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

Snack Time at UBC : A Nutrition Assessment of the The Acadia’s Snack Program

The Acadia is one of twenty-five Childcare Centres situated on the Point Grey campus currently operated by University of British Columbia. There are a total of 5 staff members (4 early childhood educators; 1 senior supervisor) and 25 children (3-5 years of age) at this location. The objective of this case study was to determine the overall nutritional adequacy of the snacks provided by the daycare centre and the impact of daycare staff knowledge and behaviors through the use of both dietary (two direct observations sessions of snack time) and ecological assessment methods (self-reported questionnaire for daycare staff). Questionnaire results indicated that staff placed a great emphasis on nutritional value when choosing snacks for children. However, observations revealed inconsistencies regarding portion control. In addition, the children were served processed snacks alongside healthier options (vegetables), leading to a perceived reduction in interest towards the healthier options, as the children needed to be coaxed to eat the vegetables. Through the questionnaire result, time allocation for preparing the snack is not an issue as staffs usually take approximately 15-30 minutes each day. There is a general awareness of providing healthier snacks to the children by the staff; however, budget is a factor when making snack purchases. The children at Acadia come from relatively educated and affluent families with adequate access to safe and sufficient amounts of food. The children are generally healthy and did not exhibit any signs of undernutrition. Based on these observations, we concluded that no nutrient deficiencies were present in the children. However, we have suggestions for how the snack program could be improved. Our first proposed intervention is based on improving the nutritional value of the snacks. Staff indicated that they already avoid providing high sugar and fat snacks. Thus, we suggest choosing low sodium and whole grain options when available. A diet low in sodium and high in whole grains have been associated with a reduced risk of chronic diet-related diseases. Our second proposed intervention would involve altering staff behavior. As communal snacks were served, staff did not have complete control over each child’s intake, creating the possibility of over- and under-consumption. We suggest snacks be pre-portioned to avoid this. Furthermore, processed snacks and healthier options were served at the same time, and children exhibited less interest in the healthier options as a result. In the future, healthier options could be served first to encourage their consumption. Disclaimer: “UBC SEEDS provides students with the opportunity to share the findings of their studies, as well as their opinions, conclusions and recommendations with the UBC community. The reader should bear in mind that this is a student project/report and is not an official document of UBC. Furthermore readers should bear in mind that these reports may not reflect the current status of activities at UBC. We urge you to contact the research persons mentioned in a report or the SEEDS Coordinator about the current status of the subject matter of a project/report.”Land and Food Systems, Faculty ofUnreviewedUndergraduat

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