RESUMEN
A recent clinical trial for adrenoleukodystrophy (ALD) showed the efficacy and safety of lentiviral vector (LV) gene transfer in hematopoietic stem progenitor cells. However, several common insertion sites (CIS) were found in patients' cells, suggesting that LV integrations conferred a selective advantage. We performed high-throughput LV integration site analysis on human hematopoietic stem progenitor cells engrafted in immunodeficient mice and found the same CISs reported in patients with ALD. Strikingly, most CISs in our experimental model and in patients with ALD cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CISs found in tumor cells from γ-retroviral vector-based clinical trials and oncogene-tagging screenings in mice always target a single gene and are contained in narrow genomic intervals. These findings imply that LV CISs are produced by an integration bias toward specific genomic regions rather than by oncogenic selection.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/terapia , Animales , Ensayos Clínicos como Asunto , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Técnicas de Transferencia de Gen , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Noqueados , Quimera por Trasplante/genética , Integración Viral/genéticaRESUMEN
The epigenetic mechanisms that enable lifelong neurogenesis from neural stem cells (NSCs) in the adult mammalian brain are poorly understood. Here, we show that JMJD3, a histone H3 lysine 27 (H3K27) demethylase, acts as a critical activator of neurogenesis from adult subventricular zone (SVZ) NSCs. JMJD3 is upregulated in neuroblasts, and Jmjd3 deletion targeted to SVZ NSCs in both developing and adult mice impairs neuronal differentiation. JMJD3 regulates neurogenic gene expression via interaction at not only promoter regions but also neurogenic enhancer elements. JMJD3 localizes at neural enhancers genome-wide in embryonic brain, and in SVZ NSCs, JMJD3 regulates the I12b enhancer of Dlx2. In Jmjd3-deleted SVZ cells, I12b remains enriched with H3K27me3 and Dlx2-dependent neurogenesis fails. These findings support a model in which JMJD3 and the poised state of key transcriptional regulatory elements comprise an epigenetic mechanism that enables the activation of neurogenic gene expression in adult NSCs throughout life.
Asunto(s)
Encéfalo/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Neurogénesis , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Células Cultivadas , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Gamma-retroviral/lentiviral vectors (γRV/LV) with self-inactivating (SIN) long terminal repeats (LTRs) and internal moderate cellular promoters pose a reduced risk of insertional mutagenesis when compared with vectors with active LTRs. Yet, in a recent LV-based clinical trial for ß-thalassemia, vector integration within the HMGA2 gene induced the formation of an aberrantly spliced mRNA form that appeared to cause clonal dominance. Using a method that we developed, cDNA linear amplification-mediated PCR, in combination with high-throughput sequencing, we conducted a whole transcriptome analysis of chimeric LV-cellular fusion transcripts in transduced human lymphoblastoid cells and primary hematopoietic stem/progenitor cells. We observed a surprising abundance of read-through transcription originating outside and inside the provirus and identified the vector sequences contributing to the aberrant splicing process. We found that SIN LV has a sharply reduced propensity to engage in aberrant splicing compared with that of vectors carrying active LTRs. Moreover, by recoding the identified vector splice sites, we reduced residual read-through transcription and demonstrated an effective strategy for improving vectors. Characterization of the mechanisms and genetic features underlying vector-induced aberrant splicing will enable the generation of safer vectors, with low impact on the cellular transcriptome.