Dystrophinexosome complex for the treatment of Duchenne muscular dystrophy (DMD).

Abstract

The invention relates to an exosome complex for use in the treatment of muscular dystrophy. In this dystrophin-exosome complex, the exosome acts as a carrier and contains a promoter with a nucleotide sequence of No. 1, the complete sequence of the dystrophin protein with a nucleotide sequence of No. 2, and a poly-α tail with a nucleotide sequence of No. 3 suitable for this complex. The muscular dystrophies treated by this invention are Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). Furthermore, the biomarkers MMP-2 and TIMP-2 are added to the surface of the exosome complex. Thanks to these biomarkers, the exosome complex targets muscle cells.

Description

1 TARIFF Dystrophin for the Treatment of Duchenne Muscular Dystrophy (DMD) EXOSOME COMPLEX Technical Field to Which the Invention Relates 5 The discovery involves a dystrophin-exosome complex that treats muscular dystrophy. It is related. The dystrophin-exosome complex in question is a promoter, the complete sequence of the dystrophin protein. It includes a sequence and a poly-a tail. Additionally, it has a carrier function within this complex. Muscle cells with MMP-2 and TIMP-2 biomarkers added to the exosome surface 10 This is the goal. The muscular dystrophies that the invention treats are Duchenne Muscular Dystrophy. These are dystrophy (DMD) and Becker muscular dystrophy (BMD). This complex enters the cell... DMD protein expression is achieved through its transport. State of the Art 15 Duchenne Muscular Dystrophy (DMD) is caused by a gene located on the X chromosome. a rare disease characterized by progressive muscle degeneration resulting from mutations It is a genetic disease and largely affects male children. Patients In most cases, symptoms appear between the ages of 2 and 5, and early motor development begins in 20 years. squamous cellulitis, frequent falls, difficulty climbing stairs, and Gowers maneuver (when getting up from the ground). Symptoms such as supporting oneself by placing hands on legs are observed. 10 to 12 years old. Around this time, patients become dependent on wheelchair use and progress In older age, respiratory support is needed due to weakening of the respiratory muscles. It can be heard. Heart muscle involvement (cardiomyopathy) is common and progresses through the disease in 25 years. In its later stages, DMD becomes one of the leading causes of morbidity and mortality. The life expectancy of patients has increased significantly in recent years with supportive treatments. With prolonged and advanced medical care, it is possible for patients to live into their 30s. However, patient profiles, individual genetic variations, and treatment have become relevant. It may vary depending on the quality of response and supportive care. Another muscle 30 Becker muscular dystrophy (BMD), which involves the development of dystrophy, is also a disease similar to DMD. Unlike DMD, in BMD dystrophin production is not completely lost, and the symptoms remain the same. Although milder, it generally appears at a later age. Symptoms of BMD symptoms such as muscle weakness, difficulty walking, difficulty climbing stairs, and frequent falls. 2 Symptoms include those affecting the hip and thigh muscles in particular, which can impact patients' daily lives. It can restrict their activities. Duchenne Muscular Dystrophy (DMD) is vital for the structural integrity of muscle cells. A progressive muscle disease resulting from a deficiency of the important dystrophin protein. It is a genetic disease characterized by degeneration. Treatment approaches, It aims to slow the progression of the disease and improve quality of life. Glucocorticoids, particularly prednisone and deflazacort, are used to increase muscle strength and respiratory function. It is effective in improving its functions [1]. However, side effects may occur with long-term use. Effects can be observed. In recent years, significant progress has been made in the field of gene therapy. It has been recorded. Delandistrogene moxeparvovec (Elevidys), adeno-associated viral vector It is a gene therapy based on a functional version of the dystrophin protein. This enables its production and is expected to be approved by the US Food and Drug Administration (FDA) in 2023. It has been confirmed [2]. In addition, antisense oligonucleotide therapies target specific exons. It promotes the skipping of the initial phase, thereby enabling the production of a functional dystrophin protein. 15 For example, eteplirsen targets the skipping of exon 51 and is used for specific DMD mutations. It has been confirmed [3]. Similarly, golodirsen (Vyondys 53) targets exon 53 and It received FDA approval in 2019 [4]. Casimersen (Amondys 45) skips exon 45. It provides and was approved in 2021 [5]. Multidisciplinary approaches, physical therapy, cardiac and supportive treatments for symptoms such as respiratory support, along with 20 patients It can significantly increase lifespan and quality of life. Although they both result from mutations in a protein on the same chromosome, for DMD The use of currently available gene therapies for BMD is limited. Becker Although there is no cure for Muscular Dystrophy (BMD), the disease affects 25% of the population. various treatment methods to slow its progression and improve quality of life Physical therapy and regular exercise programs are applied to maintain muscle strength and Corticosteroids are among the basic approaches to preventing joint stiffness. (Prednisone and Deflazacort) are used carefully to support muscle function. It can be used. Since myocardial involvement is common in BMD patients, regular 30 cardiological follow-up and heart treatment with medications such as beta-blockers or ACE inhibitors. Preserving its functions is of great importance. Experimental treatment methods These include gene therapies, exon-skipping, and myostatin inhibitors. Research is ongoing, and these treatments are not yet in routine clinical use. 3 Generally, the treatment process, carried out through a multidisciplinary approach, addresses the disease. by minimizing its effects, it helps patients lead more independent lives. It provides. Current treatments for Duchenne Muscular Dystrophy (DMD) slow disease progression by 5 years. Although significant steps have been taken to slow the spread, there are some limitations. For example, Elevidys (delandistrogene moxeparvovec) is a treatment for DMD patients aged 4 years and older. It has been developed as a promising gene therapy. However, this treatment is expensive. Therefore, it is not accessible to many families. Furthermore, the long-term effectiveness of the treatment and... More research is needed regarding its safety. Similarly, 10 Eteplirsen (Exondys 51) treatment is suitable for the exon 51 skipping in the DMD gene. It is designed for patients with mutations, and this includes approximately all DMD patients. It covers 14%. Treatment options are available for those outside this limited patient group. It does not offer any. In addition, debates continue regarding the clinical efficacy of eteplirsen. However, some studies have failed to demonstrate a significant clinical benefit. Exon 15 Skipping therapies are aimed at patient groups with specific mutations and broad DMD. It is not suitable for the entire patient population. The limitations and inadequacies of current muscular dystrophy treatment methods, DMD and They offer symptomatic treatment or treatment without providing a definitive solution for BMD. During this time, patients develop an immune response and therefore continue treatment. additionally, existing treatments are expensive and have age restrictions. These shortcomings have made it necessary to develop an alternative treatment. Brief Description and Objectives of the Invention 25 The invention involves an exosome complex for use in the treatment of muscular dystrophy. It is related to this. In this dystrophin-exosome complex, it acts as an exosome carrier and It contains a promoter with nucleotide sequence No. 1 and nucleotide sequence No. 2. to synthesize the dystrophin protein with the complete sequence of the protein and this The complex contains a poly-α tail with a sequence of 3 nucleotide units. The muscular dystrophies that the invention treats are Duchenne Muscular Dystrophy (DMD) and Becker's Dystrophy. It is Muscular Dystrophy (BMD). Also, amino acid sequence number 4 is attached to the surface of the exosome complex. 4 MMP-2 with amino acid sequence and TIMP-2 with amino acid sequence No. 5. Biomarkers have been added. Thanks to these biomarkers, the exosome complex affects muscle tissue. It targets their cells. The aim of the invention is to target specific 5-distrophin proteins that are mutated in DMD disease. Instead of limiting treatments such as skipping exons, the function of the entire protein is addressed. It provides a treatment that targets the cell. Thanks to this treatment, there are no age limits or... without limitations such as restoring the function of only the targeted exon. A gene therapy is provided. Furthermore, it can be produced using a DNA printer. It is low-cost and can be produced quickly. 10 Another aim of the invention is to provide treatment for BMD, a different type of muscular dystrophy. This is the key point. Unlike DMD, dystrophin production does not completely disappear in BMD. It is produced in smaller quantities. Therefore, although the symptoms are milder, they are definitely less severe. There is no solution. Thanks to the dystrophin-exosome complex that is the subject of the invention, 15 BMD is also treatable. Explanation of the Figures Figure 1: Muscle tissue, A) Negative control group from a DMD patient, B) Normal A) Positive control group obtained from mice and C) D2mdx 20 obtained after full dystrophin treatment. This is an image of the striated muscle tissue of the tongue muscle of a transgenic mouse. H&E, Bar: 50μm Figure 2: Muscle tissue, intracytoplasmic dystrophin expression (FITC) and γ- in myocytes. Sarcoglycan expression (Texas Red), IF, Bar: 50μm 25 Figure 3: Results of the experiment conducted to measure the effect of tail length A) 50 polyA, B) 140 polyA, C) 138 polyA, D) 220 polyA and E) 300 polyA Detailed Description of the Invention The invention is a dystrophin-exosome 30 for use in the treatment of muscle diseases. It is related to the complex. This complex contains cells isolated from mesenchymal stem cells. 5 Exosomes are used as carriers. Within these exosomes, Sequence No. 1 A promoter with a nucleotide sequence, dystrophin with nucleotide sequence No. 2. The complete sequence of the protein and a poly-A tail with Sequence No. 3 nucleotide sequence. It is located there. This complex refers to Duchenne Muscular Dystrophy (DMD). It provides treatment. Exosomes used as carriers are mesenchymal stem cells. 5 It is obtained from their cells. The invention concerns dystrophin, which is used in the treatment of muscular system diseases. exosome complex:  A promoter with nucleotide sequence number 1, 10  Full dystrophin sequence with nucleotide sequence number 2,  and a poly-α tail with a nucleotide sequence No. 3. It includes. Additionally, the invention involves the application of Sequence No. 4 15 to the surface of the exosome complex. MMP-2 with amino acid sequence No. 5 and TIMP-2 with amino acid sequence No. 5. Biomarkers have been added, and thanks to these biomarkers, the carrier exosome can be placed in the muscle tissue. This allows the targeting of muscle cells. After targeting muscle cells... The exosome complex enters the target cell and induces dystrophin expression. The complex in question carries the complete sequence of dystrophin protein Sequence No. 2 and cell 20 by enabling its expression, it causes a reaction in the gene encoding this protein. Duchenne Muscular Dystrophy (DMD) is a disease caused by mutations. It provides treatment. The invention also treats Becker Muscular Dystrophy (BMD). It is also used in treatment. 25 Preparation of the dystrophin-exosome complex: i. Initiation of dystrophin production in a laboratory setting using plasmids, ii. Production of exosomes from stem cells, ii. MMP2 with amino acid sequence No. 4 and MMP2 with amino acid sequence No. 5. Addition of TIMP2 markers onto the exosome, 30 It includes the steps involved in the process. 6 Production of dystrophin-exosome complex using DNA printer: i. Conversion of the produced plasmid into mRNA using a plasmid printer, ii. transport of converted mRNA to exosomes via electroporation, iii. obtaining the dystrophin exosome complex It includes the steps of the process. 5 Dystrophin-exosome complexes can be produced quickly thanks to their ability to be manufactured with a DNA printer. It can be done, and since the cost will be low, it is an accessible treatment. It provides. 10 Figure 1 / a shows muscle tissue from the negative control group, i.e., the group carrying the disease. A microscopic image is given. Figure 1 / b shows an image taken from a mouse in the positive control group. A microscopic image of the muscle tissue is given. Figure 1 / c shows the image after treatment. Then, a microscopic image of D2mdx mouse muscle tissue is given. Figure 1 / a In the absence of dystrophin in muscles, cellular boundaries are lost and muscle tissue becomes inflamed. and edema is observed. In Figure 1 / b, muscle tissue in normal muscle tissue is shown. The cell boundaries are well-defined, and the anatomical appearance of the fibers appears normal. (Figure) In 1 / c, dystrophin-deficient muscle tissue transforms into normal tissue after treatment. They are similar and appear to be structurally identical. 20 We kindly request that you provide us with the technical descriptions for Figure 2. Figure 2... Dystrophin was induced to emit light using FITC dye. The amount of light emission was directly proportional to the amount of dystrophin. It is proportional. The nucleus was irradiated with DAPI dye. Texas Red dye. Cytoplasm was stained with [the substance]. Dystrophin expression was observed in the DMD patient group. It is seen to be minimal. Dystrophin radiation increased in the treatment group 25 It is seen. Figure 3 shows the varying dystrophin levels depending on the use of poly-α tails of different lengths. The expression level is shown in this graph, which shows a nucleotide length of 140 adenine nucleotides. The poly-α tail appears to provide the highest expression. 30 7 REFERANSLAR [1] Manzur, A. Y., Kuntzer, T., Pike, M., & Swan, A. (2004). Glucocorticoid corticosteroids for Duchenne muscular dystrophy. The Cochrane database of systematic reviews, (2), CD003725. https: / / doi.org / 10.1002 / 14651858.CD003725.pub2 5 [2] Hoy S. M. (2023). Delandistrogene Moxeparvovec: First Approval. Drugs, 83(14), 1323–1329. https: / / doi.org / 10.1007 / s40265-023-01929-x [3] Lim, K. R., Maruyama, R., & Yokota, T. (2017). Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug design, development and therapy, 11, 533–545. https: / / doi.org / 10.2147 / DDDT.S97635 10 [4] Frank, D. E., Schnell, F. J., Akana, C., El-Husayni, S. H., Desjardins, C. A., Morgan, J., Charleston, J. S., Sardone, V., Domingos, J., Dickson, G., Straub, V., Guglieri, M., Mercuri, E., Servais, L., Muntoni, F., & SKIP-NMD Study Group (2020). Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology, 94(21), e2270–e2282. 15 https: / / doi.org / 10.1212 / WNL.0000000000009233 [5] Wilton-Clark, H., & Yokota, T. (2021). Casimersen for Duchenne muscular dystrophy. Drugs of today (Barcelona, Spain : 1998), 57(12), 707–717. https: / / doi.org / 10.1358 / dot.2021.57.12.3352740 20 25

Claims

8 REQUESTS 1. Exosome complex for use in the treatment of muscular system diseases. and its feature is:  A promoter with nucleotide sequence number 1, 5  Full dystrophin sequence with nucleotide sequence number 2,  and a poly-α tail with a nucleotide sequence No.

3. It includes.

2. According to claim 1, it is an exosome complex, characterized by the presence of a sequence on the surface of the exosome. MMP-2 has amino acid sequence No. 4 and 10 has amino acid sequence No.

5. It contains TIMP-2 biomarkers.

3. According to claim 1, it is an exosome complex and its characteristic feature is the aforementioned muscle system. The disease is Duchenne muscular dystrophy (DMD).

4. According to claim 1, it is an exosome complex and its characteristic feature is the aforementioned muscle system. The disease is Becker muscular dystrophy (BMD). 15 20 25 30

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