Development of a Genetic Therapy for CF in a CF Rabbit

描述(申请人提供)：囊性纤维化转基因小鼠模型的发展对我们理解囊性纤维化和囊性纤维化跨膜电导调节功能有重要贡献。然而，由于小鼠和人类在解剖学和生理学上的不同，对CF的病理认识和治疗方法的发展都存在局限性。在这种背景下，通过核移植成功克隆哺乳动物动物的出现，为开发像CF这样的遗传性疾病的大型动物模型打开了一扇门。最近对基于寡核苷酸的基因组基因打靶的研究表明，其中一些方法可能对作为核移植遗传物质来源的体细胞核进行修饰是有效的。因此，现在似乎有可能产生一种更合适的CF动物模型。作为老鼠模型替代品的一个候选动物是兔子，它在系统发育和解剖学上与人类的亲缘关系比老鼠更近。最近对兔CFTR(RCFTR)基因的主要部分进行了测序，现在已经提供了操纵rCFTR序列和产生转基因CF动物所需的遗传信息。小片段同源替换(SFHR)基因打靶策略是一种基于寡核苷酸的方法，以前曾被用来通过引入导致在CF患者群体中占主导地位的？F508、CFTR突变的BP缺失来修改人和小鼠的CFTR。因此，它非常适合于产生可用于核移植的转基因细胞系。该方案将利用SFHR将？F508突变导入兔胎儿成纤维细胞的rCFTR中。这些转基因供体细胞将通过荧光激活细胞分选(FACS)进行克隆浓缩，然后与去核的兔卵母细胞融合，最终用于生成CF兔。转基因供体细胞将通过聚合酶链式反应进行初步筛选，然后进行Southern杂交鉴定。移植的卵母细胞也将进行聚合酶链式反应筛选，然后在体外测试胚胎发生潜力。那些显示出胚胎发生潜力的卵母细胞将被引入代孕母亲体内。转基因CF兔的研制将极大地提高我们评估CF病理的能力，并将促进新的治疗方案的开发，以改善CF患者的生活质量。

DESCRIPTION (provided by applicant): The development of transgenic mouse models for cystic fibrosis (CF) has been an important contribution to our understanding of CF and of CF transmembrane conductance regulator (CFTR) function. However, because of the dissimilarity between mouse and human anatomy and physiology, there are limitations in what can be discerned about CF pathology and in development of CF therapies. In this context, the advent of successful mammalian animal cloning by nuclear transfer has opened the door to a host of possibilities to develop large animal models for inherited diseases like CF. Recent studies investigating oligonucleotide based genomic gene targeting suggest that some of these approaches might be effective for modifying somatic nuclei that act as the source of genetic material for nuclear transplantation. Thus it now appears possible to produce a more appropriate animal model of CF. A candidate as an alternative to the mouse model is the rabbit which is phylogenetically and anatomically more closely related to human than is the mouse. The recent sequencing of major portions of rabbit CFTR (rCFTR) gene has now provided the genetic information necessary to manipulate the rCFTR sequence and generate a transgenic CF animal. The small fragment homologous replacement (SFHR) gene targeting strategy is an oligonucleotide-based approach that has been previously used to modify both human and mouse CFTR by introducing the bp deletion that gives rise to the ?F508, CFTR mutation that predominates in the CF patient population. It is, therefore, well suited to generate transgenic cell lines that can be used for nuclear transplantation. This proposal will employ SFHR to introduce a ?F508 mutation into the rCFTR of rabbit fetal fibroblasts. These transgenic donor cells will be clonally enriched by Fluorescence Activated Cell Sorting (FACS) and then be fused with enucleated rabbit oocytes that will ultimately be used for the generation of a CF rabbit. The transgenic donor cells will be initially screened by PCR and then evaluated by Southern blot hybridization. Transplanted oocytes will also be PCR screened and then tested in vitro for embryogenic potential. Those oocytes that have demonstrated embryogenic potential will be introduced into surrogate mothers. The development of a transgenic CF rabbit will greatly enhance our ability to evaluate CF pathology and will facilitate development of new therapeutic regimens to improve the quality of life of CF patients.