For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.
Huntington's disease (HD) is a neurodegenerative disorder caused by accumulation of CAG expansions in the huntingtin (HTT) gene. Hence, decreasing the expression of mutated HTT (mtHTT) is the most upstream approach for treatment of HD. We have developed HTT gene-silencing approaches based on expression cassette-optimized artificial miRNAs (miHTTs). In the first approach, total silencing of wild-type and mtHTT was achieved by targeting exon 1. In the second approach, allele-specific silencing was induced by targeting the heterozygous single-nucleotide polymorphism (SNP) rs362331 in exon 50 or rs362307 in exon 67 linked to mtHTT. The miHTT expression cassette was optimized by embedding anti-HTT target sequences in ten pri-miRNA scaffolds and their HTT knockdown efficacy, allele selectivity, passenger strand activity, and processing patterns were analyzed in vitro. Furthermore, three scaffolds expressing miH12 targeting exon 1 were incorporated in an adeno-associated viral serotype 5 (AAV5) vector and their HTT knock-down efficiency and pre-miHTT processing were compared in the humanized transgenic Hu128/21 HD mouse model. Our data demonstrate strong allele-selective silencing of mtHTT by miSNP50 targeting rs362331 and total HTT silencing by miH12 both in vitro and in vivo. Ultimately, we show that HTT knock-down efficiency and guide strand processing can be enhanced by using different cellular pri-miRNA scaffolds.
We detected a significant excess risk for CHEK2*1100delC mutation carriers to develop a contralateral breast tumor, OR = 6.5 (95% CI 1.5-28.8, p = 0.005). The highest percentage of mutation carriers was detected among those bilateral breast cancer patients who had received radiation treatment for their first breast tumor. These results warrant prolonged medical surveillance and may indicate a clinically important interaction between CHEK2 heterozygosity and radiation in the development of contralateral breast cancer.
Introduction Radiation exposure at a young age is one of the strongest risk factors for breast cancer. Germline mutations in genes involved in the DNA-damage repair pathway (DDRP) may render women more susceptible to radiation-induced breast cancer.
Heterozygous carriers of ATM mutations are at increased risk of breast cancer. In this case-control study, we evaluated the significance of germline ATM missense variants to the risk of contralateral breast cancer (CBC). We have determined the spectrum and frequency of ATM missense variants in 443 breast cancer patients diagnosed before age 50, including 247 patients who subsequently developed CBC. Twenty-one per cent of the women with unilateral breast cancer and 17% of the women with CBC had at least one ATM germline missense variant, indicating no significant difference in variant frequency between these two groups. We have found that carriers of an ATM missense mutation, who were treated with radiotherapy for the first breast tumour, developed their second tumour on average in a 92-month interval compared to a 136-month mean interval for those CBC patients who neither received RT nor carried a germline variant, (p = 0.029). Our results indicate that the presence of ATM variants does not have a major impact on the overall risk of CBC. However, the combination of RT and (certain) ATM missense variants seems to accelerate tumour development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.