RESUMO
Retinitis pigmentosa (RP) is a group of progressive retinal degenerations of mostly monogenic inheritance, which cause blindness in about 1:3,500 individuals worldwide. Heterozygous variants in the rhodopsin (RHO) gene are the most common cause of autosomal dominant RP (adRP). Among these, missense variants at C-terminal proline 347, such as p.Pro347Ser, cause severe adRP recurrently in European affected individuals. Here, for the first time, we use CRISPR/Cas9 to selectively target the p.Pro347Ser variant while preserving the wild-type RHO allele in vitro and in a mouse model of adRP. Detailed in vitro, genomic, and biochemical characterization of the rhodopsin C-terminal editing demonstrates a safe downregulation of p.Pro347Ser expression leading to partial recovery of photoreceptor function in a transgenic mouse model treated with adeno-associated viral vectors. This study supports the safety and efficacy of CRISPR/Cas9-mediated allele-specific editing and paves the way for a permanent and precise correction of heterozygous variants in dominantly inherited retinal diseases.
Assuntos
Edição de Genes , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Rodopsina/genética , Alelos , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Terapia Genética , Humanos , Mutação INDEL , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retina/fisiopatologia , Rodopsina/metabolismoRESUMO
Strongyloidiasis is a clinical issue both in humans and in dogs. Moreover, there are concerns about its zoonotic potential. We aimed to explore Strongyloides stercoralis epidemiology in Southern Italy in humans and dogs sharing the same environment in three different settings: (1) kennels (group K); (2) livestock farms (group L) and (3) agricultural farms (group A). For humans, a commercial ELISA test was used for screening. RT-PCR on faecal samples was done for people testing positive or equivocal at serology. On dog's faecal samples, Baermann test and RT-PCR were performed. A total of 145 dogs and 139 persons were tested. Based on faecal tests in dogs and serology in humans, a S. stercoralis positivity of 4.1% and 6.5% was revealed, respectively. The sites where cases were found were different for animals and humans. In dogs the highest positivity was in group K (6.7% against 2% and 0% in L and A). Differently, in humans the proportion of positive results was similar between the groups (p = 0.883). Fifty percent (3/6) of positive dogs were healthy; the other dogs presented weight loss and/or diarrhoea. ELISA-positive persons (n=9) were all in health, but abdominal pain (37.5%), urticaria (22.2%) and asthma (22.2%) were reported, resolving after treatment with oral ivermectin 200 µg/kg. RT-PCR performed on 13 human faecal samples resulted negative. These findings suggest that strongyloidiasis is present in humans and dogs in Southern Italy, and screening in larger cohorts would be needed for more accurate estimates.
Assuntos
Strongyloides stercoralis , Estrongiloidíase , Animais , Cães , Humanos , Fezes , Itália/epidemiologia , Ivermectina/uso terapêutico , Estrongiloidíase/diagnóstico , Estrongiloidíase/epidemiologia , Estrongiloidíase/veterináriaRESUMO
Inherited retinal disorders (IRDs) affect millions of people worldwide and are a major cause of irreversible blindness. Therapies based on drugs, gene augmentation or transplantation approaches have been widely investigated and proposed. Among gene therapies for retinal degenerative diseases, the fast-evolving genome-editing CRISPR/Cas technology has emerged as a new potential treatment. The CRISPR/Cas system has been developed as a powerful genome-editing tool in ophthalmic studies and has been applied not only to gain proof of principle for gene therapies in vivo, but has also been extensively used in basic research to model diseases-in-a-dish. Indeed, the CRISPR/Cas technology has been exploited to genetically modify human induced pluripotent stem cells (iPSCs) to model retinal disorders in vitro, to test in vitro drugs and therapies and to provide a cell source for autologous transplantation. In this review, we will focus on the technological advances in iPSC-based cellular reprogramming and gene editing technologies to create human in vitro models that accurately recapitulate IRD mechanisms towards the development of treatments for retinal degenerative diseases.
Assuntos
Células-Tronco Pluripotentes Induzidas , Degeneração Retiniana , Humanos , Edição de Genes , Sistemas CRISPR-Cas/genética , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Terapia GenéticaRESUMO
Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.
Assuntos
COVID-19 , Pan troglodytes , Adenoviridae/genética , Animais , Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Pan troglodytes/genéticaRESUMO
Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Posicionamento Cromossômico/genética , Loci Gênicos/genética , HIV-1/genética , HIV-1/fisiologia , Integração Viral/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Integrase de HIV/metabolismo , Meia-Vida , Humanos , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
Retinal diseases (RD) include inherited retinal dystrophy (IRD), for example, retinitis pigmentosa and Leber's congenital amaurosis, or multifactorial forms, for example, age-related macular degeneration (AMD). IRDs are clinically and genetically heterogeneous in nature. To date, more than 200 genes are known to cause IRDs, which perturb the development, function and survival of rod and cone photoreceptors or retinal pigment epithelial cells. Conversely, AMD, the most common cause of blindness in the developed world, is an acquired disease of the macula characterised by progressive visual impairment. To date, available therapeutic approaches for RD include nutritional supplements, neurotrophic factors, antiangiogenic drugs for wet AMD and gene augmentation/interference strategy for IRDs. However, these therapies do not aim at correcting the genetic defect and result in inefficient and expensive treatments. The genome editing technology based on clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein (Cas) and an RNA that guides the Cas protein to a predetermined region of the genome, represents an attractive strategy to tackle IRDs without available cure. Indeed, CRISPR/Cas system can permanently and precisely replace or remove genetic mutations causative of a disease, representing a molecular tool to cure a genetic disorder. In this review, we will introduce the mechanism of CRISPR/Cas system, presenting an updated panel of Cas variants and delivery systems, then we will focus on applications of CRISPR/Cas genome editing in the retina, and, as emerging treatment options, in patient-derived induced pluripotent stem cells followed by transplantation of retinal progenitor cells into the eye.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Transferência de Genes/tendências , Degeneração Macular/terapia , Doenças Retinianas/terapia , Edição de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Degeneração Macular/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Retina/patologia , Retina/transplante , Doenças Retinianas/genética , Retinose Pigmentar/genética , Retinose Pigmentar/terapiaRESUMO
Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.
Assuntos
Sistemas CRISPR-Cas/genética , Moléculas de Adesão Celular/genética , Epidermólise Bolhosa Juncional/terapia , Terapia Genética , Animais , Membrana Basal/patologia , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/deficiência , Reparo do DNA/genética , DNA Complementar/genética , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa Juncional/patologia , Regulação da Expressão Gênica , Humanos , Íntrons/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Laminina/genética , Lentivirus/genética , Camundongos , Mutação , Edição de RNA/genética , CalininaRESUMO
Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.
Assuntos
Memória Imunológica , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-15/genética , Interleucina-7/genética , Selectina L/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Precursoras de Linfócitos T/citologia , Células Precursoras de Linfócitos T/transplante , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/transplante , Receptor fas/metabolismoRESUMO
The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.
Assuntos
Adenoviridae/genética , Proteínas de Bactérias/genética , Desoxirribonucleases/genética , HIV-1/genética , Cromossomos Humanos , Quebras de DNA de Cadeia Dupla , Loci Gênicos , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Transdução GenéticaRESUMO
Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.
Assuntos
Dependovirus/genética , Endonucleases/genética , Marcação de Genes , Recombinação Homóloga , Queratinócitos/metabolismo , Células-Tronco/metabolismo , Integração Viral , Animais , Linhagem Celular , Transplante de Células , Células Cultivadas , Dependovirus/metabolismo , Endonucleases/metabolismo , Vetores Genéticos , Humanos , Camundongos , Transdução Genética , Dedos de ZincoRESUMO
BACKGROUND: The genotoxicity of zidovudine has been established in experimental models. The objective of the study was to identify genotoxicity markers in cord blood cells from newborns exposed in utero to antiretroviral (ARV) combinations containing zidovudine. METHODS: Cells were investigated by karyotyping and gene expression analysis of the CD34(+) hematopoietic stem/progenitor cell (HPC) compartment. RESULTS: Karyotyping of the cord blood cells from 15 ARV-exposed newborns and 12 controls revealed a higher proportion of aneuploid cells in the exposed group (median, 18.8% [interquartile range, 10.0%-26.7%] vs 6.6% [interquartile range, 3.1%-11.7%]; P < .001). All chromosomes were involved, with a random distribution of these alterations. Gene expression profiling of CD34(+) HPCs from 7 ARV-exposed and 6 control newborns revealed that >300 genes were significantly upregulated or downregulated by at least 1.5-fold in the exposed group (P < .05 for all comparisons). Significant alterations of genes involved in cell cycle control, mitotic checkpoints, and DNA repair were identified. Although this study does not allow discrimination between the roles of each of the 3 drugs, both cytogenetic and transcriptional findings are similar to those in cellular experiments that used zidovudine alone. CONCLUSIONS: The cord blood cells, including hematopoietic stem cells, from newborns exposed in utero to a zidovudine-based ARV combination present cytogenetic and transcriptional abnormalities compatible with DNA damage.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Zidovudina/efeitos adversos , Adulto , Antígenos CD34/genética , Antígenos CD34/metabolismo , Ciclo Celular/genética , Reparo do DNA/genética , Combinação de Medicamentos , Feminino , Sangue Fetal/citologia , Sangue Fetal/fisiologia , Perfilação da Expressão Gênica/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Cariotipagem/métodos , Troca Materno-Fetal/fisiologia , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/virologia , Efeitos Tardios da Exposição Pré-Natal , Células-Tronco/metabolismo , Transcriptoma/genética , Adulto Jovem , Zidovudina/farmacocinéticaRESUMO
Antimoniate therapy, in association with allopurinol, is one of the first-line treatments of canine leishmaniasis (CanL). This study evaluates the potential adverse effects associated with aNm in the treatment of CanL through both a retrospective analysis and a long-term prospective study also aimed to investigate its efficacy. The retrospective study reviewed records of 87 dogs with CanL with at least one follow-up available during or at the end of therapy with aNm (Glucantime®) at a dose of 50 mg/kg administered subcutaneously twice a day in association with allopurinol. In total, 29.8% of dogs showed adverse effects during treatment as local reactions at the injection site (n = 6), severe systemic reaction to pain (originating from the inoculation site) with depression and anorexia (n = 4), systemic disease due to renal function worsening (n = 4), acute pancreatitis (n = 1), diarrhea (n = 5), vomiting (n = 3) and severe idiosyncratic skin reactions (n = 3). Of these dogs, 13 (14.9%) required treatment suspension. The prospective study included 16 dogs, selected among the LeishVet stages II and III CKD IRIS stage 1 (International Renal Interest Society staging of canine Chronic Kidney Disease) and treated with the same aNm plus allopurinol protocol as in the retrospective study and observed for 360 days; 2 dogs were excluded for severe reactions at the injection site. Mild and transient adverse events were reported in the other 4 dogs. The criteria used to evaluate the efficacy of treatment with aNm were as follows: a reduction in the clinical score and improvement and/or normalization of laboratory parameters, negativization of PCR on the bone marrow samples and disease-free interval time. The proportion of reduction in the clinical score reached 91.9% at D180. No animals showed clinical laboratory relapse during the whole study duration and interestingly, the PCR results showed complete negativity between D0 and D60 in 78.5% of animals. Veterinarians must be vigilant regarding the potentially serious adverse effects associated with aNm and promptly stop drug administration if unexpected clinical manifestations occur. On the other hand, they should not discard its use for CanL treatment since it is confirmed that aNm in association with allopurinol is highly effective in controlling CanL.
RESUMO
Canine strongyloidosis by Strongyloides stercoralis is a parasitic disease emerging in Europe, which represents both a veterinary clinical issue and a public health challenge because of the zoonotic potential. The disease, not yet frequent in Europe, could induce severe clinical signs in dogs; thus, an early diagnosis and appropriate treatment are desirable. The aim of the present work is to retrospectively investigate the clinical and paraclinical findings in sick dogs naturally infected by S. stercoralis, with particular attention to ultrasound (US) changes at the gastrointestinal level. Twelve dogs were included in the study. The diagnosis was made by means of larval morphological identification on faecal samples and PCR. Most dogs presented with gastrointestinal signs; diarrhea and weight loss were the most common presenting complaint. Only one dog showed respiratory signs, associated to a parasitic cutaneous nodule. Hypoproteinaemia, anaemia, leucocytosis and an increase in alpha2-globulin fraction at serum protein electrophoresis were common (>50%) but not constant findings. The most reported US picture was a fluid-filled, distended, atonic small intestine mostly associated with altered wall layering, while the wall thickness commonly associated with chronic enteritis was only rarely reported. These changes, associated with other clinical and paraclinical alterations, could increase the suspicion of canine strongyloidosis and may direct clinicians to include strongyloidosis in the differential diagnosis of dogs with diarrhea. The histological examination at the intestinal level, available in five dogs, revealed the presence of parasites from the full-thickness biopsy, but not from the endoscopic biopsy. The critical points of diagnosis in clinical practice are also discussed.
Assuntos
Doenças do Cão , Fezes , Strongyloides stercoralis , Estrongiloidíase , Animais , Cães , Doenças do Cão/parasitologia , Doenças do Cão/diagnóstico , Estrongiloidíase/veterinária , Estrongiloidíase/diagnóstico , Masculino , Feminino , Estudos Retrospectivos , Fezes/parasitologia , Strongyloides stercoralis/isolamento & purificação , Ultrassonografia/veterinária , Diarreia/veterinária , Diarreia/parasitologiaRESUMO
The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.
Assuntos
Células Epidérmicas , Epidermólise Bolhosa Juncional/terapia , Terapia Genética/métodos , Transplante de Células-Tronco , Células 3T3 , Adulto , Animais , Moléculas de Adesão Celular/genética , Células Cultivadas , Estudos de Viabilidade , Vetores Genéticos , Humanos , Masculino , Camundongos , Retroviridae , Engenharia Tecidual/métodos , CalininaRESUMO
Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and beta1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of beta1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in beta1 integrin-null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in beta1 integrin-null nerves improves sorting. Thus, increased activation of Rac1 by beta1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.
Assuntos
Axônios , Integrina beta1/fisiologia , Bainha de Mielina , Neuropeptídeos/metabolismo , Células de Schwann/citologia , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Movimento Celular , Extensões da Superfície Celular , Laminina , Camundongos , Camundongos Transgênicos , Ratos , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
6-Hydroxydopamine (6-OHDA) lesions are being used in the mouse for basic research on Parkinson's disease and L-DOPA-induced dyskinesia. We set out to compare unilateral lesion models produced by intrastriatal or intramesencephalic injections of a fixed 6-OHDA concentration (3.2 µg/µl) in C57BL/6 mice. In the first experiment, toxin injections were performed either at two striatal coordinates (1 or 2 µl per site, termed "striatum(2 × 1 µl)" and "striatum(2 × 2 µl)" models), in the medial forebrain bundle (MFB), or in the substantia nigra pars compacta (SN) (1 µl per site). All the four lesion models produced significant forelimb use asymmetry, but spontaneous turning asymmetry only occurred in the MFB and striatum(2 × 2 µl) models. After the behavioral studies, the induction of phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) by acute L-DOPA (30 mg/kg) was used as a marker of post-synaptic supersensitivity. Striatal pERK1/2 expression was sparse in the SN and striatum(2 × 1 µl) groups, but pronounced in the striatum(2 × 2 µl) and MFB-lesioned mice. In further experiments, mice with MFB and striatal(2 × 2 µl) lesions were used to compare behavioral and molecular responses to chronic L-DOPA treatment (12 days at 3 and 6 mg/kg/day). Maximally severe abnormal involuntary movements (AIMs) occurred in all MFB-lesioned mice, whereas only 35% of the mice with striatal lesions developed dyskinesia. Striatal tissue levels of dopamine were significantly lower in the dyskinetic animals (both MFB and striatum(2 × 2 µl) groups) in comparison with the non-dyskinetic ones. Noradrenaline levels were significantly reduced only in MFB lesioned animals and did not differ among the dyskinetic and non-dyskinetic cases with striatal lesions. In all groups, the L-DOPA-induced AIM scores correlated closely with the number of cells immunoreactive for tyrosine hydroxylase or FosB/∆FosB in the striatum. In conclusion, among the four lesion procedures examined here, only the MFB and striatum(2 × 2 µl) models yielded a degree of dopamine denervation sufficient to produce spontaneous postural asymmetry and molecular supersensitivity to L-DOPA. Both lesion models are suitable to reproduce L-DOPA-induced dyskinesia, although only MFB lesions yield a pronounced and widespread expression of post-synaptic supersensitivity markers in the striatum.
Assuntos
Corpo Estriado/fisiopatologia , Discinesia Induzida por Medicamentos/fisiopatologia , Levodopa/uso terapêutico , Oxidopamina/farmacologia , Doença de Parkinson/fisiopatologia , Substância Negra/fisiopatologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Contagem de Células , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Discinesia Induzida por Medicamentos/metabolismo , Imuno-Histoquímica , Levodopa/efeitos adversos , Levodopa/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
Parkinson's disease is associated with mutations in the glucocerebrosidase gene, which result in the enzyme deficiency causing Gaucher disease, the most common lysosomal storage disorder. We have performed an exhaustive literature search and found that additional lysosomal storage disorders might be associated with Parkinson's disease, based on case reports, the appearance of pathological features such as α-synuclein deposits in the brain, and substantia nigra pathology. Our findings suggest that the search for biochemical and cellular pathways that link Parkinson's disease with lysosomal storage disorders should not be limited exclusively to changes that occur in Gaucher disease, such as changes in glucocerebrosidase activity or in glucosylceramide levels, but rather include changes that might be common to a wide variety of lysosomal storage disorders. Moreover, we propose that additional genetic, epidemiological, and clinical studies should be performed to check the precise incidence of mutations in genes encoding lysosomal proteins in patients displaying Parkinson's symptoms.
Assuntos
Doença de Gaucher/epidemiologia , Doença de Gaucher/genética , Glucosilceramidase/genética , Doenças por Armazenamento dos Lisossomos/genética , Doença de Parkinson/genética , Doença de Gaucher/etiologia , Glucosilceramidase/deficiência , Humanos , Doenças por Armazenamento dos Lisossomos/epidemiologiaRESUMO
BACKGROUND: Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. METHODS: Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors. RESULTS: NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. CONCLUSIONS: Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.
Assuntos
Processamento Alternativo/genética , Fator de Ligação a CCAAT/metabolismo , Edição de Genes/métodos , Neoplasias da Próstata/genética , Animais , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.