1.1 Project Background

1.1.1 Introduction to myoblasts

This project is a study of human myoblasts, production and application of high-tech projects for the treatment of muscle atrophy, heart disease, diabetes, cancer, pain, depression, bone and joint disease, can also be used for beauty, anti- aging and other fields.

Myoblasts are muscle precursor cells, derived from the mesoderm myoblasts, self-renewal and the ability to promote regeneration of muscle fibers, is the only body with the natural fusion of cells. In the integration process, the genome of the nucleus from a normal muscle naturally passed to the mother cell in the patient's muscle cells, which lack the genetic repair diseased cells.

Myoblasts to the unique biological characteristics of the target cells for gene therapy has many advantages:

(1) The number of myoblast-rich, easy access, easy to pass by injection, for large-scale cell transplantation provided the conditions.

(2) Myoblasts to high levels of expression of the target gene product, and can be secreted recombinant protein.

(3) Myoblasts have the ability to host muscle cell fusion, which is the main destination of their post-transplant, long-term survival in muscle fibers, is conducive to protein stability, and sustained expression.

(4) Muscle blood supply, easy access to the blood circulation of expression products, reach the target organ function.

(5) Myoblasts after transplantation does not affect the physiological function of adjacent tissues, and easy to repeat muscle biopsy, the application of safety.

(6) In the body is not unlimited proliferation or the formation of tumors.

(7) natural myoblast fusion, integration period of a week or two, but lost in the integration process MHC-1 antigen, only in small doses of short-term use of immunosuppressive agents can be the same autologous , allogeneic or xenogeneic allogeneic transplantation.

(8) Small part of myoblasts (about 0.1% to 0.5%) after implantation of skeletal myoblasts as the sleep state to exist stably in the host body, when they need to re-enter the mitotic cycle to play its role in myogenic fusion.

1.1.2 Myoblast Uses

Based on more than myoblasts biological characteristics, it can play a role in the following areas:

1.1.2.1 Hereditary Myopathy

Myoblasts began in the hereditary myopathy of some gene therapy, such as Duchenne muscular dystrophy (Duchenne muscular dystrophy, DMD). DMD is the most common primary myopathy, muscle cells to enhance the patient's body lacks protein, are sex-linked recessive genetic disease, patients with skeletal muscle, cardiac muscle, smooth muscle or brain nerve cell membrane skeleton protein instability, there is no effective drugs and methods of treatment, patients usually died aged 20, died of respiratory failure.

In 1990 Peter K. Law and 9-year-old first one cases of DMD patients were treated myoblasts, cells from the donor parent of the rectus femoris muscle satellite cells, while bacteria use of immunosuppressive agents cyclosporin Su A, found that donor cells were clustered in patients with intramuscular growth, significantly improved the situation of children. Then Professor Luo Gai has conducted 280 such patients treated, some patients condition improved significantly. From 1994-1998, approved by the FDA Professor Luo muscular dystrophy research group of patients during phase II / III clinical trials, each patient received during the period from 70,000 to 150,000 U.S. dollars.

Myoblast transplantation for treatment with anti-dystrophin restoration potential of gene therapy of this disease is the main method. (① Law, PK and Law, DM Human Myoblast Genome Therapies and Devices in Regenerative Medicine. Rec Pat Regen Med 1: 88-117, 2011. ② Law, PK, et al. Myoblast transfer as a platform technology of gene therapy. Gene Ther and Molec Biol 1:345-363, 1998. ③ Law, PK, et al. Feasibility, safety, and efficacy of myoblast transfer therapy on Duchenne muscular dystrophy boys. Cell Transplantation 1:235-244, 1992. ④ Law, PK, et al. Dystrophin production induced by myoblast transfer therapy in Duchenne muscular dystrophy. Lancet, 336:114-115, 1990.)

1.1.2.2 Skeletal Muscle Injury

Muscle damage is very common in the orthopedic field, causes, including laceration, contusion and stretch injuries. Muscle damage after a while start the process of necrosis and repair two. After the inflammatory phase, granulation tissue and fibrous tissue proliferation at the same time, however, the rate of proliferation of fibrous tissue much faster than the muscle regeneration, ultimately leading to scar formation is far more than the regeneration of functional muscle. Thus, fibrosis is the process of muscle regeneration and repair a major obstacle. Currently, the myoblast-mediated gene therapy in skeletal muscle injury shows great potential.

1.1.2.3 Cardiovascular Diseases

In 1999 Peter K. Law was the first to use myoblasts to treat heart disease. (① Law, PK et al. World's first human myoblast transfer into the heart. Frontiers in Physiology A85, 2000.) Currently, the international myoblasts to treat heart disease has entered the FDA Phase III clinical trials.

Studies have shown that myoblasts can be used to treat ischemic heart disease, such as myocardial infarction and refractory heart failure. Currently 20 countries 230 cases of patients in clinical studies have shown that for patients with myocardial infarction after injection of human myoblasts may improve regional myocardial function around the infarct lesion, increased left ventricular ejection fraction, perfusion, viability, power, wall thickness, and end-diastolic and systolic blood volume, not associated with arrhythmia. This treatment may become the treatment of myocardial infarction and heart failure, the most important new ways. (① Law, PK and Law DM Human Myoblast Genome Therapies and Devices in Regenerative Medicine. Rec Pat Regen Med 1: 88-117, 2011. ② Law, PK etal Delivery of Biologics for Angiogenesis and Myogenesis in "Practical Handbook of Advanced Interventional Cardiology" ed by Nguyen, T., Colombo, A., Hu, D., Grines, CL, and Saito, S. Blackwell Futura, Malden, USA. 3rd edition, 2008, pp. 584-596. ③ Law, PK et al. Human myoblast genome therapy. J Geriatr Cardiol 3: 135-151, 2006.)

1.1.2.4 Diabetes

In 2004 Peter K. Law was the first person to use myoblasts to treat diabetes. Patients with type II diabetes is due to the lack of skeletal muscle GLUT4 gene group caused the high blood sugar. In the myoblast fusion process, the genome of the nucleus from a normal muscle naturally pass into the mother cells of patients with muscle cells, diseased cells to repair genetic lack of GLUT4 gene group. Currently, the myoblast treatment of diabetes has entered Phase I clinical trials. (① Law, PK et al. The world's first myoblast study of Type II diabetic patients. Business Briefing: North American Pharmacotherapy 2004-Issue 2. ② Ye, L. et al. Skeletal myoblast transplantation for attenuation of hyperglycemia, hyperinsulinemia, and glucose intolerance in a mouse model of type-2 diabetes mellitus. Diabetologia 52 (9) :1925-34 (IF = 6.418) 2009. ③ Ye, L. et al. Skeletal myoblast transplantation for attenuation of hyperglycaemia, hyperinsulinemia, and glucose intolerance in a mouse model of type-2 diabetes mellitus. J Cell and Molec Med 14: 323-336. (IF = 5.114), 2010.)

1.2 Necessity of Project Construction

Human myoblast genome therapy (HMGT) is an emerging treatment technologies currently in this field is blank. Construction of the project will enable China to become the first in the world with the best technology platform for the treatment of myoblasts countries, the lack of effective treatment for the treatment of diseases, as China has brought huge social and economic benefits.

Myoblast study myoblast therapy, tissue engineering and regenerative medicine foundation. With the changes in the human disease spectrum, the gradual reduction of infectious diseases, and due to their own cells, tissues or organs necrosis disease caused by more and more. These diseases only through surgery and drugs can not be solved. It is in this sense, the 20th century is the era of drug treatment, the 21st century is the era of cell therapy. Myoblasts from the current basic research and clinical application of situation analysis carried out in the next 3-5 years, will be the myoblast stage of clinical application of gold. Therefore, the use of this opportunity, the first in Asia to establish a myoblast Research and application of medical research and accelerate myoblast series of studies and clinical application development, the establishment of a series of myoblast clinical treatment methods and standards, forming a series of muscle mother cell clinical application of new technologies for improving the treatment of the disease level, with a great space for development, but also to our board the high ground in the field of cell therapy.

1.2.1 Project Advantages

(1) 55% of the tissue is human muscle cells. Human myoblast transplantation to a living, healthy, contains the entire human genome myoblasts transplanted to a genetic defect, a disease of skeletal muscle and myocardium, to guide organ regeneration, is the most mature and most advanced regenerative medicine.

(2) Since the natural myoblast fusion, allogeneic cell transplant patients for three weeks only immunosuppressive agents.

(3) Safe, not any member of tumor risk and side effects of chemicals.

(4) Wide range of applications, many muscle-derived diseases and genetic abnormalities have effect

(5) Professor Law is the world's first to carry out research scientist myoblasts, the world's most advanced technology ﹑ unique standard operating procedures (SOPs) and technical secrets, people myoblast transplantation can achieve low-cost, high purity, high energy, the purpose of an unlimited number of requirements.

(6) Professor Law holds the original patents for myoblast transfer therapy.

(7) Professor Law has a highly regarded international reputation and 40 years of experience.

(8) CTI's competent technical team.

Based on the above advanced technology and management experience, we produce myoblasts with high yield, high cell purity, high cell viability and low cost. China will become the world leader in supplying myoblast cells and myoblast transfer therapy.

1.2.2 Technical Difficulties and Innovation

Project technical difficulties:

(1) High cell viability, only the high energy of the myoblasts to survive at the injection site and differentiate into muscle cells;

(2) The purity of cultured cells, myoblasts itself is almost non-immunogenic, from fiber cells and other tissue cells have certain antigens, so the purity of the patient cells produce rejection, cells can survive the key.

This project uses the original patented technology, take 2 grams of skeletal muscle after training can be harvested 50 billion expansion of myoblasts, purity over 99%, while other international research groups only from 5-10 grams of skeletal muscle training to 800 million, the average purity of 75% of the myoblasts. While the cell number, purity and vitality of the patient's treatment is essential, and this is our innovation and technology high ground.

1.2.3 Present Situation and Trends in Related Fields

1. Currently, the domestic blank in this field from 1992 to 1995, Professor Law and Dr. Min Dan Lin, MD 301 Hospital of PLA neuromuscular disease lab has done employing female fetal muscle cells to treat muscular dystrophy clinical treatment. At present, there is still the basis of individual research work, but the technical base and R & D is very limited, in general, are blank.

Key Laboratory of domestic stem cells, but not myoblast stem cells. Professor Law owned myoblast stem cell technology than the technology mature a lot, especially for the treatment of human killer heart disease, at present, the international myoblasts to treat heart disease has entered the FDA Phase III clinical trials, and stem cells study only in its infancy, but there are many risk factors.

2. International trends are: (1) 20 countries during the treatment of heart disease with myoblasts clinical research and clinical trials, FDA in the United States has entered Phase III clinical trials, expected in the near future this treatment will likely become the treatment of myocardial infarction and heart failure the most important new ways. (2) human myoblast transplantation for the treatment of muscular dystrophy patients have access to FDA Phase III clinical trials. (3) human myoblasts for the treatment of cancer in Israel to enter the clinical stage of Phase II trials. (4) for the treatment of diabetes as a phase of clinical trials. (5) anti-aging and beauty projects have been carried out. In short, human myoblast transplantation for the treatment of this high-tech platform for a variety of human diseases will be the inevitable result, it will be for us to bring good to mankind.