Rational Design Of Responsive Polymers For Cytosolic Protein Delivery And Their Applications In CRISPR/Cas9 Gene Editing

As the most revolutionary biotechnology today,CRISPR/Cas9-based technology can target specific DNA sequences for precise editing,including gene insertion,deletion,or replacement.With its efficient gene editing capability,CRISPR/Cas9 has been widely applied in the treatment of various diseases,such as blood disorders,genetic blindness,diabetes,inflammatory diseases,cancer,etc.Among them,Cas9 ribonucleoprotein(RNP)was recognized as a promising gene editing tool due to its high specificity,safety,ease of operation and low off-target effects.The key to the function of Cas9 RNP in cells is how to enter the cells,which is also the most important and challenging work in the entire genome editing process.Currently,a variety of delivery platforms with different characteristics have been developed for intracellular delivery of Cas9 RNP,such as gold nanoparticles,lipid nanoparticles,cationic polymers.Compared with other carrier systems,cationic polymer carriers provide more options for the design and development of delivery systems because of their high delivery efficiency,structural and functional diversities,simple and rapid preparations,low costs,and have gradually become one of the most promising choices in Cas9 RNP delivery carriers.In order to further expand the options of efficient and safe carriers for the intracellular delivery and responsive release of Cas9 RNP,we designed a reactive oxygen species(ROS)degradable polypeptide carrier named PLLB3 for protein delivery and the treatment of acute liver injury mediated by Cas9 RNP.PLLB3 was obtained by modifying the biodegradable polylysine(PLL)with a ROS-responsive 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate(NBC)ligand.The treatment of PLLB3 with H2O2 can trigger the self-immolative reactions,yielding biodegradable PLL.PLLB3 can tightly bind proteins to form stable complexes with a size of about 200 nm via electrostatic,hydrophobic,nitrogen-boronate coordination and other interactions,and still maintain a high protein loading capacity in salt solutions.Intracellular delivery experiments indicated that PLLB3 can efficiently deliver multiple functional proteins including Cas9 RNP into He La cells with maintained bioactivities.The complex formed by PLLB3 and proteins is gradually disassembled to release the loaded protein under the treatment of H2O2,which is a prerequisite for achieving efficient intracellular protein delivery.By evaluating the gene editing efficiency of the PLLB3/Cas9 RNP complex,it was found that this delivery system was able to achieve specific editing of multiple targeted gene in different cells,and the efficiency is slightly higher than that of commercial Cas9 RNP delivery reagents.Finally,the experiment of Cas9 RNP targeting PUMA gene mediated treatment for acute liver injury mice revealed that the treatment group by PLLB3/Cas9 RNP complex achieved a gene editing efficiency of 10.5% in vivo.Besides,not only the levels of aspartate transaminase and alanine aminotransferase were significantly decreased by PLLB3/Cas9 RNP treatment,liver cell necrosis and hyperemia were recovered,and the survival period of mice with acute liver injury was also effectively prolonged after treatment.In summary,this study provides a simple strategy for the construction of biodegradable Cas9 RNP delivery carriers,and is expected to bring new possibilities for the treatment of diseases such as acute liver injury.On the other hand,to further improve the stability of cationic polymer carriers and their delivery efficiency in vivo,we designed a cationic polymer carrier library containing bifunctional ligands,from which carrier with the highest Cas9 RNP delivery efficiency was screened and used for treatment of psoriasis.The carrier library consists of 23 polyamidoamine dendrimers modified with different ratios of NBC and lipoic acid(LA)to generate GBLA,where the NBC and LA ligands are introduced for protein binding and reversing cross-linking,respectively to improve the complex stability.Compared with unmodified dendrimer,NBCmodified or LA-modified dendrimer,the bifunctional carriers show a higher protein delivery efficiency.In addition,GBLA is endowed with additional serum stability and responsive release due to the cross-linking between LA molecules.Moreover,carriers with the optimal delivery efficiency can be screened from the GBLA polymer library for specific proteins.Finally,the carrier with the highest delivery efficiency of Cas9 RNP was used for the gene editing treatment of mouse model with psoriasis.According to the results of evaluating sskin thickness,psoriasis area and severity index,trans epidermal water loss,skin hydration in model mice,the GBLA/Cas9 RNP complex treatment group effectively improved the symptoms of psoriasis.More importantly,the delivery system achieved 33.7% editing efficiency targeting NLRP3 gene in the psoriatic skin tissues of the model mice,and the expression levels of psoriasis-related inflammatory cytokines were also significantly downregulated in vivo.This study provides an effective strategy for gene editing-mediated psoriasis treatment and also a rich library of carrier tools for different protein therapies,allowing the selection of appropriate vectors for specific applications.Collectively,the responsive delivery system we designed and developed can deliver various proteins into cells with high efficiency and achieve responsive release intracellularly.For Cas9 RNP,these responsive delivery systems can achieve higher editing efficiency than commercial reagents for various targeting genes in different cells.Moreover,this thesis also provides new ideas and strategies for further therapeutic applications of CRISPR/Cas9 gene editing systems.