Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.
Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.
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DOI：
10.1021/acs.molpharmaceut.3c00455
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发表时间：
2023-12-04
影响因子：
4.9
通讯作者：
Nilsson, Bradley L.
中科院分区：
文献类型：
--
作者：
Jagrosse, Melissa L.;Baliga, Uday K.;Jones, Christopher W.;Russell, Jade J.;Garcia, Claudia I.;Najar, Rauf Ahmad;Rahman, Arshad;Dean, David A.;Nilsson, Bradley L.
短干扰RNA(SiRNA)寡核苷酸疗法通过访问RNA干扰(RNAi)途径来改变基因表达，在治疗一系列疾病方面具有巨大的前景；然而，它们在临床环境中的应用受到细胞递送方面的重大挑战的限制。在这里，我们报道了一系列修饰的环状两亲性细胞穿透肽(CAPS)的结构-功能研究，以确定多肽序列对(1)siRNA结合效率，(2)细胞递送和击倒效率，以及(3)内吞摄取机制的影响。评价了9个总序列Ac-C[XZ]4CG-NH2的环肽，其中X残基是疏水/芳香的(Phe、Tyr、Trp或Leu)，Z残基是带电/亲水的(Arg、Lys、Ser或Glu)，以及一个非环肽Ac-(WR)4G-NH2。环化是通过在侧翼半胱氨酸残基之间形成分子内二硫键来实现的。结合分析表明，强阳离子性质和能够参与CH-π相互作用的芳香族残基的存在导致了CAP序列与小干扰RNA最有效的相互作用。随着CAP疏水/芳香族含量的增加，Cap-siRNA结合量的增加顺序如下：His<；Phe<；Tyr<；Trp。在体外，阳离子电荷和二硫键约束的CAPS环化都可以提高siRNA的摄取。与所测试的环状、阳离子帽相比，净中性帽和非环肽显示出较低的siRNA转位效率。所有被测试的帽子都有助于有效的siRNA靶基因至少50%的敲除(与脂质体对照一样有效)，其中最好的帽子使>；80%的基因敲除。值得注意的是，基因敲除效率与CAP-siRNA内化效率没有很强的相关性，但与CAP-siRNA结合亲和力有中等程度的相关性。最后，利用小分子抑制剂和靶向敲除必需的内吞途径蛋白表明，大多数CAP-siRNA纳米颗粒通过网状蛋白和小窝蛋白介导的内吞作用促进siRNA的递送。这些结果有助于深入了解CAPS的设计原则，以促进siRNA的传递，以及这些肽将siRNA转运到细胞内的机制。这些研究还证明了多肽-siRNA结合、siRNA货物的细胞递送和功能基因敲除之间的关系的本质。这些性质之间的强烈相关性并不总是被观察到，这说明了设计用于寡核苷酸递送的最佳下一代材料的复杂性。
Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure–function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH–π interactions lead to CAP sequences that most effectively interact with siRNA. CAP–siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP–siRNA internalization efficiency but moderately correlates with CAP–siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP–siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide–siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.
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影响因子：
5.4
作者：
Mandal D;Lohan S;Sajid MI;Alhazza A;Tiwari RK;Parang K;Montazeri Aliabadi H
通讯作者：
Montazeri Aliabadi H
影响因子：
15
作者：
Hudson KL;Bartlett GJ;Diehl RC;Agirre J;Gallagher T;Kiessling LL;Woolfson DN
通讯作者：
Woolfson DN
影响因子：
12.4
作者：
Howard, Kenneth A.;Rahbek, Ulrik L.;Kjems, Jorgen
通讯作者：
Kjems, Jorgen
由siRNA结合融合蛋白介导的基因沉默被双链RNA结合结构域结构减弱。
DOI：
10.1038/mtna.2012.43
发表时间：
2012-11-13
期刊：
Molecular therapy. Nucleic acids
影响因子：
--
作者：
通讯作者：
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适体介导的siRNA传递。
影响因子：
14.9
作者：
Chu, Ted C.;Twu, Karen Y.;Ellington, Andrew D.;Levy, Matthew
通讯作者：
Levy, Matthew