TAxI-peptide targeted Cas12a ribonuclease protein nanoformulations increase genome editing in hippocampal neurons.
TAxI-peptide targeted Cas12a ribonuclease protein nanoformulations increase genome editing in hippocampal neurons.
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DOI：
10.1016/j.jconrel.2022.12.057
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发表时间：
2022-12
期刊：
影响因子：
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通讯作者：
D. Sellers;Kunwoo Lee;N. Murthy;S. Pun
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文献类型：
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作者：
D. Sellers;Kunwoo Lee;N. Murthy;S. Pun
利用簇状规则间隔短回文重复序列(CRISPR)核糖核酸酶的基因治疗方法在治疗人类疾病方面具有巨大的潜力。然而，通过整合病毒载体提供的CRISPR疗法受到核糖核酸酶功能结构性激活引起的潜在的脱靶基因组编辑的限制。因此，生物材料配方正被用于提供纯化的CRISPR组件，以提高基因组编辑方法的效率和安全性。我们先前证明了一种通过噬菌体展示鉴定的新的多肽，THY-多肽，介导重组蛋白向神经元的递送。在这份报告中，我们利用中性亲和素蛋白来制备神经元靶向的基因组编辑纳米颗粒。将生物素化的导向RNA和生物素化的三肽负载到中性亲和素蛋白上，形成靶向核糖核酸酶蛋白(RNP)复合体。TAKI-RNP络合物是多分散的，半径为14.3 nm。制备后的纳米粒稳定性好，在正常小鼠血清存在下表现出良好的稳定性。出租车-RNP纳米颗粒增加了报告小鼠的Cas12a的神经元递送，导致直接注射到海马齿状回后诱导tdTomato的表达。与胶质细胞相比，出租车-RNP纳米颗粒还提高了海马神经元的基因组编辑效率。这些研究证明，通过将生物素化的多肽和加载gRNA的Cas12a核糖核酸酶结合到蛋白质纳米颗粒中，将CRISPR靶向体内特定类型的细胞，可以将RNP纳米制剂与中性亲和素组装在一起。将CRISPR纳米颗粒输送到特定类型的细胞并控制非靶标输送以进一步减少有害的基因组编辑的可能性，对于创造治疗神经系统疾病的可行疗法至关重要。
Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.