RESUMO
Inherited retinal diseases (IRDs) are the leading cause of blindness in working-age individuals worldwide. Their genetic etiology is especially heterogenous, so the development of gene-specific therapies is unlikely to meet the medical needs of the entire patient community. Considering these challenges, a complementary strategy could be to develop therapies independent of the underlying gene variant causing retinal degeneration. As the retina is a neural tissue, it is in theory amenable to neuroprotective therapies that could help prolong cell survival or promote retinal function. Many neurotrophic factors have shown favorable results in preclinical animal models of neurodegenerative diseases, but unfortunately these findings have not yet translated into successful human clinical trials. The clinical development of these new therapies is mostly impeded by selection of pertinent clinical end points and time-to-readout, as the majority of IRDs show a relatively slow disease progression rate. Despite these challenges, several strategies have moved forward into clinical development.
RESUMO
In the field of clinical ophthalmology, many of the common visual function study end points do not effectively reflect the significant morbidity of inherited retinal diseases (IRDs) and its effect on the patient's quality of life. In the last decade, emphasis has been placed on the development and implementation of patient-performance or task-focused end points, that may have greater ability to demonstrate the improvement or preservation of the patient's quality of life provided by therapeutic interventions. This article reviews performance-based tools developed to assess functional vision, such as the multi-luminance mobility test (MLMT) or the functional low-vision observer-rated assessment (FLORA), and highlights some of the recent advancements used in clinical development for IRD or ocular interventional therapies.
Assuntos
Qualidade de Vida , Doenças Retinianas , Humanos , Doenças Retinianas/diagnóstico , Doenças Retinianas/genéticaRESUMO
BACKGROUND/OBJECTIVES: REALITY is an international observational retrospective registry of LHON patients evaluating the visual course and outcome in Leber hereditary optic neuropathy (LHON). SUBJECTS/METHODS: Demographics and visual function data were collected from medical charts of LHON patients with visual loss. The study was conducted in 11 study centres in the United States of America and Europe. The collection period extended from the presymptomatic stage to at least more than one year after onset of vision loss (chronic stage). A Locally Weighted Scatterplot Smoothing (LOWESS) local regression model was used to analyse the evolution of best-corrected visual acuity (BCVA) over time. RESULTS: 44 LHON patients were included; 27 (61%) carried the m.11778G>A ND4 mutation, 8 (18%) carried the m.3460G>A ND1 mutation, and 9 (20%) carried the m.14484T>C ND6 mutation. Fourteen (32%) patients were under 18 years old at onset of vision loss and 5 (11%) were below the age of 12. The average duration of follow-up was 32.5 months after onset of symptoms. At the last observed measure, mean BCVA was 1.46 LogMAR in ND4 patients, 1.52 LogMAR in ND1 patients, and 0.97 LogMAR in ND6 patients. The worst visual outcomes were reported in ND4 patients aged at least 15 years old at onset, with a mean BCVA of 1.55 LogMAR and no tendency for spontaneous recovery. The LOESS modelling curve depicted a severe and permanent deterioration of BCVA. CONCLUSIONS: Amongst LHON patients with the three primary mtDNA mutations, adult patients with the m.11778G>A ND4 mutation had the worst visual outcomes, consistent with prior reports.
Assuntos
Atrofia Óptica Hereditária de Leber , Adolescente , Adulto , DNA Mitocondrial/genética , Europa (Continente) , Humanos , Mutação , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Estudos RetrospectivosRESUMO
BACKGROUND: RESCUE and REVERSE were 2 Phase 3 clinical trials that assessed the efficacy and safety of intravitreal gene therapy with lenadogene nolparvovec (rAAV2/2-ND4) for the treatment of Leber hereditary optic neuropathy (LHON). RESTORE is the long-term follow-up study of subjects treated in the RESCUE and REVERSE trials. METHODS: In RESCUE and REVERSE, 76 subjects with LHON because of the m.11778 G>A mutation in the mitochondrial gene ND4 received a single unilateral intravitreal injection of lenadogene nolparvovec. After 96 weeks, 61 subjects were enrolled in the long-term follow-up study RESTORE. The best-corrected visual acuity (BCVA) was assessed over a period of up to 52 months after onset of vision loss. A locally estimated scatterplot smoothing regression model was used to analyze changes in BCVA over time. Vision-related quality of life was reported using the visual function questionnaire-25 (VFQ-25). RESULTS: The population of MT-ND4 subjects enrolled in RESTORE was representative of the combined cohorts of RESCUE and REVERSE for mean age (35.1 years) and gender distribution (79% males). There was a progressive and sustained improvement of BCVA up to 52 months after the onset of vision loss. The final mean BCVA was 1.26 logarithm of the minimal angle of resolution 48 months after the onset of vision loss. The mean VFQ-25 composite score increased by 7 points compared with baseline. CONCLUSION: The treatment effect of lenadogene nolparvovec on BCVA and vision-related quality of life observed 96 weeks (2 years) after treatment in RESCUE and REVERSE was sustained at 3 years in RESTORE, with a maximum follow-up of 52 months (4.3 years) after the onset of vision loss.
Assuntos
Terapia Genética/métodos , Atrofia Óptica Hereditária de Leber/terapia , Proteínas Recombinantes/administração & dosagem , Acuidade Visual , Campos Visuais , Adolescente , Adulto , Idoso , DNA Mitocondrial/genética , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Mutação , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/fisiopatologia , Qualidade de Vida , Fatores de Tempo , Tomografia de Coerência Óptica , Adulto JovemRESUMO
OBJECTIVE: This report presents a cross-sectional analysis of the baseline characteristics of subjects with Leber hereditary optic neuropathy enrolled in the gene therapy trials RESCUE and REVERSE, to illustrate the evolution of visual parameters over the first year after vision loss. METHODS: RESCUE and REVERSE were 2 phase III clinical trials designed to assess the efficacy of rAAV2/2-ND4 gene therapy in ND4-LHON subjects. At enrollment, subjects had vision loss for ≤6 months in RESCUE, and between 6 and 12 months in REVERSE. Functional visual parameters (best-corrected visual acuity [BCVA], contrast sensitivity [CS], and Humphrey Visual Field [HVF]) and structural parameters assessed by spectral-domain optical coherence tomography were analyzed in both cohorts before treatment. The cross-sectional analysis of functional and anatomic parameters included the baseline values collected in all eyes at 2 different visits (Screening and Inclusion). RESULTS: Seventy-six subjects were included in total, 39 in RESCUE and 37 in REVERSE. Mean BCVA was significantly worse in RESCUE subjects compared with REVERSE subjects (1.29 and 1.61 LogMAR respectively, P = 0.0029). Similarly, mean CS and HVF were significantly more impaired in REVERSE vs RESCUE subjects (P < 0.005). The cross-sectional analysis showed that the monthly decrease in BCVA, ganglion cell layer macular volume, and retinal nerve fiber layer thickness was much more pronounced in the first 6 months after onset (+0.24 LogMAR, -0.06 mm3, and -6.00 µm respectively) than between 6 and 12 months after onset (+0.02 LogMAR, -0.01 mm3, and -0.43 µm respectively). CONCLUSION: LHON progresses rapidly in the first months following onset during the subacute phase, followed by relative stabilization during the dynamic phase.
Assuntos
Terapia Genética/métodos , Atrofia Óptica Hereditária de Leber/fisiopatologia , Acuidade Visual , Campos Visuais/fisiologia , Adolescente , Adulto , Idoso , Estudos Transversais , DNA Mitocondrial/genética , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Células Ganglionares da Retina/patologia , Tomografia de Coerência Óptica/métodos , Adulto JovemRESUMO
Optogenetics may enable mutation-independent, circuit-specific restoration of neuronal function in neurological diseases. Retinitis pigmentosa is a neurodegenerative eye disease where loss of photoreceptors can lead to complete blindness. In a blind patient, we combined intraocular injection of an adeno-associated viral vector encoding ChrimsonR with light stimulation via engineered goggles. The goggles detect local changes in light intensity and project corresponding light pulses onto the retina in real time to activate optogenetically transduced retinal ganglion cells. The patient perceived, located, counted and touched different objects using the vector-treated eye alone while wearing the goggles. During visual perception, multichannel electroencephalographic recordings revealed object-related activity above the visual cortex. The patient could not visually detect any objects before injection with or without the goggles or after injection without the goggles. This is the first reported case of partial functional recovery in a neurodegenerative disease after optogenetic therapy.
Assuntos
Cegueira/fisiopatologia , Cegueira/terapia , Terapia Genética/métodos , Optogenética/métodos , Retinose Pigmentar/patologia , Ondas Encefálicas/fisiologia , Dependovirus/genética , Dispositivos de Proteção dos Olhos , Vetores Genéticos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Células Fotorreceptoras/fisiologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Visão Ocular/fisiologia , Córtex Visual/fisiologia , Percepção Visual/fisiologiaRESUMO
PURPOSE: To evaluate the efficacy of a single intravitreal injection of rAAV2/2-ND4 in subjects with visual loss from Leber hereditary optic neuropathy (LHON). DESIGN: RESCUE is a multicenter, randomized, double-masked, sham-controlled, phase 3 clinical trial. PARTICIPANTS: Subjects with the m.11778G>A mitochondrial DNA mutation and vision loss ≤6 months from onset in 1 or both eyes were included. METHODS: Each subject's right eye was randomly assigned (1:1) to treatment with rAAV2/2-ND4 (single injection of 9 × 1010 viral genomes in 90 µl) or to sham injection. The left eye received the treatment not allocated to the right eye. MAIN OUTCOME MEASURES: The primary end point was the difference of the change from baseline in best-corrected visual acuity (BCVA) between rAAV2/2-ND4-treated and sham-treated eyes at week 48. Other outcome measures included contrast sensitivity, Humphrey visual field perimetry, retinal anatomic measures, and quality of life. Follow-up extended to week 96. RESULTS: Efficacy analysis included 38 subjects. Mean age was 36.8 years, and 82% were male. Mean duration of vision loss at time of treatment was 3.6 months and 3.9 months in the rAAV2/2-ND4-treated eyes and sham-treated eyes, respectively. Mean baseline logarithm of the minimum angle of resolution (logMAR) BCVA (standard deviation) was 1.31 (0.52) in rAAV2/2-ND4-treated eyes and 1.26 (0.62) in sham-treated eyes, with a range from -0.20 to 2.51. At week 48, the difference of the change in BCVA from baseline between rAAV2/2-ND4-treated and sham-treated eyes was -0.01 logMAR (P = 0.89); the primary end point of a -0.3 logMAR (15-letter) difference was not met. The mean BCVA for both groups deteriorated over the initial weeks, reaching the worst levels at week 24, followed by a plateau phase until week 48, and then an improvement of +10 and +9 Early Treatment Diabetic Retinopathy Study letters equivalent from the plateau level in the rAAV2/2-ND4-treated and sham-treated eyes, respectively. CONCLUSIONS: At 96 weeks after unilateral injection of rAAV2/2-ND4, LHON subjects carrying the m.11778G>A mutation treated within 6 months after vision loss achieved comparable visual outcomes in the injected and uninjected eyes.
Assuntos
Terapia Genética , Atrofia Óptica Hereditária de Leber/terapia , Adolescente , Adulto , Idoso , DNA Mitocondrial/genética , Dependovirus/genética , Método Duplo-Cego , Eletrorretinografia , Feminino , Seguimentos , Vetores Genéticos , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Mutação , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/psicologia , Qualidade de Vida/psicologia , Fatores de Tempo , Resultado do Tratamento , Acuidade Visual/fisiologia , Testes de Campo Visual , Campos Visuais/fisiologia , Adulto JovemRESUMO
REVERSE is a randomized, double-masked, sham-controlled, multicenter, phase 3 clinical trial that evaluated the efficacy of a single intravitreal injection of rAAV2/2-ND4 in subjects with visual loss from Leber hereditary optic neuropathy (LHON). A total of 37 subjects carrying the m.11778G>A (MT-ND4) mutation and with duration of vision loss between 6 to 12 months were treated. Each subject's right eye was randomly assigned in a 1:1 ratio to treatment with rAAV2/2-ND4 (GS010) or sham injection. The left eye received the treatment not allocated to the right eye. Unexpectedly, sustained visual improvement was observed in both eyes over the 96-week follow-up period. At week 96, rAAV2/2-ND4-treated eyes showed a mean improvement in best-corrected visual acuity (BCVA) of -0.308 LogMAR (+15 ETDRS letters). A mean improvement of -0.259 LogMAR (+13 ETDRS letters) was observed in the sham-treated eyes. Consequently, the primary end point, defined as the difference in the change in BCVA from baseline to week 48 between the two treatment groups, was not met (P = 0.894). At week 96, 25 subjects (68%) had a clinically relevant recovery in BCVA from baseline in at least one eye, and 29 subjects (78%) had an improvement in vision in both eyes. A nonhuman primate study was conducted to investigate this bilateral improvement. Evidence of transfer of viral vector DNA from the injected eye to the anterior segment, retina, and optic nerve of the contralateral noninjected eye supports a plausible mechanistic explanation for the unexpected bilateral improvement in visual function after unilateral injection.
Assuntos
Atrofia Óptica Hereditária de Leber , Animais , Dependovirus/genética , Terapia Genética , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Tomografia de Coerência Óptica , Acuidade Visual , Campos VisuaisRESUMO
Importance: Intravitreal gene therapy is regarded as generally safe with limited mild adverse events, but its systemic effects remain to be investigated. Objective: To examine the association between immune response and intraocular inflammation after ocular gene therapy with recombinant adeno-associated virus 2 carrying the ND4 gene (rAAV2/2-ND4). Design, Setting, and Participants: This secondary analysis of an open-label, dose-escalation phase 1/2 randomized clinical trial of rAAV2/2-ND4 included data from February 13, 2014 (first patient visit), to March 30, 2017 (last patient visit at week 96), the first 2 years after injection. Patients older than 15 years with diagnosed ND4 Leber hereditary optic neuropathy (LHON) and visual acuity of at least counting fingers were enrolled in 1 of 5 cohorts. Four dose cohorts of 3 patients each were treated sequentially. An extension cohort of 3 patients received the dose of 9 × 1010 viral genomes per eye. Interventions: Patients received increasing doses of rAAV2/2-ND4 (9 × 109, 3 × 1010, 9 × 1010, and 1.8 × 1011 viral genomes per eye) as a single unilateral intravitreal injection. Patients were monitored for 96 weeks after injection; ocular examinations were performed regularly, and blood samples were collected for immunologic testing. Main Outcomes and Measures: A composite ocular inflammation score (OIS) was calculated based on grades of anterior chamber cells and flare, vitreous cells, and haze according to the Standardization of Uveitis Nomenclature. The systemic immune response was quantified by enzyme-linked immunospot (cellular immune response), enzyme-linked immunosorbent assay (IgG titers), and luciferase assay (neutralizing antibody [NAb] titers). Results: The present analysis included 15 patients (mean [SD] age, 47.9 [17.2] years; 13 men and 2 women) enrolled in the 5 cohorts of the clinical trial. Thirteen patients experienced intraocular inflammation after rAAV2/2-ND4 administration. Mild anterior chamber inflammation and vitritis were reported at all doses, and all cases were responsive to treatment. A maximum OIS of 9.5 was observed in a patient with history of idiopathic uveitis. Overall, OIS was not associated with the viral dose administered. No NAbs against AAV2 were detected in aqueous humor before treatment. Two patients tested positive for cellular immune response against AAV2 at baseline and after treatment. Humoral immune response was not apparently associated with the dose administered or with the immune status of patients at baseline. No association was found between OISs and serum NAb titers. Conclusions and Relevance: In this study, intravitreal administration of rAAV2/2-ND4 in patients with LHON was safe and well tolerated. Further investigations may shed light into the local immune response to rAAV2/2-ND4 as a potential explanation for the observed intraocular inflammation.
Assuntos
Terapia Genética/métodos , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/terapia , Parvovirinae/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Células Ganglionares da Retina/patologia , Acuidade Visual , Dependovirus , Eletrorretinografia , Feminino , Angiofluoresceinografia/métodos , Seguimentos , Fundo de Olho , Vetores Genéticos/administração & dosagem , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Atrofia Óptica Hereditária de Leber/diagnóstico , Estudos Retrospectivos , Tomografia de Coerência Óptica/métodos , Campos VisuaisAssuntos
Dependovirus/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/terapia , Visão de Cores/fisiologia , Sensibilidades de Contraste/fisiologia , Eletrorretinografia , Potenciais Evocados Visuais/fisiologia , Feminino , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/fisiopatologia , Estudos Prospectivos , Acuidade Visual/fisiologiaRESUMO
The human naive pluripotent stem cell (PSC) state, corresponding to a pre-implantation stage of development, has been difficult to capture and sustain in vitro. We report that the Hippo pathway effector YAP is nuclearly localized in the inner cell mass of human blastocysts. Overexpression of YAP in human embryonic stem cells (ESCs) and induced PSCs (iPSCs) promotes the generation of naive PSCs. Lysophosphatidic acid (LPA) can partially substitute for YAP to generate transgene-free human naive PSCs. YAP- or LPA-induced naive PSCs have a rapid clonal growth rate, a normal karyotype, the ability to form teratomas, transcriptional similarities to human pre-implantation embryos, reduced heterochromatin levels, and other hallmarks of the naive state. YAP/LPA act in part by suppressing differentiation-inducing effects of GSK3 inhibition. CRISPR/Cas9-generated YAP(-/-) cells have an impaired ability to form colonies in naive but not primed conditions. These results uncover an unexpected role for YAP in the human naive state, with implications for early human embryology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Benzamidas/farmacologia , Blastocisto/citologia , Blastocisto/metabolismo , Blastocisto/patologia , Sistemas CRISPR-Cas/genética , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Heterocromatina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Lisofosfolipídeos/farmacologia , Masculino , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Células-Tronco Pluripotentes/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) holds enormous promise for regenerative medicine. To elucidate endogenous barriers limiting this process, we systematically dissected human cellular reprogramming by combining a genome-wide RNAi screen, innovative computational methods, extensive single-hit validation, and mechanistic investigation of relevant pathways and networks. We identify reprogramming barriers, including genes involved in transcription, chromatin regulation, ubiquitination, dephosphorylation, vesicular transport, and cell adhesion. Specific a disintegrin and metalloproteinase (ADAM) proteins inhibit reprogramming, and the disintegrin domain of ADAM29 is necessary and sufficient for this function. Clathrin-mediated endocytosis can be targeted with small molecules and opposes reprogramming by positively regulating TGF-ß signaling. Genetic interaction studies of endocytosis or ubiquitination reveal that barrier pathways can act in linear, parallel, or feedforward loop architectures to antagonize reprogramming. These results provide a global view of barriers to human cellular reprogramming.
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas ADAM/metabolismo , Adesão Celular , Células-Tronco Embrionárias/metabolismo , Endocitose , Humanos , Ubiquitina/metabolismoRESUMO
Pluripotent stem cells are derived from culture of early embryos or the germline and can be induced by reprogramming of somatic cells. Barriers to reprogramming that stabilize the differentiated state and have tumor suppression functions are expected to exist. However, we have a limited understanding of what such barriers might be. To find novel barriers to reprogramming to pluripotency, we compared the transcriptional profiles of the mouse germline with pluripotent and somatic cells, in vivo and in vitro. There is a remarkable global expression of the transcriptional program for pluripotency in primordial germ cells (PGCs). We identify parallels between PGC reprogramming to pluripotency and human germ cell tumorigenesis, including the loss of LATS2, a tumor suppressor kinase of the Hippo pathway. We show that knockdown of LATS2 increases the efficiency of induction of pluripotency in human cells. LATS2 RNAi, unlike p53 RNAi, specifically enhances the generation of fully reprogrammed iPS cells without accelerating cell proliferation. We further show that LATS2 represses reprogramming in human cells by post-transcriptionally antagonizing TAZ but not YAP, two downstream effectors of the Hippo pathway. These results reveal transcriptional parallels between germ cell transformation and the generation of iPS cells and indicate that the Hippo pathway constitutes a barrier to cellular reprogramming.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes p53 , Células Germinativas/citologia , Células Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Neoplasias Embrionárias de Células Germinativas/etiologia , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Interferência de RNA , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas de Sinalização YAPRESUMO
Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chromosome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming.