Стволовые клетки и генная терапия для лечения сахарного диабета

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От редакции

Число пациентов, страдающих диабетом, растет во всем мире. Аллотрансплантация васкуляризированной поджелудочной железы или островковых клеток эффективна, но требует иммуносупрессивной терапии. Ксенотрансплантация не только клинически не применима, но и не отвечает сути проблемы. Сегодня широко распространен энтузиазм в использовании стволовых клеток и генной терапии. Теоретически это возможно, а практически еще предстоит осуществить разработки по таким направлениям, как определение возможного вида клеток, выделение гена, методика его переноса в клетки, особенности реакции клетки на уровень сахара, возможности накопления инсулина в новой клетке. Использование вируса для переноса гена инсулина (и вообще вирусного вектора для других целей) напоминает историю о троянском коне. Возможно соединение этих двух идей - перенос гена инсулина в особый вид стволовых клеток.

Научное сообщество и редакторы журналов должны тщательно оценивать как скепсис в этих исследованиях, так и чрезмерный энтузиазм. Сегодня неоправданные методы, ocнованные на доказательствах, генной инженерии и клеточной терапии в лечении пациентов не вышли из экспериментальной стадии и не дают оснований эксплуатировать надежду больных людей.

В этих чрезвычайно интересных исследованиях важно быть добросовестным и честным. Автор опре- деляет критерии приемлемости экспериментальных исследований в работе "Семь основ доверительности" (The Seven Pillars of Credibility, 2010).

Ключевые слова:сахарный диабет типа 1 и 2, эмбриональные стволовые клетки, зрелые культуры фибробластов, индуцированные плюрипотентные стволовые клетки, аутотрансплантация клеток, неостровковая регенерация

Клин. и эксперимент. хир. Журн. им. акад. Б.В. Петровского. - 2013. - № 1. - С. 97-99.

The ability to perform a successful technique in no way guarantees a satisfactory physiological response. In the 17th century there were many attempts at blood transfusion between animals and also animals and man. The results were disastrous since at that time there was no concept of blood groups nor was there a method of preventing uncontrollable clotting. Similarly the surgical transplantation of an organ from a pig to man can be achieved technically, but since virtually every protein produced by a pig differs to a greater or lesser extent to the equivalent protein in man, it is perhaps not surprising that xenografting persists as a so far unsolved problem.

The pancreas has a large reserve of function; half a pancreas is more than enough to control glucose levels for prolonged periods. The pancreas has approximately 1 million islets, each containing between 1000 and 3000 cells of which 60% are beta cells. Thus in theory to treat insulin-dependent diabetes some 300 million beta cells would be needed to maintain normal glucose control.

Developments in an understanding of genes and gene expression and the existence and clarification of the behaviour of stem cells, led to premature enthusiasm that one or both of these approaches might lead to treatment for many different diseases and especially diabetes. There are, however, important difficulties to be overcome. The insulin gene can be isolated, cloned and prepared as plasmid which can be introduced into a variety of cells, but the process is not straightforward to enable anything approaching the kind of genetic engineering that would be necessary to treat a diabetic patient. Embryonic stem cells can proliferate in culture and with appropriate feeding, can be persuaded to differentiate into any type of cell in the body but differentiation is often not straightforward and undifferentiated cells, when transplanted, would produce malignant teratomatas. Since embryonic stem cells are derived from the inner cell mass of the blastocyst there have been extremely powerful objections to their use on ethical grounds, especially by the Roman Catholic Church, since the manipulation and proliferation of embryonic stem cells involves the destruction of a potential embryo.

Yamanaka in 2006 [1] made an important advance in demonstrating that murine embryonic and adult fibroblast cultures could be persuaded to change into pluripotent stem cells. The experiments have been developed enthusiastically worldwide since the use of so-called iPS cells avoids ethical opposition and has the potential to be used as an auto-transplant for patients, iPS cells being derived from their own tissue. Therefore these cells would not be expected to be rejected. The intensity of research with iPS cells has so far not led to an effective therapy.

The beta cell is highly differentiated. Not only is the insulin gene responsible for the synthesis of insulin, but there is an extremely effective method of storing the insulin secreted into the cell in granules which release insulin in a carefully controlled manner to respond to changes in ambient glucose concentration. Unfortunately this storage and release of insulin is not fully understood and cannot therefore be reproduced. The best one could hope for is a constituative production of insulin at a steady rate which nevertheless could be of great value in therapy, particularly for patients with potentially lethal hypoglycaemic unawareness. Experimentally the insulin gene can be introduced in vitro or in vivo into cells by a variety of techniques, for example electroporation, where an electric current increases the permeability of the cell membrane whilst the gene is inserted. Most work has been with viral vectors, particularly the Lenti virus and the Adno-Associated virus. The viral vector acts as a "Trojan horse" to bring the gene into the target cell. There is an important theoretical difference between in vitro and in vivo approaches. In vitro manipulation of cells can be more accurate in assessment of gene dosage, but taking cells out of the body, manipulating them and returning them as a transplant adds concern and complexity because of the danger of infection and there is a risk of unmasking uncogenes leading to malignancy. In vivo techniques are theoretically more simple but difficult to achieve in practice. An encouraging clinical development in the treatment of Haemophilia B was reported using an Adno-Associated virus as a vector which homes to the liver when injected intravenously, a very attractive approach compared with some of the transplantation techniques suggested by in vitro gene therapy [2].

In addition to embryonic stem cells and iPS cells, there are stem cells in many tissues whose primary role is probably repair of damage or replacement of loss of cells by attrition. Stem cells in bone marrow have been used to treat leukaemia and benign haematological diseases. These bone marrow stem cells, when injected intravenously, automatically home to the bone marrow where they set up house and if successful the transplant will cure the patient. The role of mesenchymal stem cells in the bone marrow is to fashion the niche environment for the blood cell production. They can be separated from the haematological cells by their adhering to plastic surfaces in culture. After proliferation, using different feeding techniques with differentiation factors, these cells can produce cartilage, fat, bone and sometimes cells that produce insulin and have a beta cell-like phenotype. Similar mesenchymal stem cells are found in fat, amnion and umbilical cord, but they are a mixed population with idiosyncratic potential. Neonatal amnion cells have a low immunogenicity and do not express HLA Class II antigens. They secrete HLA-G which has an immunosuppressive effect. Since they would not be autologous if used therapeutically this immunosuppressive effect could be of advantage. They could also be frozen and banked in large numbers and theoretically could be given to patients having renal transplants who already require continuous immunosuppressive treatment.

We and our collaborative colleague in Singapore are studying gene therapy in rodents and pigs using both electroporition and Lenti viral vectors and attempting to obtain and develop a promoter of the human insulin gene construct to respond to surround- ing levels of blood sugar. With electroporition no virus is involved and the plasmid does not integrate so division of transfected cell dilutes the gene activity but an advantage of this technique is that large numbers of plasmids can be produced cheaply. Using the Lenti virus the insulin gene does integrate with nuclear DNA and therefore maintains undiluted gene activity after cell division but it is expensive to produce large numbers.

Our collaborative colleagues in Mansoura University, Egypt have studied mesenchymal stromal cells from, first rat bone marrow, and then human bone marrow and have succeeded in growing cells that produced insulin and glucagon [3, 4]. Sufficient active cells have been produced to cure diabetic SCID mice and in theory the bone marrow could be taken from the diabetic patient, cultured and differentiated, and then returned as a transplant to the same patient, and being an autograft should not be rejected. Whether such cells would be a target for the autoimmune damage of Type I diabetes is not known. In the Mansoura experiment about 5% of the cultured and differentiated cells produced insulin. They appeared to be glucose responsive and were physiologically active for at least three months.

Auto-transplantation of cells, cultured or engineered in vitro, is an important and not well- researched topic. There seems to be a tendency for cells to function best if they remain in close proximity to each other, perhaps they interact to reinforce physiological activity. Sites suggested for transplantation areunder the renal capsule, intraperitoneal, intraportal, in a scaffold, intrahepatic, intrapancreatic, intramuscular, subcutaneous, intravenous, intrabone marrow and intratesticular. The use of one site does not preclude the use of another. Ex vivo culture, manipulation and re-transplantation would require a bespoke and therefore very expensive treatment, individual to each patient and the management and care of the cells outside the body would come under strict, regulatory control to make sure they complied with GMP requirements.

Currently it is extremely difficult to produce sufficient cells to give adequate therapy and persistent gene expression and protein synthesis has not been achieved. Progress of both stem cell and genetic engineering experiments has given rise to unreasonable expectastions of potential rewards and have led to an overwhelming temptation for fraudulent results to be reported and actual treatment to be offered to desperate patients who have failed with conventional therapy. In addition to deliberate fraud, there have been many examples of poorly conducted and controlled experiments. We have suggested a need to satisfy "The Seven Pillars of Credibility" for an acceptance of experimental data [5]:

1. Cure of hyperglycaemia.

2. Response to glucose tolerance test.

3. Evidence of appropriate C-peptide secretion.

4. Weight gain.

5. Prompt return of diabetes when the transfecting gene and/or insulin producing cells are removed.

6. No islet regeneration of stereptozocin treated animals and no regeneration of pancreas in pancreatectomised animals.

7. Presence of insulin storage granules in the treated cells.

It is important that the credibility and ethical behaviour of research work in this area is scrutinised by colleagues and journal editors from a sceptical point of view to minimise the cruel raising of premature hopes for sick and desperate patients and permit legitimate research to progress in a proper scientific manner.

Reference

1. Takahashi K., Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors // J. Cell. - 2006. - Vol. 126. - Р. 663-676.

2. Gabr M.M., Sobh M.M., Zakaria M.M. et al. Transplantation of insulin-producing clusters derived from adult bone marrow stem cells to treat diabetes in rats // Ex. Clin. Transplant. - 2008. - Vol. 6. - Р. 236-243.

3. Gabr M.M., Zakaria M.M., Ghoneim M.A. et al. Insulin-producing islet-like clusters derived from adult human bone marrow mesenchymal stem cells cure chemically-induced diabetes in nude mice // Cell Transplant. - 2013. - Vol. 22 (1). - Р. 133-145.

4. Nathwani A., Edward G.D., Tuddenham M.B. et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B // N. Eng. J. Med. - 2011. - Vol. 365, N 25. - Р. 2357-2365.

5. Calne R.Y., Ghoneim M. Novel Diabetes Therapy: The Seven Pillars of Credibility. Treatment Strategies - Gene Therapy for Diabetes, 2010.

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ГЛАВНЫЙ РЕДАКТОР
ГЛАВНЫЙ РЕДАКТОР
Дземешкевич Сергей Леонидович
Доктор медицинских наук, профессор (Москва, Россия)

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