GOODCELLS · Development of a proof of principle model for the therapeutical use of induced Pluripotent Stem (iPS) cells
7РП — „Хора“ (Действия „Мария Кюри“)
- Период
- 2011-07-20 → 2013-07-19
- Финансиране от ЕС
- 230 980 €
- Участници
- 1
- Схема
- MC-IEF
Линиите свързват координатора с партньорите.
Накратко на български
Индуцираните प्लурипотентни стволови клетки се използват за коригиране на генетичната мутация, причиняваща кистична фиброза. Разработването на този модел помага за създаването на ефективно лечение чрез комбиниране на генна и клетъчна терапия.
Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.
Резултати накратко
Development of a proof of principle model for the therapeutical use of induced Pluripotent Stem (iPS) cells.
The overall goal of this proposal was to develop an efficient treatment for Cystic Fibrosis (CF), the main genetic cause of death in Caucasian children, by combining techniques of gene and cellular therapy (Fig. 1). To this end, human and mouse induced Pluripotent Stem Cells (iPSCs) carrying the p.F508del mutation in the CFTR gene were produced from homozygous subjects. iPSC lines were generated by retroviral transduction of the c-Myc, Klf4, Oct4, and Sox2 transcription factors (Objective 1). The newly created lines have been maintained in continuous culture for up to 6 months showing expression of stemness markers such as Nanog, Oct-4, Sox2 and SSEA1 (Fig. 2) and a normal karyotype. Differentiation properties of both human and mouse iPSCs were demonstrated by embryoid bodies and teratoma formation, respectively. The correction of the p.F508del allele of the CFTR gene in iPSCs (Objective 2) relied on TALEN-based homologous recombination mediated by a targeting vector based on piggyback technology. TALENs pairs and targeting vectors were nucleofected into CF-iPSCs. Candidate positive clones were identified by negative selection with Ganciclovir and positive selection with puromycin. Bona fide targeted clones containing a corrected CFTR allele were validated by PCR analysis and sequencing (Fig. 3). Once, repaired iPSC lines were well characterized, a two-step in vitro differentiation protocol was established (Objective 3). It mimics the natural process of lung embryonic development, and is able to produce foregut endodermal cells reproducibly (Fig. 4). Finally, we have started a series of cell transplantation experiments aimed to define the conditions required for lung regeneration (Objective 4). The results are very preliminary, but after intravenous injection, iPSCs expressing Infrared fluorescent protein (IRFP) have a clear tendency to home into the lungs (Fig. 5). [NOTE: See figures in the attached document]
Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз
Цел на проекта
Cystic Fibrosis is a hereditary disease produced by the absence or malfunctioning of the Cystic Fibrosis Transmembrane Conductase Regulator (CFTR) gene. To date over 1,200 alterations in the DNA composition of the CFTR gene have been detected, although the deletion of the phenylalanine in position 508 (ΔF508) is responsible for more than 70% of the cases described in the European Population. CF is a degenerative disease, which can be considered as the main genetic cause of death in Caucasian children. Its first manifestations occur in early childhood, generally affecting the respiratory tract, and later extending to other organs.The identification and isolation of the gene responsible for the disease raised great expectations of finding a treatment. However, such hopes have yet to be realized. Different attempts to develop effective gene therapy protocols have not provided satisfactory results.In the current circumstances, I believe that the best strategy to develop an effective treatment for CF is to use autologous grafts of healthy lung epithelium progenitors, derived, in vitro, from human induced pluripotent stem (iPS) cells.The method that I propose will be developed simultaneously in human and mouse cells, and includes the following steps: (I) Production of iPS cells from keratinocytes obtained from ΔF508 CF patients and ΔF508 mutant mice; (II) Correction of the mutation in the iPS cells; (III) in vitro differentiation of repaired iPS (iPSr) cells up to a state compatible with their functional integration into the pulmonary epithelium; (IV) Transplantation of the differentiated cells into suitable receptors.Steps I through III will be carried out in parallel with human and mouse cells. Step IV will serve as a preclinical study and will be performed only in mice. During the process we plan to: 1) incorporate new, less aggressive de-differentiation methods, 2) improve homologous recombination efficiency in iPS cells, 3) determine the optimal differ""
Оригинален текст от CORDIS (на английски).
Участници
- FUNDACIO INSTITUT D'INVESTIGACIO SANITARIA ILLES BALEARS · PalmaКоординаторИспания
Връзки
Данни: CORDIS, © Европейски съюз