以羟基磷灰石为基础的生物相容性纳米载体绕过了病毒转导,实现了多能性标记基因的持续沉默,证明了小鼠胚胎干细胞的理想分化。

IF 4.3 3区 材料科学 Q1 ENGINEERING, ELECTRICAL & ELECTRONIC ACS Applied Electronic Materials Pub Date : 2024-07-04 DOI:10.1002/jgm.3716
Pranjita Zantye, Asha Dahiya, Meenal Kowshik, Sutapa Roy Ramanan, Indrani Talukdar
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引用次数: 0

摘要

背景:将多能干细胞分化成所需的细胞系是再生医学和细胞疗法的关键环节。虽然 RNA 干扰(RNAi)技术在这方面得到了广泛应用,但长期沉默导致分化的靶基因的方法仍然是一个挑战。用 RNAi 技术持续敲除靶基因的效率往往不高,原因包括传递效率低、方案诱导毒性以及与病毒载体相关的安全问题。早些时候,我们建立了八精氨酸功能化羟基磷灰石纳米载体(R8HNPs),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)。虽然我们证明了目标基因的极佳敲除效率,但尚未实现导致分化的持续基因沉默:为了利用 R8HNP 建立一种持续的非病毒基因沉默方案,我们研究了多种 siRNA 递送方法:粘附细胞双递送(Adh-D)、悬浮递送后粘附递送(Susp + Adh)、悬浮液中单次递送(Susp-S)和悬浮液中多次递送(Susp-R)。通过反转录酶-PCR、荧光激活细胞分选和免疫荧光技术分析了多能标记基因持续敲除后的分化情况。此外,还测试了重复暴露 R8HNP 对细胞活力的影响:结果:在测试的各种方案中,重复悬浮递送 R8HNP-siRNA 共轭物能最有效地长期敲除目标基因。长期沉默多能性标记基因导致 R1 ESCs 主要向胚外和外胚层系分化。细胞对反复暴露于 R8HNPs 表现出极好的耐受性:研究结果表明,R8HNPs 是一种前景广阔、生物相容性好的非病毒替代品,可用于长期基因沉默和获得用于治疗的分化细胞。
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Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells

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.

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