背景:多能干细胞分化为所需谱系是再生医学和基于细胞的治疗的关键方面。尽管RNA干扰(RNAi)技术被广泛用于此,导致分化的靶基因的长期沉默方法仍然是一个挑战。由于递送效率低,RNAi对靶基因的持续敲除通常效率低下。方案引起与病毒载体相关的毒性和安全性问题。早些时候,我们建立了八精氨酸功能化的羟基磷灰石纳米载体(R8HNP),用于在小鼠胚胎干细胞中递送针对多能性标记基因的小干扰RNA(siRNA)。尽管我们证明了靶基因的优秀敲除效率,导致分化的持续基因沉默尚未实现.
方法:为了建立使用R8HNP的持续非病毒基因沉默方案,我们研究了siRNA递送的各种方法:粘附细胞的双重递送(Adh-D),悬浮递送,然后粘附递送(Susp+Adh),暂停单交货(Susp-S)和暂停多交货(Susp-R)。通过逆转录酶-PCR分析了多能标记基因的持续敲除,然后进行分化。荧光激活细胞分选和免疫荧光技术。还测试了重复暴露R8HNP对细胞活力的影响。
结果:在测试的方案中,通过重复悬浮递送R8HNP-siRNA缀合物,获得了长时间最有效的靶基因敲低.多能性标记基因的长期沉默导致R1ESC主要向胚胎外和外胚层谱系分化。细胞对R8HNP的重复暴露表现出优异的耐受性。
结论:结果表明,R8HNP是有希望的,生物相容性用于延长基因沉默和获得用于治疗的分化细胞的非病毒替代品。
BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (
RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by
RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved.
METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested.
RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs.
CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.