Enhanced Gene Delivery and CRISPR/Cas9 Homology-Directed Repair in Serum by Minimally Succinylated Polyethylenimine

Abstract

Gene therapy aims to treat patients by altering or controlling gene expression. The field of gene therapy has had increasing success in recent years primarily using viral-based approaches; however, there is still significant interest toward the use of polymeric materials due to their potential as flexible, low-cost scaffolds for gene delivery that do not suffer the mutagenesis and immunogenicity concerns of viral vectors. To address the challenges of efficiency and biocompatibility, a series of zwitterion-like polyethylenimine derivatives (zPEIs) were produced via the succinylation of 2–11.5% of polyethylenimine (PEI) amines. With increasing modification, zPEI polyplexes exhibited decreased serum-protein aggregation and dissociated more easily in the presence of a competitor polyanion when compared to unmodified PEI. Surprisingly, the gene delivery mediated in the presence of serum showed that succinylation of as few as 2% of PEI amines resulted in transgene expression 260- to 480-fold higher than that of unmodified PEI and 50- to 65-fold higher than that of commercial PEI-PEG2k in HEK293 and HeLa cells, respectively. Remarkably, the same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. In addition, we show that 2% succinylation does not significantly decrease polymer/DNA binding ability or serum protein interaction to a significant extent, yet this small modification is still sufficient to provide a remarkable increase in transgene expression and gene knock-in in the presence of serum.

Targeted delivery of RNAi to cancer cells using RNA-ligand displaying exosome

“…The endolysosomal trap could also hamper the reproducibility of in vivo experiments. To address these issues, most nonviral therapy using polymers or lipids as carriers incorporate membrane fusogenic 145 and endosomolytic peptides 146 or chemical modifications of polymers 8 , 147 . These modifications aim to protect the cargo from endolysosomal degradation and enhance delivery 8 , 148 .…”

“…These modifications aim to protect the cargo from endolysosomal degradation and enhance delivery 8 , 148 . Some molecular modifications/post-polymerization modifications could induce receptor-mediated endocytosis 147 . Even though the literature reports orchestrating chemicals to withstand a lysosomal environment, the overall success rate, with some exception 149 , 150 , in vivo , is still insufficient to translate the work into the clinic.…”

Tuning the charge density and crosslinking of precise amphiphilic oligo(ethanamino)amides for efficient and biocompatible gene delivery

“…9 Polyethyleneimine (PEI) is the most widely investigated cationic polymer for gene delivery, with many protonable secondary and tertiary amine groups in the polymeric backbone, it can effectively complex with nucleic acids through electrostatic interactions and realize efficient transfection. However, the high charge density of PEI also introduces instability under serum and physiological fluids, 10,11 resulting in poor biocompatibility. [12][13][14][15] In recent years, Wagner and co-workers have developed a library of oligomers through a solid phase peptide synthesis strategy based on artificial amino acids, such as succinoyltetraethylene pentamine (Stp), succinoyl-pentaethylene hexamine (Sph), etc.…”

Cationic Micelles Outperform Linear Polymers for Delivery of Antisense Oligonucleotides in Serum: An Exploration of Polymer Architecture, Cationic Moieties, and Cell Addition Order

“…Serum, a component of blood composed of many types of proteins, often causes aggregation, can cause burst release of payloads, and/or binds nonspecifically to delivery systems, causing issues with both in vitro and in vivo delivery. 1,5 Some previous methods of improving serum stability involve the incorporation of hydrophilic sheath layers, such as PEG, 6,7 carbohydrate, 8,9 or zwitterionic 10,11 decrease interactions of the delivery system with the payloads or polyplex interactions with the cells and often impacting performance. 6 Micelles, composed of self-assembled diblock polymers, offer a defined architecture containing a multivalent polycation brush for delivery systems that binds nucleic acid payloads via pre-formed nanoparticles.…”

“…For example, serum instability of formulations is a huge issue with nonviral formulations. Serum, a component of blood composed of many types of proteins, often causes aggregation, can cause burst release of payloads, and/or binds nonspecifically to delivery systems, causing issues with both in vitro and in vivo delivery. , Some previous methods of improving serum stability involve the incorporation of hydrophilic sheath layers, such as PEG, , carbohydrate, , or zwitterionic , moieties. However, these methods of improving serum stability can decrease interactions of the delivery system with the payloads or polyplex interactions with the cells and often impacting performance …”