Lipid nanoparticle-mediated efficient delivery of CRISPR/Cas9 for tumor therapy

The emerging CRISPR/Cas9 system represents a promising platform for genome editing. However, its low transfection efficiency is a major problem hampering the application of the gene-editing potential of CRISPR/Cas9. Herein, by screening a pool of more than 56 kinds of agents, we constructed a novel polyethylene glycol phospholipid-modified cationic lipid nanoparticle (PLNP)-based delivery system that can condense and encapsulate a Cas9/single-guide RNA (sgRNA) plasmid (DNA) to form a core–shell structure (PLNP/DNA) that mediated up to 47.4% successful transfection of Cas9/sgPLK-1 plasmids in A375 cells in vitro. An intratumor injection of Cas9/sgPLK-1 plasmids into melanoma tumor-bearing mice resulted in significant downregulation of Polo-like kinase 1 (PLK-1) protein and suppression of the tumor growth (>67%) in vivo. This approach provides a versatile method that could be used for delivering the CRISPR/Cas9 system with high efficiency and safety both in vitro and in vivo. A material for delivering CRISPR–Cas9 to the nuclei of cells has been developed by researchers in China. CRISPR–Cas9 is a powerful gene editing system found in bacteria. Scientists have recently harnessed it to edit genes in mammalian cells and even human embryos, opening the door to a host of revolutionary medical treatments. But the plasmid encoding CRISPR–Cas9 is a large nucleic acid, which limits the efficiency with which it can enter target cells. Now, Xingyu Jiang from the National Center for Nanoscience and Technology, Beijing, and colleagues have demonstrated a versatile method for delivering CRISPR–Cas9 efficiently and safely. After screening more than 56 agents, they constructed polyethylene glycol phospholipid–modified cationic lipid nanoparticles to encapsulate CRISPR–Cas9, which allowed nanoparticles to be delivered to melanoma cells with an efficiency of 47%. Our work contributes to synthesize a vehicle based on lipid nanoparticles, which can effectively deliver Cas9/sgRNA-fused plasmid DNA in vitro and in vivo. This approach mediated successful transfection of Cas9/sgRNA plasmids in multiple cell lines in vitro. The vehicle carrying Cas9/sgRNA targeting PLK-1 resulted in significant down-regulation of PLK-1 protein and suppression of melanoma growth in vivo.

[1]  F. Dosio,et al.  Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential , 2006, International journal of nanomedicine.

[2]  E. Lander,et al.  Development and Applications of CRISPR-Cas 9 for Genome Engineering , 2015 .

[3]  James E. DiCarlo,et al.  Supplementary Materials for RNA-Guided Human Genome Engineering via Cas 9 , 2012 .

[4]  Robert Langer,et al.  CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling , 2014, Cell.

[5]  Henk-Jan Guchelaar,et al.  Liposomal drug formulations in cancer therapy: 15 years along the road. , 2012, Drug discovery today.

[6]  A. Nel,et al.  Correction to Use of a Lipid-Coated Mesoporous Silica Nanoparticle Platform for Synergistic Gemcitabine and Paclitaxel Delivery to Human Pancreatic Cancer in Mice , 2016, ACS nano.

[7]  Jessica Zucman-Rossi,et al.  Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas , 2015, Nature Genetics.

[8]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[9]  Baoquan Ding,et al.  Tunable Rigidity of (Polymeric Core)–(Lipid Shell) Nanoparticles for Regulated Cellular Uptake , 2015, Advanced materials.

[10]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[11]  Liangfang Zhang,et al.  Hydrogel Containing Nanoparticle-Stabilized Liposomes for Topical Antimicrobial Delivery , 2014, ACS nano.

[12]  Qiang Zhang,et al.  Synergistic inhibition of breast cancer by co-delivery of VEGF siRNA and paclitaxel via vapreotide-modified core-shell nanoparticles. , 2014, Biomaterials.

[13]  Gert Storm,et al.  Sheddable Coatings for Long-Circulating Nanoparticles , 2007, Pharmaceutical Research.

[14]  Xiaojun Zhu,et al.  Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos , 2013, Cell Research.

[15]  John G. Doench,et al.  Ophir Shalem Genome-Scale CRISPR-Cas 9 Knockout Screening in Human Cells , 2014 .

[16]  Xingyu Jiang,et al.  Gene regulation with carbon-based siRNA conjugates for cancer therapy. , 2016, Biomaterials.

[17]  Yu-cheng Tseng,et al.  Lipid-based systemic delivery of siRNA. , 2009, Advanced drug delivery reviews.

[18]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

[19]  J. Keith Joung,et al.  Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo , 2014, Nature Biotechnology.

[20]  Xingyu Jiang,et al.  Microfluidic Synthesis of Hybrid Nanoparticles with Controlled Lipid Layers: Understanding Flexibility-Regulated Cell-Nanoparticle Interaction. , 2015, ACS nano.

[21]  Daniel G. Anderson,et al.  Genome editing with Cas 9 in adult mice corrects a disease mutation and phenotype Citation , 2014 .

[22]  R. Ketteler,et al.  A CRISPR CASe for high-throughput silencing , 2013, Front. Genet..

[23]  Chao Wang,et al.  Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. , 2015, Angewandte Chemie.

[24]  Hao Yin,et al.  CRISPR-mediated direct mutation of cancer genes in the mouse liver , 2014, Nature.

[25]  Bo Zhang,et al.  Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish , 2013, Nucleic acids research.

[26]  Jürgen Bereiter-Hahn,et al.  Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells. , 2002, Journal of the National Cancer Institute.

[27]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[28]  Sarah Seifert,et al.  Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape , 2013, Nature Biotechnology.