rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Abstract Background Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. Methods For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. Results The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Conclusions Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation

The literature is increasing more slowly for humanized mouse models than for GWASs and HWGS or novel mouse models.

<p>Interrogation of the PubMed literature database (<a href="http://www.ncbi.nlm.nih.gov/pubmed" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed</a>) reveals a faster growing body of literature related to GWASs and HWGS (white bars) or novel mouse models (grey bars) than to HuMMs (black bars). Interrogation of the database was done using the online search option from EndNote (<a href="http://www.endnote.com/" target="_blank">http://www.endnote.com/</a>). Individual numbers of entries for the search terms “genome wide association studies” and “human whole genome sequencing” were added together for the figure. Search terms for novel mouse models were “novel knockout mouse”, “novel knockin mouse”, and “novel knock-in mouse”. The entries for the search term “humanized mouse models” were not restricted to genetic mouse models but included xenograft mouse models as well. Search terms were interrogated in “all fields” per year.</p

High-throughput generation of regulatory allele series.

<p>(A) Human BAC RP11-144P8 was retrofitted seven times to generate the different regulatory variants (column 1). The method of retrofitting (column 2), targeting (column 3), and variant screening (column 4) is presented for each variant. Also given are the number of ESC clones isolated after electroporation (column 5), the number of correctly targeted clones after PCR validation using assays an average of 6 kb, and a maximum of 11 kb, apart (column 6), and the resulting percentage of correctly targeted clones (column 7). (B) Species-specific reverse transcriptase PCR demonstrates transcription from the human BAC in germline animals from four of the strains generated by the high-throughput approach. One-step reverse transcription PCR reactions were performed using oligonucleotides specific for human <i>NR2E1</i>, mouse <i>Nr2e1</i>, and mouse <i>Gapdh</i>. The results show, as expected, expression of the human <i>NR2E1</i> gene in adult eye, forebrain, and midbrain, but not in adult lung, heart, and liver. Marker, 100-bp ladder; positive control (Ctl+), human RNA for human <i>NR2E1</i> assay and mouse RNA for mouse <i>Nr2e1</i> and <i>Gapdh</i> assays; negative control (Ctl−), human RNA for mouse <i>Nr2e1</i> and <i>Gapdh</i> assays and mouse RNA for human <i>NR2E1</i> assay.</p

What explains between-school differences in rates of smoking?

BACKGROUND: Schools have the potential to influence their pupils' behaviour through the school's social organisation and culture (non-formal school characteristics), as well as through the formal curriculum. This paper examines whether these school characteristics (which include a measure of quality of social relationships) can account for school differences in smoking rates. METHODS: This study uses a longitudinal survey involving 5,092 pupils in 24 Scottish schools. Pupils' smoking (at age 15/16), cognitive measures, attitude to school and pupils' rating of teacher pupil relationships (at age 13/14) were linked to school level data comprising teacher assessed quality of pupil-staff relationships, school level deprivation, staying on rates and attendance. Analysis involved multi-level modelling. RESULTS: Overall, 25% of males and 39% of females reported smoking, with rates by school ranging from 8% to 33% for males and from 28% to 49% for females. When individual socio-economic and socio-cultural factors were controlled for there was still a large school effect for males and a smaller (but correlated) school effect for females at 15/16 years. For girls their school effect was explained by their rating of teacher-pupil relationships and attitude to school. These variables were also significant in predicting smoking among boys. However, the school effect for boys was most radically attenuated and became insignificant when the interaction between poor quality of teacher - pupil relationships and school level affluence was fitted, explaining 82% of the variance between schools. In addition, researchers' rating of the schools' focus on caring and inclusiveness was also significantly associated with both male and female smoking rates. CONCLUSION: School-level characteristics have an impact on male and female pupils' rates of smoking up to 15/16 years of age. The size of the school effect is greater for males at this age. The social environment of schools, in particular the quality of teacher-pupil relationships, pupils' attitude to school and the school's focus on caring and inclusiveness, can influence both boys' and girls' smoking. This provides support for the school-wide or "Health Promoting School" approach to smoking prevention

rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Background: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. Methods: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. Results: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Conclusions: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.Medicine, Faculty ofOther UBCNon UBCMedical Genetics, Department ofPsychiatry, Department ofReviewedFacult

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development

BACKGROUND: Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. RESULTS: In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. CONCLUSIONS: In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1770-3) contains supplementary material, which is available to authorized users