RESUMO
Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.
Assuntos
Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Meduloblastoma/genética , Análise de Sequência de DNA/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Feminino , Genoma/genética , Histonas/metabolismo , Humanos , Meduloblastoma/patologia , Camundongos , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Criança , Cromossomos Humanos Par 9/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Meduloblastoma/classificação , Meduloblastoma/patologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Assuntos
Neoplasias Cerebelares/genética , Genoma Humano/genética , Meduloblastoma/genética , Envelhecimento/genética , Sequência de Aminoácidos , Transformação Celular Neoplásica , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/patologia , Criança , Cromatina/metabolismo , Cromossomos Humanos/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Proteínas Hedgehog/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/genética , Humanos , Meduloblastoma/classificação , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Metilação , Mutação/genética , Taxa de Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatases/genética , Poliploidia , Receptores de Superfície Celular/genética , Análise de Sequência de RNA , Transdução de Sinais , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells and that hamper the identification of early cancer-driving events among bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVVs) by which we could efficiently induce hepatocellular carcinoma (HCC) in three different mouse models. By virtue of the LVV's replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of four previously unknown liver cancer-associated genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. The newly identified genes are likely to play a role in human cancer because they are upregulated, amplified and/or deleted in human HCCs and can predict clinical outcomes of patients.
Assuntos
Carcinoma Hepatocelular/genética , Lentivirus/genética , Neoplasias Hepáticas/genética , Mutagênese Insercional , Oncogenes , Animais , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Vetores Genéticos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/deficiência , Pré-Albumina/genética , Receptor de Interferon alfa e beta/deficiênciaRESUMO
Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.
Assuntos
Fator IX/genética , Vetores Genéticos/administração & dosagem , Hemofilia B/terapia , Lentivirus/genética , Animais , Arginina/metabolismo , Códon , Modelos Animais de Doenças , Fator IX/metabolismo , Terapia Genética , Vetores Genéticos/uso terapêutico , Hemofilia B/patologia , Células Hep G2 , Humanos , Leucina/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
OBJECTIVES: The rapid early-phase decay of plasma HIV-1 RNA during integrase inhibitor-based therapy is not fully understood. The accumulation of biologically active episomal HIV-1 cDNAs, following aborted integration, could contribute to antiviral potency in vivo. METHODS: This prospective, controlled clinical observation study explored raltegravir's impact on the dynamics of HIV-1 RNA in plasma, and concentrations of total HIV-1 cDNA, episomal 2-long terminal repeat (LTR) circles and HIV-1 integrants in peripheral blood mononuclear cells (PBMC). Individuals starting therapy with two nucleoside reverse transcriptase inhibitors plus either raltegravir (raltegravir group; nâ=â10 patients) or boosted protease inhibitor/non-nucleoside reverse transcriptase inhibitor (control group; nâ=â10 patients) were followed for 48 weeks. RESULTS: Suppression of HIV-1 RNA (<50 copies/mL) was reached earlier (5/10 versus 0/10 at week 4; 8/10 versus 4/10 at week 12) on raltegravir. Significant total HIV-1 cDNA reductions in PBMC were reached by day 99 and persisted until day 330, with median factors of decrease of 7.2 and 8.9, respectively. Broad inter-individual variations, yet no treatment-associated differences, were noted for HIV-1 cDNA concentrations. Despite reductions in HIV-1 RNA (â¼3 log) and total HIV-1 cDNA (â¼1 log), concentrations of integrants and 2-LTR circles remained largely unchanged. CONCLUSIONS: These results extend the previously reported early benefit of raltegravir on the decline of plasma viraemia to treatment-naive patients. The modest treatment-associated, yet group-independent, decline in total HIV-1 cDNA load and the lack of significant changes in integrated and episomal HIV-1 cDNA suggest that most integrated DNA is archival and targeting of HIV reservoirs other than PBMC may underlie beneficial effects of raltegravir.
Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores de Integrase de HIV/farmacologia , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Pirrolidinonas/farmacologia , Pirrolidinonas/uso terapêutico , Adulto , Idoso , Feminino , Repetição Terminal Longa de HIV , HIV-1/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Provírus , RNA Viral , Raltegravir Potássico , Carga Viral , Integração Viral , Adulto JovemRESUMO
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.
Assuntos
Antígenos CD34/imunologia , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Vetores Genéticos , Lentivirus/genética , Transdução Genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Células da Medula Óssea/imunologia , Camundongos , Camundongos KnockoutRESUMO
UNLABELLED: Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. CONCLUSION: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.
Assuntos
Epitopos , Vetores Genéticos/genética , Hepatócitos , Tolerância Imunológica/genética , Integrases/genética , Lentivirus/enzimologia , Animais , Células Cultivadas , Dano ao DNA , Feminino , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RiscoRESUMO
X-linked chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by a defect in the gp91(phox) gene. In an effort to treat X-CGD, we investigated the safety and efficacy of gene therapy using a retroviral vector, MT-gp91. Two X-CGD patients received autologous CD34(+) cells transduced with MT-gp91 after a conditioning regimen consisting of fludarabine and busulfan. The level of gene-marked cells was highest at day 21 (8.3 and 11.7% in peripheral blood cells) but decreased to 0.08 and 0.5%, respectively, 3 years after gene transfer. The level of functionally corrected cells, as determined by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assay, reached a peak at day 17 (6.5% patient 1 (P1) and 14.3% patient 2 (P2) of total granulocytes) and declined to 0.05% (P1) and 0.21% (P2), 3 years later. Some retroviral vectors were found to have integrated within or close to the proto-oncogenes MDS1-EVI1, PRDM16, and CCND2; however, no abnormal cell expansion or related hematological malignancy was observed. Overall, the gene transfer procedure did not produce any serious adverse effects and was able to convert a significant fraction of blood cells to biologically functional cells, albeit for a short period of time.
Assuntos
Terapia Genética , Vetores Genéticos , Doença Granulomatosa Crônica/terapia , Retroviridae/genética , Adolescente , Criança , Perfilação da Expressão Gênica , Vetores Genéticos/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , Transdução Genética , Resultado do Tratamento , Integração ViralRESUMO
Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Mitose/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , DNA Satélite/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Ratos , Sequências Repetidas Terminais/genética , Fatores de Transcrição/genética , Integração Viral/genéticaRESUMO
Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.
Assuntos
Anemia de Fanconi/terapia , Terapia Genética/métodos , Congressos como Assunto , Células-Tronco Hematopoéticas/citologia , Humanos , Transplante de Células-Tronco/métodosRESUMO
Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.
Assuntos
Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Genoma , Integração Viral , Animais , Mapeamento Cromossômico , Redes Reguladoras de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Primatas , Transplantes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapiaRESUMO
In order to restore or to introduce a gene function integrating viral vector systems are used to genetically modify hematopoietic stem cells. The occurrence of immortalized cell clones after transduction in vitro (Blood 106:3932-3939, 2005) and clonal dominance as well as leukemia in preclinical (Nat. Med. 12:401- 409, 2006; Blood 106:2530-2533, 2005; Science 308:1171-1174, 2005; Science 296:497, 2002; Blood 107:3865-3867, 2006) and clinical (Nat. Med. 12:401-409, 2006; Science 302:415-419, 2003; J. Clin. Invest. 118:3143-3150, 2008) gene therapy trials revealed that the nondirected integration of a vector may be associated with serious side effects. By means of the linear amplification-mediated PCR (LAM-PCR) (Blood 100:2737-2743, 2002; Nat. Methods 4:1051-1057, 2007) it is possible to identify miscellaneous vector-genome junctions in one sample, each unique for one integration clone down to the single cell level. Thus this method allows to determine the clonality of a genetically modified hematopoietic repopulation as well as to sequence the vector integration sites and therefore to analyze the integration site distribution and the influence of the vector integration site on the cell fate. The recognition of the integration site sequence corresponding to a specific clone allows the tracking of an individual clone in various samples.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Reação em Cadeia da Polimerase/métodos , Retroviridae/genética , Integração Viral , Sequência de Bases , Primers do DNARESUMO
Integrating viral gene transfer vectors are commonly used gene delivery tools in clinical gene therapy trials providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones. To date, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vitro and in vivo. We have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by non-restrictive linear amplification-mediated PCR (nrLAM-PCR) and LAM-PCR. MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol. Here, we present an updated protocol of (nr)LAM-PCR for the analysis of lentiviral IS using a double-barcode system and followed by deep sequencing using the MiSeq device.
Assuntos
Técnicas de Transferência de Genes , Lentivirus/genética , Integração Viral/genética , Terapia Genética/métodos , Vetores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.
Assuntos
Terapia Genética , Hemofilia B/terapia , Lentivirus/genética , Fígado/patologia , Animais , Coagulação Sanguínea , Modelos Animais de Doenças , Cães , Feminino , Vetores Genéticos/metabolismo , Camundongos Endogâmicos C57BL , Mutagênicos/toxicidade , Fatores de Tempo , Transdução Genética , TransgenesRESUMO
Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3'- and 5'- sequences adjacent to the integrated lentiviral vector.
Assuntos
DNA/análise , Reação em Cadeia da Polimerase/métodos , DNA/genética , Vetores Genéticos/análise , Vetores Genéticos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lentivirus/genética , Retroviridae/genéticaRESUMO
Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor (hGDNF) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Lentivirus , Fármacos Neuroprotetores/metabolismo , Doença de Parkinson/terapia , Animais , Sobrevivência Celular/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Adolescente , Adulto , Animais , Sequência de Bases , Compostos de Bifenilo/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Criança , Pré-Escolar , RNA Helicases DEAD-box/genética , Variações do Número de Cópias de DNA/genética , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Fatores de Transcrição Kruppel-Like/genética , Masculino , Meduloblastoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Transplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Receptores Patched , Receptor Patched-1 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/uso terapêutico , Receptores de Superfície Celular/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Receptor Smoothened , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Adulto Jovem , Proteína Gli2 com Dedos de ZincoRESUMO
Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.
Assuntos
Gammaretrovirus/genética , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/virologia , Lentivirus/genética , Sequências Repetidas Terminais/genética , Animais , Células Clonais/citologia , Células Clonais/metabolismo , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Cinética , Camundongos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.