgical removal of an aganglionic or poorly ganglionic area in a subject. 74. The use according to clause 72 or 73, wherein the surgical removal of the aganglionic or poorly ganglionic area is via pull-through surgery. 75. The use according to any one of clauses 61 to 74, wherein the intestinal neuropathy is enteric hypoganglionosis. 76. The use according to any one of clauses 61 to 75, wherein the intestinal neuropathy is Hirschsprung's disease (HSCR). 77. The use according to clause 76, wherein the subject is suffering from short-field HSCR. 78. The use according to clause 76 or 77, wherein the HSCR is sporadic HSCR. 79. The use according to any one of clauses 76 to 78, wherein HSCR is associated with reduced expression or activity of the RET receptor. 80. The use according to clause 79, wherein the HSCR is associated with a mutation in the RET gene. 81. The use according to any one of clauses 61 to 80, wherein intestinal neurogenesis comprises the production of enteric neurons and / or enteric glial cells. 82. The use according to clause 81, wherein enteric neurogenesis comprises the production of enteric neurons and enteric glial cells. 83. The use according to clause 81 or 82, wherein the production of enteric neuronal and / or glial cells comprises proliferation of enteric neuronal and / or enteric glial precursors. 84. The use according to any one of clauses 61 to 83, wherein the method corrects an imbalance of nitrergic and cholinergic neuronal subtypes located upstream of an aganglionic or ganglionic-poor area. 85. The use according to any one of clauses 61 to 84, wherein the medicament restores distal colon motility in a subject. 86. The use according to any one of clauses 61 to 85, wherein the medicinal product restores the proportion of lymphocytes and / or myeloid immune cells in the distal colon of the subject. 87. The use according to any one of clauses 61 to 86, wherein the human subject is under 5 years of age or under 1 year of age. 88. The use according to clause 87, wherein the human subject is less than 6 months of age. 89. The use according to any one of clauses 61 to 88, wherein the medicinal product is for administration to the rectum and / or sigmoid colon. 90. The use according to clause 89, wherein the medicinal product is for administration to the rectosigmoid region.

[0011] Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-limiting description of specific embodiments, given by way of example only with reference to the accompanying drawings. [Brief description of the drawings]

[0012] In the accompanying drawings: [Figure 1]Figure 1A-H shows the setting of GDNF therapy parameters in HolTg / Tg mice. (Figure 1A-B) Distribution of 10 μl methylene blue enemas in the colon of HolTg / Tg pups at P4 (Figure 1A) and P8 (Figure 1B). (Figure 1C) Effect of GDNF concentration on survival rate of HolTg / Tg pups receiving 10 μl enemas once a day between P4 and P8. The amount shown corresponds to the total amount of GDNF administered on each day. (Figure 1D) Effect of treatment time frame (P4-P8 vs. P8-P12), duration (1d, 5d, or 10d; starting at P4), and frequency (once or twice a day for 5 days) on survival rate of HolTg / Tg pups treated with GDNF enemas (the amount of GDNF administered per single enema was kept constant at 10 μg in 10 μl). (Figure 1E) Survival of HolTg / Tg pups administered a 10 μl enema containing the indicated neurotrophic molecules (noggin, endothelin-3, or the serotonin receptor agonist RS67506; all at a final concentration of 1 μg / μl) once daily between P4 and P8. (Figure 1F) Effect of food consistency (regular chow vs. gel chow) on survival of HolTg / Tg pups receiving daily GDNF enemas (10 μg in 10 μl) between P4 and P8. (Figure 1G) Effect of co-administration of ascorbic acid (Vit.C; 100 μM final concentration), serotonin (5-HT; 1 μg / μl final concentration), and endothelin-3 (ET3; 1 μg / μl final concentration) on survival of HolTg / Tg pups receiving GDNF enemas (10 μg in 10 μl) once daily between P4 and P8. (Figure 1H) Neuronal density (expressed as % of surface area) in the colon and associated health status of P20 HolTg / Tg mice receiving daily GDNF enemas (10 μg in 10 μl) between P4 and P8. [Diagram 2]Figure 2A-E shows that GDNF enemas rescue aganglionic megacolon in HSCR mouse models. Daily administration of GDNF enemas to HolTg / Tg (Figure 2A), EdnrbS-l / sl (Figure 2B), and TashTTg / Tg (Figure 2C) mice between P4 and P8 positively affected both megacolon symptoms (i.e., distal obstruction and accumulation of fecal material in the colon, growth retardation, lethargy, and hunchback) and survival rates (Mantel-Cox statistical test, comparing GDNF-treated and non-treated groups in the case of HolTg / Tg and EdnrbS-l / sl, or comparing GDNF-treated and PBS-treated groups in the case of TashTTg / Tg). (Figure 2D) Whole-mount immunofluorescence staining of colonic muscle strips from P20 mice shows that 5 days of GDNF treatment induces myenteric ganglia containing HuC / D+ neurons and Sox10+ glia in otherwise aganglionic regions of HolTg / Tg mice. Dashed outlines indicate the area occupied by extrinsic nerve fibers. Below each representative image is a schematic of the average neuronal density of each colonic subregion (represented by cylinders) along the length of the colon. Neuronal density is expressed as a percentage of the area occupied by HuC / D+ cells at the level of the myenteric plexus in the intestinal wall in a single confocal slice (n = 6 mice per group; 3 fields per subregion). Neuronal density in each distal colonic subregion is also shown as a numerical value. (Figure 2E) Immunofluorescence analysis of distal colon muscularis from P20 mice that received intraperitoneal injections of EdU during 5 days of GDNF treatment shows that a subset of induced myenteric neurons (arrowheads) and glia (arrows) were generated from dividing precursors during the treatment period. Dashed outlines indicate the area occupied by a single ganglion. (Figure 2F) Quantification of EdU incorporation in myenteric neurons and glia in the distal colon. Results are expressed as the number of EdU+ cells per mm2 (n = 3 WT and 3 GDNF-treated HolTg / Tg mice; 2–7 fields per animal; ***P<0.001; ****P<0.0001; one-way ANOVA with post-hoc Sidak's test). All images shown represent z-stack projections at the level of the myenteric plexus. Scale bars, 100 µm (Figure 2D) and 50 µm (Figure 2E). [Diagram 3] Figure 3 shows an overview of the myenteric and submucosal plexuses in the distal colon of WT, untreated HolTg / Tg, or GDNF-treated HolTg / Tg mice at P20. Immunofluorescence analysis of TuJ1+ neuronal structures in the myenteric (upper panel) and submucosal (lower panel) plexuses. Arrows point to GDNF-induced ganglia containing neuronal bodies. Inset is a magnified view of the GDNF-induced ganglion within the dashed box. GDNF-treated mice received 10 µg of GDNF in a 10 µL enema once daily from P4–P8. All images show z-stack projections representing observations obtained from three mice. Scale bars, 100 µm (large panel) and 50 µm (inset). [Figure 4] Figure 4A-C shows the analysis of myenteric ganglion size and neuronal density in the colon of P20 HolTg / Tg and TashTTg / Tg mice treated or not with GDNF between P4-P8. (Figure 4A) Analysis of myenteric ganglion size in HolTg / Tg mice. (Figure 4B) Analysis of neuronal density in TashTTg / Tg mice. The average neuronal density for each colonic subregion (represented by cylinders) is shown along the length of the colon. Neuronal density is expressed as a percentage of the area occupied by HuC / D+ cells at the level of the myenteric plexus in the intestinal wall in a single focal plane (n=6 mice per group; 3 fields per subregion). Neuronal density in each distal colonic subregion is also shown as a numerical value. (Figure 5C) Analysis of myenteric ganglion size in TashTTg / Tg mice. [Diagram 5]Figure 5A-C show analysis of EdU incorporation in myenteric and submucosal ganglia in colons from P20 WT and GDNF-treated HolTg / Tg mice administered EdU between P4-P8. (Figure 5A) Example of EdU incorporation in z-stack projections of submucosal neurons (arrowheads) and glia (arrows) in the distal colon. Dashed outlines indicate the area occupied by a single ganglion. Scale bar, 50 µm. (Figure 5B) Quantitative analysis of EdU incorporation in submucosal, neuronal (HuC / D+) and glial (SOX10+) ganglia in the middle and distal colon. Results are expressed as number of EdU+ cells per mm2 (n = 3 WT and 3 GDNF-treated HolTg / Tg mice; 2-7 fields per animal; *P < 0.05; one-way ANOVA with post hoc Sidak's test). (Figure 5C) Quantitative analysis of EdU incorporation in neurons (HuC / D+) and glia (SOX10+) in the intermuscular (left panel) and submucosal (right panel) regions in the distal colon. Results are expressed as the percentage of EdU+ cells per ganglion (n=3 WT and 3 GDNF-treated HolTg / Tg mice; 2–7 fields per animal; ***P<0.001; ****P<0.0001; one-way ANOVA with post-hoc Sidak's test). GDNF-treated mice received 10 μg GDNF in a 10 μL enema once daily from P4–P8. [Figure 6]Figure 6A-K shows the phenotypic and functional characterization of the ENS induced by GDNF in P20 HolTg / Tg mice. (Figure 6A-B) Immunofluorescence-based quantitative analysis of GDNF-induced myenteric ganglia from the distal colon of HolTg / Tg mice shows the WT-like neuronal (HuC / D+) to glial (Sox10+) ratio (Figure 6A), as well as the percentage of nitrergic (nNOS+HuC / D+) and cholinergic (ChAT+HuC / D+) neurons (Figure 6B) (n = 6 mice per group; 3 fields per animal). (Figure 6C) Analysis of GDNF-induced myenteric ganglia not only shows the presence of nitrergic and cholinergic neurons, but also other neuronal subtypes expressing either tyrosine hydroxylase (TH), substance P (SubP), calretinin (CalR), or vasoactive intestinal peptide (VIP). All images are single focal planes representing observations obtained from three mice per subtype marker, arrows point to examples of the indicated neuronal subtypes. Scale bar, 20 µm. (Figure 6D) In ​​vivo bead latency test shows partial recovery of colonic motility in some HolTg / Tg mice treated with GDNF (n = 8–9 mice per group, *P < 0.05; one-way ANOVA with post-hoc Sidak test). Note that we limited the time to expel the glass bead after rectal insertion to an upper limit of 30 min to simplify the analysis without affecting statistical significance. (Figure 6E) Ex vivo analysis of electric field stimulation and drug modulation patterns of contraction-relaxation of longitudinal smooth muscle in organ baths equipped with force transducers. Contraction strength (expressed in g / s) is calculated from the difference from baseline of area under the curve (AUC) values ​​obtained after electric field stimulation. The mean ΔAUC values ​​of the HolTg / Tg+GDNF group are divided into two subgroups according to the contractile response.Approximately half (4 of 7) of the colonic muscle strips from HolTg / Tg mice treated with GDNF displayed a WT-like response pattern to sequential inhibition of nitrergic and cholinergic signaling (with L-NAME and atropine, respectively), whereas the other half (3 of 7) remained poorly responsive similar to muscle strips from untreated HolTg / Tg mice (n = 6 WT and HolTg / Tg, n = 7 HolTg / Tg+GDNF; **P<0.01; ****P<0.0001; two-way ANOVA with post-hoc Tukey's test). (FIG. 6F) Ex vivo measurement of distal colonic mucosal permeability to FITC-labeled 4 kDa dextran (FD4) in Ussing chambers indicates increased permeability in the HolTg / Tg colon, with two-thirds (4 of 6) of HolTg / Tg mice treated with GDNF on the way to recovery (n=6 mice per group; *P<0.05; **P<0.01; one-way ANOVA with post hoc Sidak test). (Figure 6G-I) H&E staining of transverse sections of distal colon shows that GDNF treatment rescues both the increase in smooth muscle thickness (see brackets in Figure 6G, and quantification in Figure 6H) and the increase in the number of neutrophils in the submucosa (see asterisks in Figure 6G, and quantification in Figure 6I) (n = 6 mice per group; scale bar, 150 μm; *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA with post hoc Sidak test). (Figure 6J-K) Microbiome profiling shows that GDNF treatment at least partially rescues the microbiota imbalance typically observed in untreated HolTg / Tg mice (n = 5 mice per group). Bar histograms (Figure 6J) show the average relative abundance of 16S rRNA gene sequences at the genus level (*P<0.05; one-way ANOVA with post-hoc Tukey test). Beta diversity comparisons with 95% confidence interval ellipses (Figure 6K) are based on nonmetric multidimensional scaling (NMDS) of Bray-Curtis dissimilarities of relative abundance of operational taxonomic units between samples (P<0.001; PERMANOVA). [Figure 7]Figure 7A-B show the percentages of nitrergic and cholinergic myenteric neurons in the proximal and mid-colon of WT, untreated HolTg / Tg, or GDNF-treated HolTg / Tg mice at P20. (Figure 7A) Qualitative analysis of the percentages of nitrergic (left panel) and cholinergic (right panel) neurons. Scale bar, 50 µm. (Figure 7B) Quantitative analysis of the percentages of nitrergic (left panel) and cholinergic (right panel) neurons (n ​​= 3 WT and 3 GDNF-treated HolTg / Tg mice; 3 fields per animal; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; one-way ANOVA with post-hoc Sidak's test). GDNF-treated mice received 10 μg of GDNF in a 10 μL enema once daily from P4–P8. [Figure 8] Figure 8A-B show supporting information on in vivo and ex vivo analysis of motility in the distal colon of WT, untreated HolTg / Tg, or GDNF-treated HolTg / Tg mice at P20. (Figure 8A) Correlation between neuronal density in the distal colon at P20 and bead ejection time in GDNF-treated HolTg / Tg mice (Supporting Figure 6D). (Figure 8B) Examples of electric field stimulation and drug modulation patterns of longitudinal smooth muscle contraction-relaxation in organ baths with force transducers (Supporting Figure 6E). In responsive tissues, electric field stimulation (EFS) causes contraction of colonic muscle, contraction that can be slightly increased by L-NAME-mediated inhibition of nitrergic signaling and robustly countered by atropine-mediated inhibition of cholinergic signaling. GDNF-treated mice received 10 μg of GDNF in a 10 μL enema once daily from P4–P8. [Figure 9]Figure 9A-B show analysis of smooth muscle thickness in the distal colon of WT, untreated HolTg / Tg, or GDNF-treated HolTg / Tg mice at P20. (Figure 9A) Representative H&E stained cross sections of different colonic regions, smooth muscle thickness is indicated by red brackets. Scale bar, 150 μm. (Figure 9B) Average muscle thickness in each colonic region (n = 6 mice per group; **P<0.01; ***P<0.001; one-way ANOVA with post hoc Tukey test). GDNF-treated mice received 10 μg GDNF in a 10 μL enema once daily from P4-P8. [Figure 10]Figure 10A-I show that exogenous Schwann cell precursors (SCPs) are the source of GDNF-induced neurons and glia in the aganglionic colon. (Figure 10A) Western blot analysis of GDNF distribution in different subregions of the GI tract from WT, HolTg / Tg, and GDNF-treated HolTg / Tg mice at P8. Endogenous GDNF (eGDNF) is normally restricted to the ileum in all mice, while recombinant GDNF (rGDNF) is exclusively detected in the distal colon of GDNF-treated HolTg / Tg mice. In GDNF-treated HolTg / Tg, eGDNF is expressed in all subregions of the colon. Anti-GDNF blots shown represent observations from three mice per group. (Figure 10B-C) Time course analysis of the distribution of a 6xHis-tagged version of GDNF (HisGDNF) used for enema treatment of HolTg / Tg mice between P4 and P8. Anti-His and anti-RET double staining of the distal colon at 2-day intervals shows that both HisGDNF and RET accumulate submucosally during treatment (Figure 10B). Both are detected in induced myenteric neurons near extrinsic nerve fibers, at P8 (Figure 10C). All images show z-stack projections representing observations obtained from three mice. Scale bar, 20 µm. (Figure 10D) Representative images from a 10-h long time-lapse recording of aganglionic colonic tissue from a HolTg / Tg;G4-RFP mouse showing that SCP-like cells are dividing (arrows) and migrating (arrowheads) on extrinsic nerve fibers. Explants were prepared from P4 distal colon and pre-cultured with GDNF for 72 h, followed by intravital imaging with a confocal microscope in the continued presence of GDNF. Images are 50 μm thick z-stack projections representing observations from three explants. Scale bar, 100 μm. (Figure 10E-F) Anti-SOX10 and anti-Ki67 double labeling shows that exposure to GDNF for 96 h significantly increases the SCP proliferation rate in distal colon explants prepared from P4HolTg / Tg mice. The image in Figure 10E is a single focal plane representing observations from three explants. Scale bar, 50 μm.Each value in Figure 10F corresponds to the average percentage of Ki67+SCP (i.e., (Ki67+SOX10+ / SOX10+) × 100) calculated from a minimum of three fields per explant (**P < 0.01; two-tailed Student's t test). (Figure 10G-H) Immunofluorescence analysis of myenteric ganglia in the distal colon of P20 HolTg / Tg;Dhh-CreTg / +;R26YFP / + mice that received GDNF enema and EdU via intraperitoneal injection between P4 and P8. Four categories of induced neurons are detected: 1) SCP-derived (Dhh+ lineage) and EdU positive (solid gray arrowhead), 2) SCP-derived and EdU negative (empty gray arrowhead), 3) unknown origin and EdU positive (solid white arrowhead), 4) unknown origin and EdU negative (empty white arrowhead). Images in Figure 10G are single focal planes representing observations from three mice. Scale bar, 50 μm. The relative proportions of the four categories of induced neurons per ganglion plotted in Figure 10H correspond to the average calculated from a minimum of three fields per mouse. Dashed outlines indicate areas occupied by either extrinsic nerve fibers (Figure 10E), or extrinsic nerve fibers and adjacent single ganglia (Figures 10C and G). FIG. 10I: Schwann cells in the aganglionic distal colon of HolTg / Tg mice express neuronal cell adhesion molecule (NCAM) but not RET. Immunofluorescence analysis of NCAM and RET expression in extrinsic nerve fibers (delineated by dashed lines) from the distal colon of untreated HolTg / Tg mice at P20. NCAM, but not RET, is expressed in SOX10+ Schwann cells and putative enteric glial / ENS precursors (arrows). DAPI, 4',6-diamidino-2-phenylindole. Images shown are single focal planes representing observations from three mice. Scale bar, 50 μm. [Figure 11]Figure 11 shows the analysis of GDNF distribution in multiple tissues of HolTg / Tg mice treated with GDNF at P20. Western blot analysis of α-tubulin normalized levels of endogenous GDNF (eGDNF) and recombinant GDNF (rGDNF) in different tissues of P20 HolTg / Tg mice receiving 10 μg GDNF in a 10 μL enema once daily from P4-P8. The blots shown represent observations from three mice. [Figure 12] FIG. 12 shows a time course analysis of HisGDNF distribution and RET expression in colonic smooth muscle of P4-P8 HolTg / Tg mice treated with HisGDNF. Immunofluorescence analysis of HisGDNF distribution and RET expression in the distal colonic muscularis of HolTg / Tg mice treated with HisGDNF. White arrowheads point to RET+ neurons that also stain positive for HisGDNF. All images show a single focal plane representing observations from three mice. Scale bar, 20 μm. [Figure 13]Figure 13A-D shows analysis of SCP-derived neurogenesis in myenteric and submucosal ganglia of Dhh-CreTg / +;R26YFP / + and HolTg / Tg;Dhh-CreTg / +;R26YFP / + mice at P20. (Figure 13A) Analysis of myenteric neurons (HuC / D+) and YFP expression in the proximal colon of Dhh-CreTg / +;R26YFP / + (control) and HolTg / Tg;Dhh-CreTg / +;R26YFP / + (HolTg / Tg) mice. Yellow arrowheads point to SCP-derived neurons. (Figure 13B) Quantitative analysis of myenteric neurons (HuC / D+) and YFP expression in the proximal and mid-colon of Dhh-CreTg / +;R26YFP / + (control) and HolTg / Tg;Dhh-CreTg / +;R26YFP / + (HolTg / Tg) mice (n=3 control and 3 HolTg / Tg mice; 3 fields per animal; *P<0.05; one-way ANOVA with post-hoc Sidak's test). (Figure 13C) Analysis of submucosal neurons (HuC / D+) and YFP expression in the distal colon of HolTg / Tg;Dhh-CreTg / +;R26YFP / + (HolTg / Tg) mice treated with GDNF between P4 and P8. Neurons of either SCP (grey arrowheads) or unknown (white arrowheads) origin are detected. (Figure 13D) Analysis of RET-expressing myenteric neurons (HuC / D+) and YFP expression in the distal colon of HolTg / Tg;Dhh-CreTg / +;R26YFP / + (HolTg / Tg) mice treated with GDNF between P4 and P8. RET is expressed in a subset of neurons, regardless of their SCP (RET+, filled gray arrowhead; RET-, empty gray arrowhead) or non-SCP (white arrowhead) origin. All images shown are z-stack projections representing observations obtained from three mice. Scale bar, 50 µm. Dashed outline indicates the area occupied by a single ganglion. [Figure 14]Figure 14A-H shows ex vivo preclinical studies of GDNF therapy on aganglionic colon explants from HolTg / Tg mice and human HSCR patients. (Figure 14A-C) Immunofluorescence-based analysis of explants prepared from the distal colon of P4 HolTg / Tg mice and cultured for 96 h in the presence of GDNF and EdU (+GDNF) or EdU alone (control). Although new HuC / D+ neurons could be induced by GDNF under these ex vivo culture conditions, the total number of neurons per explant was variable (Figure 14A), these neurons, if present, formed only very small ganglia (Figure 14B), and they were less likely to show EdU incorporation than SCPs (see arrowheads in Figure 14B and quantification in Figure 14C) (n = 7 explants per condition; P < 0.05; **P < 0.01; ***P < 0.001; two-tailed Mann-Whitney U test). (Figure 14D-G) Immunofluorescence-based analysis of explants prepared from samples of aganglionic colon resected from human HSCR patients and cultured for 96 h in the presence of GDNF and EdU (+GDNF) or EdU alone (control). In all human explants cultured under these conditions, GDNF treatment leads to robust incorporation of EdU into SOX10+SCPs but not into HuC / D+neurons (Figure 14D-E). Significant numbers of HuC / D+neurons could be induced by GDNF in a subset of explants derived from patients less than 3 months old at the time of surgery (Figure 14G) (Figure 14F) (n = 12 explants per condition; *P < 0.05; two-tailed Mann-Whitney U test). (FIG. 14H) Neurons were detected in human explants from patients ≥3 months old at the time of surgery by extended culture in the presence of GDNF for a total of 7 days, including some neurons that incorporated EdU (arrowheads). All images shown represent z-stack projections at the level of the myenteric plexus. Scale bars, 50 μm (FIGS. 14B and H) and 100 μm (FIGS. 14D and F). Dashed outlines indicate the area occupied by extrinsic nerve fibers. [Figure 15]Figure 15A-B show analysis of neurogenesis and SCP proliferation in distal colon explants prepared from P4 HolTg / Tg mice and cultured for 96 h in the presence or absence of GDNF. Representative images of HuC / D+ neurons (Figure 15A), and EdU+SOX10+ proliferating SCPs (arrows in Figure 15B) in distal colon explants from HolTg / Tg mice cultured in the presence of GDNF and EdU (+GDNF) or EdU alone (control). Images shown are single focal planes representing observations obtained from seven mice. Scale bar, 50 µm. Dashed outline indicates the area occupied by extrinsic nerve fibers. [Figure 16] Figure 16 shows marker analysis of GDNF-induced neurons in sigmoid colon explants prepared from HSCR patients and cultured in the presence of GDNF for 96 hours. Immunofluorescence analysis showing that human GDNF-induced neurons are closely associated with extrinsic nerves and express βIII-tubulin (TuJ1), RET, PGP9.5, and PHOX2B (Supporting Figure 14F). Arrowheads point to the round / ovoid nuclei of PGP9.5+ neurons. Images shown are single focal planes representing observations from three human samples. Scale bars, 100 μm (top panels), 50 μm (middle panels), and 25 μm (bottom panels). [Figure 17] Figure 17A shows the amino acid sequence of human GDNF isoform 1 (UniProtKB Accession No. P39905, SEQ ID NO:1), with the sequence corresponding to the signal peptide (residues 1-19) underlined, the sequence corresponding to the propeptide (residues 20-75) in italics, and the sequence corresponding to the mature polypeptide (residues 78-211) in bold. Figures 17B-C show the nucleotide sequence of a cDNA encoding human GDNF isoform 1 (RefSeq Accession No. NM_000514.4, SEQ ID NO:2), with the sequence encoding the signal peptide (nucleotides 562-618) underlined, the sequence encoding the propeptide (nucleotides 619-786) in italics, and the sequence encoding the mature polypeptide (nucleotides 793-1194) in bold.

[0013] Disclosure of the Invention In the context of describing the technology (particularly in the context of the claims which follow), use of the terms "a" and "an" and "the" and similar referents are intended to be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0014] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise noted.

[0015] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0016] Any and all examples provided herein, or the use of exemplary language (such as, for example, etc.), are intended merely to better illustrate embodiments of the claimed technology, and do not pose a limitation on scope unless otherwise claimed.

[0017] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0018] As used herein, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation for error of the device or method being used to determine the value, or encompasses a value close to the recited value, e.g., within 10% of the recited value (or range of values).

[0019] The recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. Every subset of values ​​within a range is also incorporated herein as if it were individually recited herein.

[0020] Where features or aspects of the disclosure are described in terms of a Markush group or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual components or subgroups of components of the Markush group or list of alternatives.

[0021] Unless specifically defined otherwise, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0022] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), TA Brown (editor), Essential Molecular Biology, A Practical Approach, Volumes 1 and 2, IRL Press (1991), D M Glover and B D Hames (editors), DNA Cloning, A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and FM Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all revised editions to date), Ed Harlow and David Lane (editors), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J E Coligan et al. al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all current editions), and other sources.

[0023] In the study described herein, we show that in a HSCR mouse model, administration of an appropriate dosage of recombinant GDNF in the distal colon via rectal enema can induce the permanent formation of new functional enteric neurons and glia, as well as the restoration of colonic motility, in the aganglionic colon. Gene lineage tracing indicates that SCPs located in extrinsic nerve fibers are one source of these newly generated enteric neurons and glia. It has further been shown that GDNF can stimulate neurogenesis in cultured explants of aganglionic colon from human HSCR patients. Thus, due to its ability to stimulate Schwann cell migration and proliferation in a RET-independent but GFRα1-dependent manner, particularly through its alternative receptor neuronal cell adhesion molecule (NCAM), GDNF appeared to be a primary candidate for the postnatal reactivation of ENS precursors in the aganglionic area. These results provide evidence that administration of recombinant GDNF to the colon (e.g., distal colon) can be used to treat intestinal neuropathy (e.g., ENS deficiencies such as HSCR), i.e., ameliorate one or more of the pathological features of intestinal neuropathy in human patients.

[0024] Thus, in a first aspect, the disclosure provides a method for inducing enteric neurogenesis in aganglionic or hypoganglionic areas of the distal colon of a human subject suffering from an enteric neuropathy (e.g., Hirschsprung's disease (HSCR) or enteric hypoganglionosis), the method comprising administering to the distal colon of the subject a pharmaceutical composition comprising an effective dose of a glial cell line-derived neurotrophic factor (GDNF) polypeptide and a pharma- ceutical acceptable carrier.

[0025] The present disclosure also provides a method for restoring distal colon motility and / or epithelial barrier in a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), the method comprising administering to the distal colon of the subject a pharmaceutical composition comprising an effective dose of a GDNF polypeptide and a pharma- ceutical acceptable carrier.

[0026] The present disclosure also provides use of a pharmaceutical composition comprising a human GDNF polypeptide and a pharma- ceutically acceptable carrier for inducing intestinal neurogenesis in aganglionic or hypoganglionic areas of the distal colon of a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), wherein the composition is for administration to the distal colon of the subject.

[0027] The present disclosure also provides use of a pharmaceutical composition comprising a human GDNF polypeptide and a pharma- ceutical acceptable carrier for restoring distal colon motility in a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), wherein the composition is for administration to the distal colon of the subject.

[0028] The present disclosure also provides use of a pharmaceutical composition comprising a human GDNF polypeptide and a pharma- ceutical acceptable carrier for the manufacture of a medicament for inducing intestinal neurogenesis in aganglionic or hypoganglionic areas of the distal colon of a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), wherein the medicament is for administration to the distal colon of the subject.

[0029] The present disclosure also provides the use of a pharmaceutical composition comprising a human GDNF polypeptide and a pharma- ceutical acceptable carrier for the manufacture of a medicament for restoring distal colon motility in a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), wherein the medicament is for administration to the distal colon of the subject.

[0030] The present disclosure also provides a pharmaceutical composition for inducing intestinal neurogenesis in aganglionic or hypoganglionic areas of the distal colon of a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), the composition comprising a human GDNF polypeptide and a pharma- ceutical acceptable carrier, the pharmaceutical composition being for administration to the distal colon of the subject.

[0031] The present disclosure also provides a pharmaceutical composition for restoring distal colon motility in a human subject suffering from intestinal neuropathy (e.g., HSCR or enteric hypoganglionosis), the composition comprising a human GDNF polypeptide and a pharma- ceutical acceptable carrier, the pharmaceutical composition being for administration to the distal colon of the subject.

[0032] The term "distal colon" as used herein refers to the last three parts of the colon, namely the descending colon, the sigmoid colon, and the rectum. In one embodiment, the pharmaceutical composition is administered to or is for administration to the rectum and / or the sigmoid colon. In one embodiment, the pharmaceutical composition is administered to or is for administration to the rectosigmoid region, which includes the last part of the sigmoid colon and the beginning of the rectum. Those skilled in the art will understand that the pharmaceutical composition can be administered directly to the distal colon, or can be administered to a site distant from the distal colon, but using a suitable means that provides for delivery of the pharmaceutical composition (and more specifically, the human GDNF polypeptide) to the distal colon. For example, the pharmaceutical composition may include a coating that is specifically degraded under the conditions of the distal colon (e.g., pH, enzymatic environment, bacterial environment, etc.), and thus, although the pharmaceutical composition may be administered to other areas of the gastro-intestinal system, the human GDNF polypeptide will only be released when the pharmaceutical composition reaches the colon, and more specifically, the distal colon. Approaches for colon-specific drug delivery are well known in the art (see, e.g., Philip et al., Oman Med J. 2010 Apr;25(2):79-87; Lee et al., Pharmaceutics. 2020 Jan;12(1):68), including pH-dependent systems (e.g., using pH-dependent polymers), receptor-mediated systems, magnetic-driven systems, delayed or time-dependent systems, microbially triggered drug delivery systems (e.g., including sugar-based polymers that can be degraded by enzymes produced by the colonic microflora, such as glucoronidase, xylosidase, arabinosidase, galactosidase, etc.), pressure-controlled colonic delivery capsules (drug release triggered by the higher pressure encountered in the colon), osmotically controlled drug delivery, and any combination of these approaches (e.g., Colon Targeted Delivery System (CODES™), using a combined approach of pH-dependent and microbially triggered drug delivery).

[0033] The term "enteric neuropathy" as used herein refers to diseases associated with abnormalities in the ENS, including abnormal development of the ENS, for example, abnormalities in the number of neurons (hypopangionosis, aganglionosis), and / or abnormal differentiation of neurons. Examples of enteric neuropathy include enteric ganglion cell abnormalities, such as HSCR and enteric hypoganglionosis. In one embodiment, the enteric neuropathy is HSCR. In another embodiment, the enteric neuropathy is enteric hypoganglionosis.

[0034] As used herein, the phrase "inducing enteric neurogenesis" refers to an increase in the production of enteric neurons and / or enteric glial cells compared to before treatment with a composition comprising a human GDNF polypeptide. The enteric nervous system comprises various types of neurons, including enteric primary afferent neurons (EPANs), excitatory circular motoneurons, inhibitory circular motoneurons, longitudinal motoneurons, ascending interneurons, descending interneurons, secretomotor and vasomotor neurons, and intestinofugal neurons, as well as enteric glial cells (EGCs), which provide structural support to neurons and contribute to their maintenance, survival, and function (Costa et al., Gut 2000; (Suppl IV) 47: iv15-iv19; De Giorgio et al., American Journal of Physiology-Gastrointestinal and Liver Physiology, Vol. 303, No. 8: G887-G893, 2012).

[0035] The production of one or more of these cell types may be induced by administration / use of a composition comprising a human GDNF polypeptide. In one embodiment, the production of EGCs, preferably Sox10-expressing EGCs, is induced by administration / use of a composition comprising a human GDNF polypeptide. In one embodiment, the administration / use of a composition comprising a human GDNF polypeptide restores the intestinal neuron / glial cell ratio in the colon (e.g., distal colon) of the patient. In a further embodiment, the intestinal neuron / glial cell ratio in the colon (e.g., distal colon) of the patient is at least 0.5, e.g., 0.5-1.5. In another embodiment, the administration / use of a composition comprising a human GDNF polypeptide restores the intestinal neuron / glial cell ratio in the colon (e.g., distal colon) of the patient, e.g., 0.5-1.5. + ) and cholinergic (ChAT + ) restores the proportion of neurons.

[0036] In another embodiment, administration / use of a composition comprising a human GDNF polypeptide reduces infiltration of inflammatory or immune cells (e.g., neutrophils) in the colon (e.g., distal colon). In another embodiment, administration / use of a composition comprising a human GDNF polypeptide restores (partially or completely) the proportion of immune cells in the colon (e.g., distal colon).

[0037] The term "human GDNF polypeptide" as used herein refers to native mature human GDNF protein, or a functional variant or fragment thereof that retains the biological activity of native mature human GDNF protein, e.g., the ability to bind to GDNF receptors (particularly "rearranged during transfection" (RET) proto-oncogene and / or neuronal cell adhesion molecule (NCAM) receptors) and trigger a signal in cells expressing GDNF receptors (e.g., RET and / or NCAM). The amino acid sequence of native human GDNF protein (isoform 1, canonical sequence) is shown in Figure 17A (SEQ ID NO: 1), with the sequence corresponding to the mature protein (residues 78-211) highlighted in bold. GDNF precursor protein is processed into a mature secreted form that exists as a homodimer. Each GDNF monomer contains seven conserved cysteine ​​residues, including Cys-101, which is used for interchain disulfide bridges, and others that are involved in intermolecular ring formation known as the cysteine-knot configuration.

[0038] In one embodiment, the human GDNF polypeptide is a recombinant human GDNF polypeptide. When referring to a protein or polypeptide, the term "recombinant" refers to a protein or polypeptide molecule that is not isolated from a natural source (e.g., a biological sample), e.g., expressed from a recombinant nucleic acid construct that is produced by means of molecular biology techniques. Thus, referring to a nucleic acid construct as "recombinant" indicates that the nucleic acid molecule is manipulated using genetic engineering, i.e., by human intervention. Recombinant nucleic acid constructs can be introduced into host cells, for example, by transformation (e.g., transduction or transfection).

[0039] A functional variant or fragment of a native mature human GDNF protein may contain one or more amino acid substitutions, deletions, and / or additions compared to the native mature human GDNF protein, and may have a biological activity that is lower, similar, or higher than that of the native mature human GDNF protein. In one embodiment, the functional variant or fragment has an activity that is similar (e.g., 90% to 110%) or higher (e.g., more than 110%) than that of the native mature human GDNF protein. In one embodiment, the variant comprises one or more conservative substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is replaced with another amino acid with the same chemical or physical properties. For example, a conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp-Glu or vice versa), an amino acid having a non-polar side chain substituted for another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid having a polar side chain substituted for another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.). In another embodiment, the variant can comprise the amino acid sequence of a native GDNF protein or polypeptide with at least one non-conservative amino acid substitution. Preferably, the non-conservative amino acid substitution improves the activity of the variant compared to that of the native mature human GDNF protein. In one embodiment, the human GDNF polypeptide has the ability to bind to the RET receptor. In one embodiment, the human GDNF polypeptide has the ability to bind to the NCAM receptor.

[0040] In one embodiment, the human GDNF polypeptide comprises at least 10, 15, or 20 amino acids (e.g., consecutive amino acids) from the mature human native GDNF protein. In one embodiment, the human GDNF polypeptide comprises the sequence ETTYDKILKNLSRNR (gliafin, SEQ ID NO: 3), which corresponds to residues 153-167 of SEQ ID NO: 1 and is the predicted binding domain of human GDNF to the NCAM receptor (see Nielsen et al., J Neurosci. 2009 Sep 9;29(36):11360-11376). In other embodiments, the human GDNF polypeptide comprises at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids (e.g., consecutive amino acids) from the mature human native GDNF protein.

[0041] In one embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 50%, 60%, or 70% identical to residues 78-211 of the sequence shown in Figure 178A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 80% identical to residues 78-211 of the sequence shown in Figure 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 85% identical to residues 78-211 of the sequence shown in Figure 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 90% identical to residues 78-211 of the sequence shown in Figure 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 95% identical to residues 78-211 of the sequence shown in Figure 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 98% identical to residues 78-211 of the sequence shown in FIG. 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises an amino acid sequence that is at least 99% identical to residues 78-211 of the sequence shown in FIG. 17A (SEQ ID NO:1). In another embodiment, the human GDNF polypeptide comprises or consists of residues 78-211 of the sequence shown in FIG. 17A (SEQ ID NO:1). "Identity" refers to the sequence identity between two polypeptides. Identity can be determined by comparing each position in the aligned sequences. Methods for determining percent identity are known in the art, and several tools and programs are available for aligning amino acid sequences and determining percent identity, including EMBOSS Needle, ClustalW, SIM, DIALIGN, and the like.As used herein, a given percentage of identity with respect to a particular subject sequence or a particular portion thereof may be defined as the percentage of amino acids in a candidate derivative sequence that are identical to those in the subject sequence (or a particular portion thereof) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity generated by the Smith Waterman algorithm (Smith & Waterman, J. Mol. Biol. 147:195-7 (1981)) using the BLOSUM substitution matrix (Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992)) as the similarity measure. The "% identity value" is determined by dividing the number of matching identical amino acids by the sequence length for which the percent identity is reported.

[0042] The covalent modification of human GDNF polypeptide is included within the scope of the present disclosure.For example, the natural glycosylation pattern of human GDNF polypeptide can be modified (Beck et al., Curr.Pharm.Biotechnol.9:482-501,2008; Walsh, Drug Discov.Today 15:773-780,2010), and human GDNF polypeptide can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patent No. 4,640,835, U.S. Patent No. 4,496,689, U.S. Patent No. 4,301,144, U.S. Patent No. 4,670,417, U.S. Patent No. 4,791,192, or U.S. Patent No. 4,179,337. Human GDNF polypeptides may contain one or more modifications that confer additional biological properties to the polypeptide, such as protease resistance, plasma protein binding, increased plasma half-life, tissue or intracellular penetration, etc. Such modifications include, for example, fatty acids (e.g., C6-C 18These include covalent attachment of the molecule / moiety to a polypeptide such as ribonuclease, ...

[0043] Human GDNF polypeptide can also be conjugated to one or more therapeutic or active agents (e.g., drugs, or another polypeptide to form a fusion polypeptide).Any method known in the art for conjugating human GDNF polypeptide to another moiety (e.g., active agent) can be used, including those methods described by Hunter et al. (1962) Nature, 144:945, David et al. (1974) Biochemistry, 13:1014, Pain et al. (1981) J.Immunol.Meth., 40:219, Nygren, J.Histochem.and Cytochem., 30:407 (1982), and Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0044] In one embodiment, the effective dose of recombinant GDNF polypeptide administered to or for administration to a human subject corresponds to a dose of about 5 μg to about 20 μg in a mouse pup, a range shown to be effective in the studies described herein. A 10 μl enema containing recombinant GDNF solution was administered to the mouse pup. It is estimated that 10 μl corresponds to the volume required to fill the distal colon and rectum of the pup. Thus, administration of 5 μg of GDNF in a mouse pup is achieved by administering 10 μl of a 0.5 μg / μl GDNF solution, and administration of 20 μg of GDNF in a mouse pup is achieved by administering 10 μl of a 2.0 μg / μl GDNF solution. The volume required to fill the distal colon and rectum of a human infant can be estimated using the following formula: 10 ml x infant's body weight (kg). Thus, a dose of 5 μg GDNF in a mouse corresponds to about 5 mg per kg in a human infant, and a dose of 20 μg GDNF in a mouse corresponds to about 20 mg per kg in a human infant. Thus, in one embodiment, an effective dose of recombinant GDNF polypeptide administered to or for administration to a human subject is about 5 mg to about 20 mg per kg, preferably about 10 mg to about 15 mg per kg. In one embodiment, the recombinant GDNF polypeptide is administered or for administration via a composition (solution or gel) of 0.5 mg / ml to 2 mg / ml.

[0045] Formulations for rectal / distal colon administration may be presented as suppositories, which can be prepared by mixing the subject composition with one or more suitable non-irritating carriers, including, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylate, which are solid at room temperature but liquid at body temperature, and thus will melt in the appropriate body cavity and release the composition containing GDNF.More recently, liquid suppositories have been developed.Liquid suppositories typically contain thermosensitive and / or mucoadhesive polymers, such as poloxamer, Carbopol® (crosslinked polyacrylic acid polymer), sodium alginate, polycarbophil, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose, and methylcellulose.

[0046] Formulations for rectal / distal colon administration may also be presented as enemas, liquid drug solutions, suspensions, or emulsions that are injected into the rectum and distal colon. The liquid in which the GDNF polypeptide is diluted may be, for example, water or a saline solution.

[0047] Formulations for rectal / distal colon administration can also be in the form of rectal foams or gels. Rectal gels are semi-solid formulations that contain a solvent entrapped within a polymer network to create a viscous consistency. The viscosity of the gel can be modified by the addition of co-solvents (e.g., glycerin and propylene glycol) and electrolytes. Foams contain a hydrophilic liquid continuous phase containing a foaming agent and a gaseous dispersed phase distributed throughout. Following rectal administration, they transition from a foam state to a liquid or semi-solid state on the mucosal surface. Foaming agents are typically amphiphilic substances that are important in the generation and stabilization of foams. The molecules contain a hydrophilic component that is soluble in the aqueous phase and a hydrophobic component that forms micelles to minimize contact with the aqueous phase.

[0048] Administration to the rectum / distal colon can be performed using currently available endoscopes or dedicated catheters designed for rectal administration or injection of medications or liquids into the distal colon wall, and can be safely placed and comfortably retained within the rectum for repeated use.

[0049] Compositions comprising GDNF polypeptides can be administered according to any suitable dosing regimen, e.g., four times a day, twice a day, once a day, twice a week, once a week, etc. Treatment can be carried out for any suitable period of time, e.g., one week, two weeks, three weeks or more, to achieve the desired effect.

[0050] In one embodiment, the above-mentioned treatment comprises the use / administration of more than one active / therapeutic agent (i.e., combination), one of which is the above-mentioned pharmaceutical composition comprising GDNF polypeptide. The combination of therapeutic agents and / or compositions can be administered in any conventional dosage form, or can be co-administered (e.g., sequentially, simultaneously at different times). Co-administration in the context of the present disclosure refers to the use of more than one therapy in the course of treatment coordinated to achieve improved clinical outcomes. The pharmaceutical composition comprising GDNF polypeptide as described herein can be used in combination with other therapies or drugs, for example, analgesics or anti-inflammatory agents. The pharmaceutical composition comprising GDNF polypeptide as described herein can also be used in combination with agents that stimulate ENS precursor proliferation, such as neurotrophic molecules.

[0051] In one embodiment, the above-mentioned treatment with a composition comprising a GDNF polypeptide may be performed in combination with surgery (e.g., Swenson, Soave, or Duhamel type pull-through surgery). Especially for newborns with HSCR, clinicians often recommend trying a trial of daily enema treatment before surgery. Adding recombinant GDNF to the enema may increase the chances that children with HSCR will respond well to enema therapy before surgery. Thus, in one embodiment, the above-mentioned treatment with a composition comprising a GDNF polypeptide is performed before pull-through surgery. Saline enemas are also commonly used for children with HSCR after pull-through surgery. It is believed that post-operative problems in HSCR patients are at least partially due to hypoganglionosis remaining in the distal gut, the so-called "passing zone". Thus, adding recombinant GDNF to the enema may be useful to correct the hypoganglionosis in the distal gut remaining after surgery. Thus, in another embodiment, the above-mentioned treatment with a composition comprising a GDNF polypeptide is performed after pull-through surgery.

[0052] In one embodiment, the above-mentioned treatment with a composition comprising a GDNF polypeptide is administered in combination with ENS stem cell-based therapies being considered for the treatment of HSCR, and GDNF may be a useful adjunct to these therapies to promote engraftment.

[0053] HSCR is clinically subdivided into short-range (S-HSCR) and long-range (L-HSCR). S-HSCR, occurring in >80% of cases, refers to the absence of ENS in the rectum and sigmoid colon. L-HSCR refers to the aganglionicity of longer regions of the distal intestine. The methods / uses described herein are for the treatment of human patients suffering from S-HSCR or L-HSCR. In one embodiment, the methods / uses described herein are for the treatment of human patients suffering from S-HSCR. In one embodiment, the methods / uses described herein are for the treatment of human patients suffering from L-HSCR.

[0054] The HSCR patient can be an adult or a pediatric patient. In one embodiment, the HSCR patient is a pediatric patient, preferably a patient under 5, 4, 3, or 2 years old, more preferably a patient under 1 year old or under 6 months old. In another embodiment, the patient is male.

[0055] HSCR is associated with mutations in RET, EDNRB, SOX10, PHOX2B, and ZFHX1B, as well as Down's syndrome or trisomy 21 (collagen VI-associated HSCR). There is also a significant gender disparity, with a male-female ratio as high as 5:1. In one embodiment, the HSCR is collagen VI-associated HSCR. In another embodiment, the HSCR is EDNRB mutation-associated HSCR. In one embodiment, the HSCR is a male-biased HSCR. In one embodiment, the HSCR is a RET mutation-associated HSCR, for example, an HSCR associated with a mutation that reduces RET expression and / or activity in cells from the distal colon. The mutation can be in RET or a protein involved in RET signaling. In one embodiment, the mutation is in the RET protein. Mutations in the RET gene on chromosome 10q11.2 have been shown to account for 50% of familial cases of HSCR and 15-20% of sporadic cases, most of which (about 75%) were associated with L-HSCR.

[0056] In another embodiment, the HSCR is not RET mutation-associated HSCR.

[0057] Mode for carrying out the invention The invention is illustrated in further detail by the following non-limiting examples.

[0058] Example 1: Materials and Methods Mice. Holstein (Tg[Sox3-GFP,Tyr]HolNpln), TashT (Tg[SRY-YFP,Tyr]TashTNpln), and G4-RFP (Gata4p[5kb]-RFP) strains were as previously described (all maintained in the FVB / N genetic background) ( 39 , 40 , 54 ), whereas the Piebald-lethal (Ednrb s-l ; JAX stock #000308; C3H / HeJ-C57BL / 6 mixed background) and Dhh-Cre (Tg[Dhh-cre]1Mejr; JAX stock #012929; FVB / N background) were obtained from The Jackson Laboratory. Other mouse strains used were R26 [Floxed Stop]YFP (Gt[ROSA]26Sor tm1(EYFP)Cos ; provided by F. Costantini (Columbia University, USA) and maintained on an FVB / N background) (80).

[0059] For all enema treatments, mutant pups were identified at P3 via pigmentation-based genotyping. Unless otherwise specified (see Figure 1), 10 μl enemas consisting of a 1 μg / μl solution of recombinant human mature GDNF (Peprotech catalog #450-10) diluted in PBS were administered daily between P4 and P8. Clinical grade GDNF (Medgenesis Therapeutix Inc., Canada) used for some experiments and a previously described 6XHis-tagged version (53) had similar efficiency. Other tested molecules (1 μg / μl solution in 10 μl enema) included serotonin receptor (5-HT4R) agonist RS67506 (R&D Systems, Catalog #0990), Noggin (Sigma, Catalog #SRP4675), Endothelin-3 (Sigma, Catalog #E9137), Serotonin (Sigma, Catalog #H9523), and L-Ascorbic Acid (Sigma, Catalog #A4403). Enemas were administered using a 24-gauge gavage needle (Fine Science Tools, Canada) attached to a micropipette. The head of the gavage needle was introduced into the rectum just beyond the anus (pre-lubricated with Vaseline™) and the enema was injected over a few seconds. Pups were then returned to their mothers and either sacrificed at P20 for tissue analysis or checked daily to follow survival.

[0060] For EdU incorporation assays, mouse pups were given an intraperitoneal injection of 10 μl of 10 mM EdU solution (ThermoFisher Scientific, catalog #C10337) once a day for 5 days (P4–P8) during GDNF enema treatment.

[0061] Tissue processing. All intestinal tissues to be labeled were cut longitudinally along the mesentery, washed with PBS, pinned onto Sylgard-coated Petri dishes, fixed in 4% PFA overnight at 4° C., and finally microdissected to separate the longitudinal / circular muscles (containing the ganglia of intermyenteric neurons) from the submucosa / mucosal layer (containing the ganglia of submucosal neurons). For experiments involving ex vivo analysis of living tissues, the longitudinal / circular muscles and the submucosa / mucosal layers were similarly microdissected but not fixed. Instead, the procedure was performed in ice-cold oxygenated Kreb's solution (0.187 g / L NaH2PO4-2H2O, 6.84 g / L NaCl, 0.35 g / L KCl, 2.10 g / L NaHCO3, 1.98 g / L glucose, 0.368 g / L CaCl2-2H2O, 0.244 g / L MgCl2-6H2O).For histological analysis, PFA-fixed intestinal tissue was prepared as above, but without further microdissection, and fixed full thickness intestinal segments were then embedded in paraffin and sectioned transversely at 10 μm.

[0062] Tissue labeling and imaging. For immunofluorescence staining, microdissected whole tissues were permeabilized in blocking solution (10% FBS and 1% Triton™ X-100 in PBS) for 2 h and then sequentially incubated with specific primary antibodies (overnight at 4°C) and relevant secondary antibodies (2 h at room temperature), both diluted in blocking solution that was also used to wash the tissue between all steps. All antibodies and dilution factors are listed in Table 1. EdU was detected using the Invitrogen Click-iT EdU Imaging Kit (ThermoFisher Scientific, catalog #C10337) according to the manufacturer's instructions. For histological analysis, full-thickness intestinal tissue cross sections were stained with hematoxylin and eosin (H&E) as previously described (83). [Table 1]

[0063] All immunofluorescence images were acquired using either a 20x or 60x objective on a confocal microscope (either Nikon A1R or Zeiss710), except for H&E stained sections, which were imaged using a 10x objective using an Infinity-2 camera (Lumenera Corporation) mounted on a Leica DM2000 microscope (Leica Microsystems Canada). Image analysis was performed with ImageJ, using the "multipoint" function for cell counting and the "polygon selection" function for surface area calculation.

[0064] In vivo and ex vivo analysis of colonic motility. In vivo analysis of distal colonic motility in P20 mice was performed using the bead latency test. Mice were anesthetized with 2% isoflurane and a 2 mm glass bead (Sigma, Cat#1.04014) was inserted into the distal colon over a distance of 0.5 cm from the anus using a probe. Each mouse was then isolated in a mouse cage without food and water and the time required to expel the glass bead was monitored, which was a surrogate for distal colonic transit. The maximum time to expel the bead was 30 min.

[0065] For ex vivo analysis of colonic motility, strips of in vivo muscle from the most distal colon (1 cm from the anus) of P20 mice were prepared as described above and attached longitudinally in a Schuler organ bath (Harvard apparatus) filled with oxygenated Kreb's solution. The muscle strips were first stretched by preloading them with a tension of 2 g for 60 min, and then longitudinal muscle contraction / relaxation was continuously recorded using a myograph (Narco Biosystems Inc., model F-60) attached to a computer with BIOPAC student Lab 4.0.2 software (BIOPAC Systems Inc.). Electric field stimulation (EFS) was applied using a voltage stimulator (BIOPAC Systems Inc., model BSL MP36 / 35) connected to electrodes, using parameters (12 V, 20 Hz, 10 s train duration, and 300 μs stimulation pulse duration) that activate enteric neurons without directly activating the muscle. This procedure was repeated three times with a 10 min washout period between stimulations. To characterize the nitrergic and cholinergic components of the EFS-induced contractile responses, N-nitro-L-arginine methyl ester (L-NAME; Sigma, catalog #N5751) and atropine (Sigma, catalog #A01132) were added to the Krebs solution at final concentrations of 0.5 μM and 1 μM, respectively. The area under the curve (AUC) during each EFS-induced response was measured and data were expressed as ΔAUC (corresponding to the difference between the AUC measured for 20 s after stimulation and the AUC measured for 20 s before stimulation).

[0066] Ex vivo analysis of paracellular permeability. Vivo mucosal areas from the most distal colon (1 cm from the anus) of P20 mice were prepared as described above and separated by 0.5 cm 2The exposed surface area of ​​each well was then attached to an Ussing chamber (Warner Instruments, Model U-9926). Each chamber contained 5 ml of DMEM / F12 medium (Wisent, Cat #319-085-CL), which was maintained at 37°C and continuously oxygenated (95% O2 / 5% CO2). After a 30 min equilibration period, 200 μl of the upper medium was replaced with 200 μl of a 1 mg / ml solution of fluorescein isothiocyanate-conjugated dextran 4 kDa (FD4; Sigma, Cat #60842-46-8). The fluorescence intensity of a 150 μl basolateral aliquot, which reflects paracellular transport from the luminal surface, was then measured every 30 min over a 3 h period using a fluorometer (TECAN, Model Infinite M1000). The fluorescence intensity was finally converted to the amount of FD4 by comparison with a standard curve, and the average value over a 3-h period was used to calculate paracellular permeability, which was expressed as FD4 in ng per mucosal surface area per minute (ng / cm). 2 / min).

[0067] Microbiome analysis. Stool isolation, microbiome sequencing, and data analysis were performed as previously described (38). Briefly, mice were sacrificed at P20 and their feces were collected directly from the colon (3 feces per mouse). Bacterial DNA was then extracted using the QIAamp® Fast DNA Stool Mini Kit (QIAGEN, catalog #51604) and the V5-V6 region of the 16S rRNA gene was PCR amplified with a collection of barcoded primers as previously described (84). Uncorrected sequences generated using an Illumina MiSeq sequencer were aligned and processed using the MOTHUR pipeline (85), and then the biome files were transferred to R (87) to generate graphs of relative taxon abundance and beta diversity using the BIOM package (86).

[0068] Western blot analysis. Organs from P8 or P20 mice were weighed and lysed in RIPA buffer (containing 25 mM Tris-HCL [pH 7.6], 150 mM sodium chloride, 1% Nonidet-P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1x Roche Complete protease inhibitors) using 1 mL per 100 mg of tissue. Samples were then sonicated on ice and centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatants were saved for Western blot analysis. Equal volumes of samples were separated by electrophoresis in 18% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to Immun-blot® PVDF membranes (Bio-Rad, Cat. (1620177). Membranes were then incubated in blocking solution (5% nonfat milk and 0.1% Tween® 20 in TBS) followed by incubation with either mouse anti-GDNF (Santa Cruz Biotechnology, Cat. #sc-13147, 1:500 dilution) or rabbit anti-α-tubulin (Abcam, Cat. #ab176560, 1:70,000 dilution) primary antibodies, then the relevant horseradish peroxidase-conjugated secondary antibodies, all diluted in blocking solution. Each incubation was performed for 60 min at room temperature and interspersed by washing three times with blocking solution. Finally, the membranes were incubated with Immobilon Western Chemiluminescence HRP Substrate (Millipore Proteins were visualized using a Fluorescence Imaging Kit (Sigma, catalog #WBKLS0050) and a Fusion FX imaging system (Vilber).

[0069] Ex vivo time-lapse imaging and culture of mouse aganglionic colon tissue. For time-lapse imaging, P4Hol Tg / TgStrips of viable muscle from the last 1 cm of distal colon from ;G4-RFP double transgenic pups were prepared as described above and pinned onto Sylgard-coated 35 mm ibidi μ-dishes (ibidi, catalog (81156). Muscle strips were cultured in suspension in DMEM / F12 medium (Wisent, catalog #319-085-CL) supplemented with 10% FBS and 100 IU / ml antibiotic-antimycotic with or without 5 μg / ml GDNF under standard culture conditions (37 °C, 5% CO2). After 72 h of culture, each Petri dish was placed in a microscope incubation chamber (Okolab) for 10 h under the same culture conditions and image stacks (250 μm thick) of RFP-labeled extrinsic nerves and SCPs were acquired every 10 min using a 20x objective on a Nikon A1R confocal unit as previously described (39).

[0070] For ex vivo induction of neurogenesis, P4Hol Tg / Tg Strips of viable muscle from the last 1 cm of the distal colon from pups were prepared and cultured as described above, with the notable exception that cultures were performed for longer periods in the presence of 0.5 μM EdU. After 96 h of culture, tissues were fixed in PFA and processed for immunofluorescence and EdU labeling.

[0071] Culture of human aganglionic colon tissue. Human samples of sigmoid colon were obtained from 12 HSCR patients who underwent Swenson-type surgical resection of the aganglionic segment, confirmed by histopathological analysis. Nine patients (seven boys and two girls, age 28-1638 days at surgery) were recruited at the Centre Hospitalier Universitaire Sainte-Justine (Montreal, Canada) and three patients (two boys and one girl, age 300-1177 days at surgery) were recruited at the Children's Hospital of Philadelphia. After surgery, full thickness colon tissue was placed in ice-cold Krebs solution or Belzer UW Cold Storage Solution (Bridge to Life Ltd.) and immediately transported to the relevant laboratory. Strips of viable muscle were then prepared as described above and cut into smaller sections of 0.5 cm × 0.5 cm. At least one of these pieces was immediately fixed and kept for validation of aganglionosis via immunofluorescence, while the others were cultured for 96 h as described above to induce neurogenesis in mouse tissue. Samples from two patients (age 86-1638 days at the time of surgery) were further cultured for 7 days under the same conditions. At the end of the culture period, all tissues were fixed in PFA and processed for immunofluorescence and EdU labeling.

[0072] Profiling of immune cell populations in P20 colon. Holstein mice were treated or not with GDNF by enema (10 μg GDNF (10 μl of 1 μg / μl) administered daily for 5 consecutive days between P4 and P8). On P20, colons of GDNF-treated Holstein and control (untreated Holstein and WT) mice were microdissected, washed in cold RPMI medium to remove feces, cut into small pieces, mechanically dissociated, and filtered (40 μm). Dissociated cells were then collected by centrifugation, resuspended in 1 ml ice-cold RPMI containing 2% FBS, and counted in a hemocytometer. For all experimental and control groups, 1 × 10 6Isolated cells were incubated with a cocktail of 14 fluorescently labeled antibodies specific for either lymphoid or myeloid cell populations for 1 hour on ice, after which quantitative profiling was performed using an LSR Fortessa cytometer (BD Biosciences) and FlowJo software (FlowJo LLC).

[0073] Statistics. All experiments were performed with a minimum of three biological replicates. When relevant, the exact number of independent replicates (n) and the statistical test used to calculate P values ​​are included in the figures and / or legends. P values ​​were determined using GraphPad Prism6, except for microbiome data, which were analyzed with R software (The R Foundation for Statistical Computing, Vienna, Austria).

[0074] Example 2: GDNF enemas rescue aganglionosis in three mouse models of short-zone HSCR Using three mouse models of short-zone HSCR, we first tested whether GDNF enemas in young mice could improve intestinal neuronal numbers and restore function in the distal aganglionic intestine. The strains selected were Holstein (Holstein- ... Tg / Tg ; a model of Trisomy21 [collagen VI]-associated HSCR) ( 39 ), TashT ( TashT Tg / Tg ; a model of male-biased HSCR) (40), and Piebald-Lethal (Ednrb s-l / / s-l The enema volume required to fill the entire colon, the concentration of GDNF homodimer (30 kDa), the treatment time window, and the duration and frequency of therapy were determined using Holt et al. Tg / TgThe survival rate was determined in pups (Figure 1A-D). Administration of 100 μg of GDNF was observed to reduce survival rate compared to saline controls (Figure 1C), suggesting that such a dose was toxic or exacerbated the disease. GDNF administration at 1 μg or 25 μg had no significant effect on survival rate, with 10 or 15 μg shown to be optimal for improving survival. Of note, the treatment chosen (i.e., daily administration of 10 μg of GDNF in PBS as a 10 μl enema for 5 consecutive days between postnatal days [P]4 and P8) did not significantly affect survival in control Holstein mice. Tg / Tg At P28, the maximum age for mouse survival, approximately half of the Hol Tg / Tg GDNF treatment prevented death from megacolon in mice (Figure 2A). Most animals that survived to P28 reached adult age after GDNF treatment, and the mice evaluated were able to breed (two tested breeding pairs were fertile). The few males that were able to survive beyond P56 (adult reference age) eventually died from megacolon at P68, P97, P101, and P180, and females eventually died either from megacolon at P63 or from dystocia at P138 and P250. Importantly, the same GDNF enema treatment also ...B). s-l / / s-l Over 60% of mice (Figure 2B), and all male TashT Tg / Tg The GDNF-treated male TashT mice were compared with controls to determine whether they had a normal immune response, and whether they had a normal immune response. Tg / Tg The mice were kept for over a year and appeared healthy with no signs of tumors or other adverse effects. Saline enemas reduce intestinal inflammation in children with HSCR, so Hol Tg / Tg , Ednr.b. s-l / s-l , and TashT Tg / Tg Separate groups of mice were treated with PBS via enema, but PBS alone did not improve survival (Figure 2A-C). Holstein-Hull endothelin-3 (Hull 100) mice were treated with Noggin homodimer (46 kDa), Endothelin-3 monomer (26 kDa), or the serotonin receptor (5-HT4R) agonist RS67506 (454.41 g / mol) (10 μg each in 10 μl PBS) in PBS. Tg / TgEnema treatment of mice also failed to increase the mean lifespan (Figure 1E). Collectively, these data suggest that GDNF enemas may benefit mice with HSCR-like distal intestinal aganglionosis due to Holstein, TashT, or Piebald-lethal mutations.

[0075] In mutant mouse strains that were clearly GDNF-responsive, variability in response to GDNF enemas was observed (Figure 2A-C). Either by replacing standard chow with a gel diet (Figure 1F) or by combining GDNF with other molecules that have been shown to stimulate ENS precursor proliferation, such as vitamin C (44), serotonin (45), or endothelin-3 (46), GDNF-treated Holter mice were significantly increased in response to GDNF enemas. Tg / Tg Attempts to increase the overall survival rate of the animals were unsuccessful (Fig. 1G). Tg / Tg GDNF enema responsiveness may be in part dependent on intestinal damage or inflammation, which also reduces lifespan in HSCR mouse models without GDNF treatment (47).

[0076] To determine how GDNF enemas improve survival in HSCR mouse models, we tested the hypothesis that the improved survival was driven by postnatal intestinal neurogenesis in distal colonic aganglionosis. Tg / Tg Since mice generally reach the P20 stage without enema treatment, P20 animals were analyzed (Figure 2A). + Myenteric interstitial neurons are abundant in the WT distal colon and express Hol Tg / Tg It was absent from the last cm of the colon (Fig. 2D). + Enteric glia were also abundant in WT myenteric ganglia. Tg / Tg In the distal colon, mainly SOX10 + SOX10 is expressed in thick extrinsic nerve fibers (Fig. 2D) where SCPs are present. +Notably, GDNF-treated Hol cells Tg / Tg Distal colon from animals contains numerous HuC / D neurons organized in ganglia between the circular and longitudinal smooth muscles. + Neurons and SOX10 + These myenteric ganglia contained glia (Figure 2D). + Tuj1 labeling further revealed that the GDNF-induced neuronal ganglia formed interconnected networks in both the myenteric and submucosal plexuses (Figure 3). Tg / Tg and male TashT Tg / Tg Quantification of myenteric neuron density in the entire colon of mice showed that the GDNF effect was most pronounced in the distal colon (i.e., the final 3 cm), with minor effects in the proximal colon (Figures 2D and 5A-C). Tg / Tg The increased neuronal density in the central colon of mice (Figure 2D) was primarily due to the expansion of pre-existing myenteric ganglia, as evidenced by a higher proportion of large ganglia (i.e., >50 neurons) (Figure 4A). Tg / Tg In the distal colon, a region where mice normally lack any enteric neurons, GDNF-treated Hol Tg / Tg Mice had a mean neuronal density that was 40% of that of WT mice (5.2 ± 2.2% vs. 13.1 ± 3.6%) (Figure 2D). Furthermore, all GDNF-treated Hol Tg / Tg Although the mice had neurons in the distal colon upon isolation, some mice had very low neuronal density (2.3% or less) in the distal colon, and these mice often had megacolon (Figure 1H). Notably, GDNF-treated TashT mice Tg / TgIn males, neuronal density in the distal-most colon increased from 36% (4.7 ± 3.8% vs. 13.1 ± 3.6% neuronal density) to 108% (14.2 ± 4.3% vs. 13.1 ± 3.6% neuronal density) of WT levels (Figure 4B-C), suggesting that in some models it is possible to completely restore normal intestinal neuronal numbers by GDNF enemas.

[0077] Importantly, EdU labeling confirmed that GDNF induced proliferation of neuronal and glial precursors during the 5-day treatment from P4 to P8. Tg / Tg Immunofluorescence staining of the colon revealed abundant EdU in both the myenteric and submucosal ganglia. + HuC / D + (putative neurons) and EdU + SOX10 + (putative glial or neuronal / glial precursors) were revealed (Figures 2E-F and 5A-C). + The percentage of cells varied considerably from ganglion to ganglion (Figure 5C). + Only a few ganglia were complete with neurons, and such ganglia were always very small (i.e., composed of three neurons). Collectively, these results suggest that GDNF enemas induce the proliferation of precursors in the distal colon, with some of the dividing cells clustering into new ganglia expressing neuronal and glial markers.

[0078] Example 3: GDNF-induced ENS is morphologically and functionally similar to WT Hol Tg / Tg Focusing on the model, we next determined to what extent GDNF-induced ENS in the distal colon resembled WT at P20. GDNF-induced myenteric ganglia (mean neuron-to-glia ratio in ENS of 0.77 ± 0.09 neurons / glia) were statistically similar to WT (0.96 ± 0.06 neurons / glia, P = 0.16) (Figure 6A).+ (choline acetyltransferase) and nNOS + The relative proportions of major myenteric neuronal subtypes, including TH (neuronal nitric oxide synthase) neurons, were also highly similar to WT (Figure 6B-C). + (tyrosine hydroxylase) dopaminergic neurons, CalR + (Calretinin) Excitatory motor neuron, VIP + (vasoactive intestinal peptide) inhibitory motor neurons, and SubP + Many other neuronal subtypes were detected, including (substance P) excitatory motor neurons (Figure 6C). Tg / Tg In the mouse proximal and mid-colon (Figure 7A-B), GDNF treatment also corrected the imbalance of nitrergic (increased) and cholinergic (decreased) neuronal subtypes observed upstream of the aganglionic zone in both HSCR mouse models and human patients (38, 48-51).

[0079] To evaluate the function of the myenteric ganglion induced by GDNF at P20, we analyzed colonic motility in vivo using the bead latency test. Tg / Tg Mice did not expel glass beads inserted into the rectum during the 30-min observation period, whereas Holtmann-treated mice did not expel glass beads inserted into the rectum during the 30-min observation period. Tg / Tg A subset of GDNF-treated Holt mice expelled beads at 10–21 min, a time that was slightly slower than that of WT mice (range, 2–8 min) (Figure 6D). Tg / TgAnalysis of neuronal density in mice revealed a robust inverse correlation between the time to expel a bead and neuronal density in the distal colon (Figure 8A). We also assessed colonic motility ex vivo using strips of distal colonic muscularis externa from P20 animals mounted on force transducers in an organ bath. This system allows contractions of WT colonic muscle induced by electric field stimulation to be slightly increased by inhibition of nitric oxide synthase with L-NAME (nitro-L-arginine methyl ester) and then robustly counteracted by inhibition of cholinergic signaling with atropine (a muscarinic receptor antagonist). Similar to the in vivo data, GDNF-treated HoloLens-treated mice showed robust inverse correlation between the time to expel a bead and neuronal density (Figure 8A). Tg / Tg Strips of colon muscle from mice showed one of two distinct response patterns. Some Hol muscles treated with GDNF showed Tg / Tg The colonic response was similar to that of WT (4 / 7 mice), whereas the others were similar to that of untreated Hol Tg / Tg The response was similar to that of the GDNF-responsive and GDNF-unresponsive Holt cells (3 / 7 mice) (Figs. 6E and 8B). Two distinct groups appear to exist, GDNF-responsive and GDNF-unresponsive Holt cells (Fig. 8C). Tg / Tg The mean area under the curve (ΔAUC) of the contractile response was graphed for each mouse individually (Figure 6E). To indirectly test the function of GDNF-induced submucosal ganglia at P20, epithelial permeability to a small fluorescently labeled dextran molecule (FD4) was analyzed in Ussing chambers. Again, GDNF-treated Holter Tg / Tg Colon tissue from mice was cultured in a 10-well plate, with mucosal strips either impermeable to FD4, as in WT tissue, or as control Hol from mice not treated with GDNF. Tg / Tg The tissues either showed two distinct response types, either permeable to FD4 or not ( Fig. 6F ).

[0080] To complement the ENS analysis, we evaluated other HSCR-associated intestinal anomalies. Tg / Tg The mouse colon had thicker smooth muscles and more neutrophils than WT mice, but the GDNF-treated Hol Tg / TgMouse colons were similar to WT (Figures 6G-I and 9A-B). Similarly, fecal microbiome profiling showed that P20Hol Tg / Tg The mouse colon showed dysbiosis, but Hol Tg / Tg The mean abundance of several bacterial genera in the mouse colon (e.g., Desulfovibrio, Escherichia, Helicobate, Mucispirillum, Oscillospira, Parabacteroides, and Sutterella) was indistinguishable from WT after GDNF treatment (Figure 6J). A notable exception was the abundance of Bacteroides, which was significantly higher than that of H. Tg / Tg The beta diversity analysis therefore indicates that the microbial community structure is significantly lower in WT, Hol Tg / Tg , and Hol treated with GDNF Tg / Tg This suggests that it differs between mice ( Fig. 6K ).

[0081] Example 4: SCPs in the extrinsic nerve are targets of GDNF in the aganglionic colon To clarify how GDNF induces intestinal neurogenesis, GDNF distribution in the intestine was first assessed via Western blot, taking advantage of the size difference between recombinant (15 kDa) and endogenous (20 kDa, glycosylated) GDNF proteins in SDS-PAGE gels. Tg / Tg Consistent with the increased epithelial permeability in the distal colon of mice (Figure 6F), recombinant GDNF protein significantly increased the permeability of Holter cells treated with GDNF on P8. Tg / TgRecombinant GDNF enema was detected in the distal colon but not in the proximal colon (Figure 10A). Surprisingly, endogenous GDNF is normally only detected in the ileum, but recombinant GDNF enema caused a robust increase in endogenous GDNF throughout the entire colon (Figure 10A). Consistent with the short half-life of GDNF (34-92 h) (52), recombinant GDNF was no longer detectable in the colon or any other tissues at P20, supporting the idea that the administered GDNF was acting primarily during the treatment period (Figure 11). To test for possible GDNF autoregulatory loops and to assess the precise location of recombinant GDNF during treatment, Holt et al. Tg / Tg GDNF treatment of mice was repeated, but this time with GDNF( His A time course analysis of the distal colon 2 h after GDNF treatment on P4, P6, and P8 revealed that Hol Tg / Tg We found that recombinant GDNF accumulated over time in the colonic submucosa (Figure 10B), smooth muscle, and a subset of enteric neurons (Figures 10C and 12) in mice. Interestingly, levels of the main GDNF receptor, RET, were also increased (Figures 10B and 12), supporting the hypothesis that a GDNF-RET autoregulatory loop may be activated in GDNF-treated colons and enhance RET signaling even if initial RET levels are low (e.g., in patients with mutations that reduce RET levels and / or activity). Notably, His Both GDNF and RET were upregulated in Hol Tg / Tg Mouse (Figure 10C) and Ednrb s-l / s-l It was detected in neurons induced near the extrinsic nerve in mice.

[0082] We next assessed whether nerve-associated Schwann cells might be ENS precursors that target GDNF. To test this hypothesis, we first Tg / TgWe used live explants of the distal colon muscularis externa from G4-RFP double transgenic pups to assess the response of Schwann cells in extrinsic nerve fibers to GDNF. In these mice, neural crest inducers, including SCPs, are marked by RFP fluorescence (54). Time-lapse imaging of explants after 72 h of culture in the presence or absence of GDNF (5 μg / ml) suggested that GDNF stimulates both the migration and proliferation of SCPs along the extrinsic nerve (Figure S10D). The effect of GDNF on the proliferation of these SCPs was confirmed via immunofluorescence after 96 h of ex vivo culture. Ki67 in the extrinsic nerve + SOX10 + Double-positive cells tripled upon exposure to GDNF (5.5 ± 0.4% without GDNF vs. 15.2 ± 2.1% with GDNF) (Figure 10E,F). Some of the GDNF-induced neurons expressing RET are derived from Schwann cells, but RET may not be required to activate these precursors in the aganglionic intestine, since GDNF signaling in Schwann cells is instead mediated by NCAM (35). In line with this, the data reported in Figure 10I show expression of NCAM, but not RET, in Schwann cells of extrinsic nerves in the aganglionic mouse colon. These results suggest that GDNF therapy can be used even in patients with reduced RET levels and / or activity, e.g., those with mutations in RET or the RET signaling pathway.

[0083] To further demonstrate that the SCP is the source of GDNF-induced neurons and glia, we transfected the Schwann lineage-specific Cre driver-transgene Dhh-Cre and the Rosa26Cre reporter allele R26 [Floxed Stop]YFP We used in vivo genetic lineage tracing using the Holstein [FVB / N] mutant background. Tg / + ;R26 YFP / + and Hol Tg / Tg ;Dhh-Cre Tg / + ;R26 YFP / +Animals were cultured using a 3-fold increase in YFP expression in the proximal and mid-colon. + There were lower than expected numbers of neurons and glia derived from the SCP. In a mixed C57BL / 6-129Sv genetic background at 1 month of age, the SCP was previously reported to contribute 20% of neurons in the colon (29), but in a pure FVB / N genetic background at P20, it was found to contribute only 5–7% of myenteric neurons, and this contribution increased to 10–11% in the presence of the homozygous Holstein mutation (Figure S13A,B). Notably, the SCP-derived (YFP + ) cells were treated with GDNF Tg / Tg ;Dhh-Cre Tg / + ;R26 YFP / + These neurons constituted 34% of the neurons in the distal colon in Dhh animals (Figure 10G,H). Based on their cellular origin (YFP fluorescence) and / or EdU incorporation, we identified four subgroups of myenteric neurons that were induced by daily administration of EdU during GDNF treatment from P4 to P8 (Figure 10G,H, and 14C). + Although we confirmed that the SCP is the source of GDNF-induced neurons and glia in both the myenteric (Fig. S10G,H) and submucosal (Fig. S13C) plexuses, we also reveal that the majority of induced neurons (66%) were YFP negative, suggesting a non-SCP origin (i.e., non-Dhh-expressing cell type). Furthermore, the majority of induced neurons (62%), regardless of cellular origin, did not incorporate EdU (Fig. S10G,H), raising the possibility that neurogenesis could result from trans-differentiation (i.e., direct differentiation of a postmitotic cell into another type of specialized cell) instead of requiring proliferation of progenitor cells.

[0084] Example 5: GDNF can induce new neurons ex vivo in human aganglionic colon To test whether GDNF can induce new enteric neurons in human tissue, an ex vivo model was needed. GDNF treatment for 96 hours ex vivo increased the number of enteric neurons in all Holt cells. Tg / TgAlthough we induced neurons in distal colon aganglionic tissue, we found that neurogenesis was much less efficient than in vivo. Only 3-67 neurons were found in these 1 cm long explants (Figures 14A and 15A). Furthermore, induced neurons rarely clustered into ganglia, and such ganglia were always very small (Figure 14B). In sharp contrast to the widespread EdU incorporation into SCP (Figures 14C and 15), EdU incorporation into induced neurons was also minimal (less than 20% HuC / D in explant cultures). + Cells) (Figure 14B, C). P4Hol cells cultured without GDNF Tg / Tg Control aganglionic tissue from mice remained completely devoid of neurons (Figs. 14A, C, and 15), supporting the idea that GDNF can induce neurogenesis ex vivo in the aganglionic distal colon, but with less efficiency than that which occurred with GDNF enemas in vivo.

[0085] Finally, we tested whether GDNF could induce neurogenesis in aganglionic human colonic muscle from children who underwent Swenson pull-through surgery to resect the aganglionic distal intestine. The cohort consisted of 12 children. The epidemiological characteristics were typical of HSCR (i.e., mostly sporadic, male predilection, short-segmented) (Table 2). Patients who underwent Soave surgery were not enrolled because this approach does not resect the muscularis externa from the most distal colon. For each subject, full-thickness aganglionic colon resected on the day of surgery was collected and immediately microdissected to remove the mucosal and submucosal layers. The remaining muscularis externa was cut into small pieces and cultured with or without GDNF. Exposure to GDNF for 96 h increased neurogenesis in 9 / 9 human tissues with EdU added to the medium. +The proportion of SCPs was significantly increased (Figure 14D,E). Most importantly, new neurons expressing HuC / D, βIII-tubulin (Tuj1), RET, PGP9.5, and PHOX2B were also detected in three HSCR explants (Figure 14F,G, and 16). These three explants were from the youngest (28-44 days of age) children in our cohort (Figure 14G and Table 2). Two of these children had sporadic HSCR with unknown genetic causes. The third child had a RET mutation associated with MEN2A syndrome (Table 2). For slightly older children, we tested whether longer GDNF treatment could be beneficial. Very interestingly, by culturing distal colons from slightly older children for 7 days (n=2; 86 and 1638 days of age at the time of surgery), neurons that incorporated EdU in association with extrinsic nerve fibers were identified (Figure 14H). Collectively, the data provide compelling evidence that observations from mice can be extended to humans. [Table 2]

[0086] Example 6: Effects of GDNF on immune cell populations in the colon of Holstein mice Multicolor flow cytometry analysis revealed that untreated Hol Tg / Tg The abnormal proportions of immune cells observed in the colon of mice are generally normalized to the proportion of WT immune cells upon GDNF treatment. For the majority of markers, density plots show complete rescue (B cells, CD73 + T cells, CD44 + CD62L + T cells, RORy + T cells, CX3CR1 + MHC-II + macrophages), or at least partial rescue (CD4 + T cells, CD39 + T cells, CD25 + T cells, Ly-6C + CD64 + Monocytes, F4-80 +MHC-II + Macrophage, CD11b + CD103 - The results are summarized in Table 3. [Table 3]

[0087] While the present invention has been described herein with reference to specific embodiments above, modifications may be made without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term that is substantially equivalent to the phrase "including but not limited to." The singular forms "a," "an," and "the" include the corresponding plural referents unless the context clearly dictates otherwise.

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Claims

1. A pharmaceutical composition for inducing enteric neurogenesis in aganglionic or poorly ganglionated areas of the distal colon of a human subject suffering from enteric neuropathy, comprising a recombinant glial cell line-derived neurotrophic factor (GDNF) polypeptide and a pharma- ceutical acceptable carrier, said composition being for administration to the distal colon of said subject, and wherein the dose of recombinant GDNF polypeptide administered is from about 10 mg to about 15 mg per kg.

2. The pharmaceutical composition for use according to claim 1, wherein the GDNF polypeptide comprises an amino acid sequence having at least 90% identity to amino acids 78-211 of SEQ ID NO:

1.

3. The pharmaceutical composition for use according to claim 2, wherein said GDNF polypeptide comprises an amino acid sequence having at least 95% identity to amino acids 78-211 of SEQ ID NO:

1.

4. The pharmaceutical composition for use according to claim 3, wherein the GDNF polypeptide comprises amino acids 78-211 of SEQ ID NO:

1.

5. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the recombinant GDNF polypeptide is at a concentration of 0.5 mg / ml to 2 mg / ml.

6. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein said pharma- ceutically acceptable carrier is a saline solution or a gelling agent.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, wherein said pharmaceutical composition is for rectal administration via an enema.

8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein said pharmaceutical composition is for administration by injection into the distal colon wall.

9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein said pharmaceutical composition is for administration once a day up to four times a day.

10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein said pharmaceutical composition is for administration on at least two consecutive days.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the pharmaceutical composition is for use prior to surgical removal of the aganglionic or poorly ganglionated area in the subject.

12. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the pharmaceutical composition is for use after surgical removal of the aganglionic or poorly ganglionated area in the subject.

13. 13. The pharmaceutical composition for use according to claim 11 or 12, wherein the surgical removal of the aganglionic or poorly ganglionated areas is through a pull-through procedure.

14. The pharmaceutical composition for use according to any one of claims 1 to 13, wherein the intestinal neuropathy is enteric hypoganglionosis.

15. The pharmaceutical composition for use according to any one of claims 1 to 14, wherein said intestinal neuropathy is Hirschsprung's disease (HSCR).

16. 16. The pharmaceutical composition for use according to claim 15, wherein the subject is suffering from short-range HSCR.

17. 17. The pharmaceutical composition for use according to claim 15 or 16, wherein the HSCR is sporadic HSCR.

18. The pharmaceutical composition for use according to any one of claims 15 to 17, wherein said HSCR is associated with a reduced expression or activity of the RET receptor.

19. The pharmaceutical composition for use according to claim 18, wherein said HSCR is associated with a mutation in the RET gene.

20. The pharmaceutical composition for use according to any one of claims 1 to 19, wherein said enteric neurogenesis comprises the production of enteric neurons and / or enteric glial cells.

21. 21. The pharmaceutical composition for use according to claim 20, wherein said enteric neurogenesis comprises the production of enteric neurons and enteric glial cells.

22. 22. The pharmaceutical composition for use according to claim 20 or 21, wherein the production of enteric neuronal and / or glial cells comprises proliferation of enteric neuronal and / or enteric glial precursors.

23. 23. The pharmaceutical composition for use according to any one of claims 1 to 22, wherein the pharmaceutical composition (a) corrects an imbalance in nitrergic and cholinergic neuronal subtypes located upstream of the aganglionic or poorly ganglionic areas, (b) restores distal colon motility in the subject, and / or (c) restores the proportion of lymphoid and / or myeloid immune cells in the distal colon of the subject.

24. The pharmaceutical composition for use according to any one of claims 1 to 23, wherein the human subject is under 5 years of age.

25. 25. The pharmaceutical composition for use according to claim 24, wherein the human subject is under 6 months of age.

26. A pharmaceutical composition for use according to any one of claims 1 to 25, wherein said pharmaceutical composition is for administration to the rectum and / or sigmoid colon.

27. 27. The pharmaceutical composition for use according to claim 26, wherein said pharmaceutical composition is for administration to the rectosigmoid region.

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