CN-122003495-A - Mesoderm angioblast composition

Abstract

Disclosed is a mesodermal angioblast composition, wherein the Mesodermal Angioblasts (MAB) are engineered to express truncated collectins.

Inventors

• Giulio Kosu
• Francesco Gali

Assignees

• 曼彻斯特大学

Dates

Publication Date

20260508

Application Date

20240821

Priority Date

20230822

Claims (20)

1. 1. A mesodermal angioblast composition, wherein the Mesodermal Angioblast (MAB) is engineered to express a truncated collectin.
2. 2. The composition of claim 1, wherein the donor from which the MAB is derived is: (a) Subject suffering from muscular dystrophy, or (B) Healthy subjects.
3. 3. The composition of claim 2, wherein the muscular dystrophy is muscular dystrophy characterized by abnormal calcium homeostasis.
4. 4. The composition of any one of the preceding claims, wherein the truncated collectin is a truncated collectin having a length of 4-40% of the length of the natural collectin based on the number of amino acid residues.
5. 5. The composition of any one of the preceding claims, wherein the truncated collectin is a truncated collectin comprising from 1 to 12 domains each derived from a domain present in a native collectin.
6. 6. The composition of claim 5, wherein each of said one or more domains of said truncated collectin has at least 90% sequence identity to a corresponding domain present in a native collectin.
7. 7. The composition of claim 5 or 6, wherein each of the plurality of domains is derived from a domain present in the C-terminal portion of the native collectin.
8. 8. The composition of any one of the preceding claims, wherein the truncated collectin is an A0B0 collectin isoform.
9. 9. The composition of any one of the preceding claims, wherein the truncated collectin: (a) Is capable of binding to alpha-dystrophin proteoglycans; (b) Failure to induce aggregation and/or phosphorylation of acetylcholine receptor (AChR), and/or (C) Cytoplasmic calcium spikes can be reduced or eliminated when expressed in DMD muscle fiber cultures.
10. 10. The composition of any one of the preceding claims, wherein the MAB is engineered to additionally express small nuclear RNA (snRNA) capable of inducing exon skipping in a dystrophin pre-mRNA transcript.
11. 11. The composition of claim 10, wherein the snRNA is a modified uridine 7 (U7) -enriched snRNA.
12. 12. The composition of claim 11, wherein the snRNA sequence consists of the RNA sequence of SEQ ID No.1 or comprises the RNA sequence of SEQ ID No. 1.
13. 13. The composition of any one of claims 10 to 12, wherein the snRNA comprises a base sequence that specifically hybridizes to a region of a dystrophin pre-mRNA transcript, thereby inducing skipping of one or more dystrophin exons.
14. 14. The composition of claim 13, wherein the snRNA induces skipping of dystrophin exon 51.
15. 15. The composition of any one of claims 10 to 14, wherein the MAB is engineered to express a plurality of different snrnas.
16. 16. A composition according to any one of claims 1 to 15 for use as a medicament.
17. 17. The composition of claim 16 for use in the treatment of muscular dystrophy.
18. 18. The composition for use of claim 16 or 17, wherein the composition is self-administered.
19. 19. The composition for use of any one of claims 16 to 18, wherein the composition is in the form of an injectable cell suspension.
20. 20. A method of producing the composition of any one of claims 1 to 19, comprising transducing a MAB in vitro with a vector expressing a truncated collectin.

Description

Mesoderm angioblast composition Technical Field The present invention relates generally to ex vivo gene therapy, and in particular to compositions comprising genetically engineered mesodermal angioblasts (mesoangioblast), methods of producing the same, and uses for treating muscular dystrophies, including duchenne muscular dystrophy (Duchenne Muscular Dystrophy, DMD) and behcet muscular dystrophy (Becker Muscular Dystrophy, BMD). Background Muscular Dystrophy (MD) is a genetic disease that affects skeletal muscle and often the heart muscle. They vary greatly in terms of age of onset, severity and the muscle groups that are predominantly affected. They all lack effective therapies and steroids represent the only treatment capable of delaying the progression of the disease, despite serious side effects. Duchenne Muscular Dystrophy (DMD) is one of the most common and most severe muscular dystrophies, affecting about one-fourth of newborns. It is characterized by progressive atrophy of skeletal and cardiac muscles, resulting in variable but progressive muscle weakness, which limits patient mobility and affects cardiac and respiratory functions over the following years. DMD is caused by different mutations in the dystrophin gene located on the X chromosome. In 90% of cases, the mutation results in a change in the reading frame of the mRNA, thereby preventing dystrophin production. The in-frame deletion produces a shorter but partially functional dystrophin protein, which is associated with milder Belleville Muscular Dystrophy (BMD). Dystrophin and proteins associated with dystrophin-related glycoprotein complexes (e.g., myoglycans) have a critical role in the interaction of muscle cells with the basement membrane and provide elastic resistance to the muscle membrane during contraction. In the absence of dystrophin and related proteins, the membrane is more susceptible to damage, resulting in calcium influx. This influx can be readily measured by dyes such as Fura2 that become fluorescent when bound to calcium ions. Depending on the duration of the inflow, this may appear as a spike in fluorescence or as a more sustained rise. If unregulated, calcium influx leads to excessive shrinkage, proteolysis and fibrosis. After myofibrosis, satellite cells, resident myogenic stem/progenitor cells, and, to a minimum, mesenchymal cells such as pericytes are regenerated. In humans, adult myogenic cells have limited self-renewal potential, and in DMD, the continuous regenerative cycle ultimately leads to cell population depletion. Muscle degeneration is accompanied by chronic inflammation, which progressively results in the accumulation of dense connective and adipose tissue that replaces muscle fibers, rendering any therapy ineffective at this stage. After muscular dystrophy proteins have been cloned for more than 30 years, there is still no cure for DMD. Many therapeutic approaches have entered the clinical field, but none have achieved significant and long-lasting clinical efficacy. Thus, there is a considerable clinical need for improved treatment methods for DMD and associated muscular dystrophy. Disclosure of Invention The inventors found that Mesodermal Angioblasts (MAB) expressing truncated collectin block calcium spikes in a mouse model of muscular dystrophy. Thus, according to a first aspect of the invention there is provided a Mesodermal Angioblast (MAB) composition wherein the MAB is engineered to express a truncated collectin (truncated agrin protein). Furthermore, the inventors have found that expression of micronuclear RNAs (snrnas) in MABs induces exon skipping in dystrophin pre-mRNA transcripts. Notably, the inventors have unexpectedly found that when used in combination, this strategy provides synergistic therapeutic effects within a therapeutically effective range, resulting in rich dystrophin protein production. Thus, in some embodiments, the MAB is engineered to additionally express snRNA capable of inducing exon skipping in the dystrophin pre-mRNA transcript. Thus, in these embodiments, the MAB is engineered to express: (i) Truncated collectin, and (Ii) Small nuclear RNAs (snrnas) capable of inducing exon skipping in dystrophin pre-mRNA transcripts. Mesoderm angioblast MAB is a progenitor cell initially identified in the dorsal aorta of the developing embryo. They also exist as vascular-related cell populations in post-partum skeletal muscle. MAB has properties similar to those of adult pericytes and may represent an in vitro derivative of adult pericytes. When derived from embryonic tissue (including skeletal and cardiac muscles, bones and cartilage), they are capable of differentiating into several mesodermal lineages. Human skeletal muscle-derived MAB is defined by the expression of the pericyte markers NG2 proteoglycan and alkaline phosphatase (ALP) and the absence of endothelial markers. This makes it promising for isolation from freshly dissociated alp+ cells. Its differentiation potential is limite