Inducible transgenic expression of tripeptidyl peptidase 1 in a mouse model of late-infantile neuronal ceroid lipofuscinosis

Late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease of children caused by mutations resulting in loss of activity of the lysosomal protease, tripeptidyl peptidase 1 (TPP1). While Tpp1-targeted mouse models of LINCL exist, the goal of this study was to create a transgenic mouse with inducible TPP1 to benchmark treatment approaches, evaluate efficacy of treatment at different stages of disease, and to provide insights into the pathobiology of disease. A construct containing a loxP-flanked stop cassette inserted between the chicken-actin promoter and a sequence encoding murine TPP1 (TgLSL-TPP1) was integrated into the ROSA26 locus in mice by homologous recombination. Tested in both transfected CHO cells and in transgenic mice, the TgLSL-TPP1 did not express TPP1 until cre-mediated removal of the LSL cassette, which resulted in supraphysiological levels of TPP1 activity. We tested four cre/ERT2 transgenes to allow tamoxifen-inducible removal of the LSL cassette and subsequent TPP1 expression at any stage of disease. However, two of the cre/ERT2 driver transgenes had significant cre activity in the absence of tamoxifen, while cre-mediated recombination could not be induced by tamoxifen by two others. These results highlight potential problems with the use of cre/ERT2 transgenes in applications that are sensitive to low levels of basal cre expression. However, the germline-recombined mouse transgenic that constitutively overexpresses TPP1 will allow long-term evaluation of overexposure to the enzyme and in cell culture, the inducible transgene may be a useful tool in biomarker discovery projects.

晚发性婴儿型神经元蜡样脂褐质沉积症（Late-infantile neuronal ceroid lipofuscinosis, LINCL）是一种致命的儿童神经退行性疾病，由导致溶酶体蛋白酶三肽肽酶1（tripeptidyl peptidase 1, TPP1）活性丧失的突变引发。尽管已有针对Tpp1的LINCL小鼠模型，但本研究的目标是构建一种可诱导表达TPP1的转基因小鼠，以此为治疗方案建立基准评估体系，评估疾病不同阶段的治疗效果，并为阐明该疾病的病理生物学机制提供见解。研究人员将一段两侧带有loxP位点的终止盒插入鸡肌动蛋白启动子与编码小鼠TPP1的序列之间，通过同源重组将该构建体（TgLSL-TPP1）整合至小鼠的ROSA26基因座中。在转染的中国仓鼠卵巢（CHO）细胞与转基因小鼠中均验证发现，TgLSL-TPP1仅在cre介导切除LSL盒后才会表达TPP1，此时可达到超生理水平的TPP1活性。我们测试了四种cre/ERT2转基因，以实现他莫昔芬诱导型切除LSL盒，进而在疾病任意阶段启动TPP1表达。但其中两种cre/ERT2驱动转基因在未添加他莫昔芬时即存在显著的cre活性，而另外两种则无法通过他莫昔芬诱导cre介导的重组。这些结果凸显了在对基础cre表达水平敏感的应用中使用cre/ERT2转基因可能存在的潜在问题。不过，组成型过表达TPP1的种系重组转基因小鼠可用于长期评估该酶的过度暴露效应；而在细胞培养实验中，该可诱导转基因或可成为生物标志物发现项目中的实用工具。