Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system.

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(ß-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Highly branched poly(ß-amino ester) delivery of minicircle DNA for transfection of neurodegenerative disease related cells.

Current therapies for most neurodegenerative disorders are only symptomatic in nature and do not change the course of the disease. Gene therapy plays an important role in disease modifying therapeutic strategies. Herein, we have designed and optimized a series of highly branched poly(ß-amino ester)s (HPAEs) containing biodegradable disulfide units in the HPAE backbone (HPAESS) and guanidine moieties (HPAESG) at the extremities. The optimized polymers are used to deliver minicircle DNA to multipotent adipose derived stem cells (ADSCs) and astrocytes, and high transfection efficiency is achieved (77% in human ADSCs and 52% in primary astrocytes) whilst preserving over 90% cell viability. Furthermore, the top-performing candidate mediates high levels of nerve growth factor (NGF) secretion from astrocytes, causing neurite outgrowth from a model neuron cell line. This synergistic gene delivery system provides a viable method for highly efficient non-viral transfection of ADSCs and astrocytes.

Brushlike Cationic Polymers with Low Charge Density for Gene Delivery.

Using a combined synthesis approach comprising reversible addition-fragmentation transfer polymerization and ring opening reaction, a series of poly glycidyl methacrylate (polyGMA) polymers were designed and synthesized for gene delivery. These polymers characterized by low cationic charge respective to established gene delivery vectors such as PEI were studied to further elucidate the key structure-activity parameters that mediate efficient and biocompatible gene delivery. Compared to PEI, these brushlike polymers facilitated markedly improved safety and gene delivery efficiency.

A new developing class of gene delivery: messenger RNA-based therapeutics.

Gene therapy has long been held as having the potential to become a front line treatment for various genetic disorders. However, the direct delivery of nucleic acids to correct a genetic disorder has numerous limitations owing to the inability of naked nucleic acids (DNA and RNA) to traverse the cell membrane. Recently, messenger RNA (mRNA) based delivery has become a more attractive alternative to DNA due to the relatively easier transfection process, higher efficiency and safety profile. As with all gene therapies, the central challenge that remains is the efficient delivery of nucleic acids intracellularly. This review presents the recent progress in mRNA delivery, focusing on comparing the advantages and limitations of non-viral based delivery vectors.

Thermo- and pH-Responsive, Coacervate-Forming Hyperbranched Poly(ß-amino ester)s for Selective Cell Binding.

We report a new type of thermo- and pH-responsive, coacervate-forming highly degradable polymer-hyperbranched poly(ß-amino esters) (HPAEs) and its selective cell binding behaviors. The HPAEs were synthesized from 5-amino-1-pentanol (S5) and trimethylolpropane ethoxylate triacrylate (TMPETA) via an A2+B3 type Michael addition. The existence of multiple hydrogen bond pairs as well as tertiary amines makes the S5-TMPETA polymers manifest temperature- and pH-dependent phase transition. By varying the length of the ethylene glycol (EG) spacers in the TMPETA, polymer molecular weight, concentration, and pH value, the phase transition of the S5-TMPETA can be easily tuned in aqueous and buffer solutions, as evidenced by UV-vis spectroscopy and DLS measurements. Especially, the S5-TMPETA prepared from S5 and trimethylolpropane ethoxylate triacrylate 692 (S5-TMPETA692) shows a lower critical solution temperature (LCST) around 33 °C, above which the S5-TMPTEA can form coacervate particles able to encapsulate functional molecules effectively. Importantly, when incubation with HeLa cells, the S5-TMPTETA692 exhibits a temperature- and pH-responsive selective cell binding behaviors. In addition, the S5-TMPETA are highly hydrolyzable and elicit negligible cytotoxicity. This new type of "smart" polymer should find use in a variety of biomedical applications.

Star Poly(ß-amino esters) Obtained from the Combination of Linear Poly(ß-amino esters) and Polyethylenimine.

Composed of a three-dimensional structure with a central core and multiple radiating linear "arms", star polymers represent a significant type of branched macromolecular architectures. Due to the spatially defined core-shell-periphery architecture, star polymers have demonstrated their superiority in a variety of biomedical applications such as drug/gene delivery, molecular imaging, antibacterial agents, and so on. In this paper, we report the successful synthesis of a new type of star-shape poly(ß-amino esters) with low molecular weight PEI as core and linear PAE (LPAE) as arms. This new star-PAE exhibits low cytotoxicity and high gene transfection efficacy. Star-PAE achieved between 264-fold and 14781-fold higher gene transfection efficiency of primary rat adipose derived mesenchymal stem cells in comparison with studies performed with the individual PEI and LPAE, respectively. The results suggest that star-PAE is a promising nonviral gene delivery vector.

Development of Branched Poly(5-Amino-1-pentanol-co-1,4-butanediol Diacrylate) with High Gene Transfection Potency Across Diverse Cell Types.

One of the most significant challenges in the development of polymer materials for gene delivery is to understand how topological structure influences their transfection properties. Poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32) has proven to be the top-performing gene delivery vector developed to date. Here, we report the development of branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) as a novel gene vector and elucidate how the topological structure affects gene delivery properties. We found that the branched structure has a big impact on gene transfection efficiency resulting in a superior transfection efficiency of HC32 in comparison to C32 with a linear structure. Mechanistic investigations illustrated that the branched structure enhanced DNA binding, leading to the formation of toroidal polyplexes with smaller size and higher cationic charge. Importantly, the branched structure offers HC32 a larger chemical space for terminal functionalization (e.g., guanidinylation) to further enhance the transfection. Moreover, the optimized HC32 is capable of transfecting a diverse range of cell types including cells that are known to be difficult to transfect such as stem cells and astrocytes with high efficiency. Our study provides a new insight into the rational design of poly(ß-amino ester) (PAE) based polymers for gene delivery.

Highly Branched Poly(ß-amino esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight.

A successful polymeric gene delivery vector is denoted by both transfection efficiency and biocompatibility. However, the existing vectors with combined high efficacy and minimal toxicity still fall short. The most widely used polyethylene imine (PEI), polyamidoamine (PAMAM) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) suffer from the correlation: either too toxic or little effective. Here, we demonstrate that with highly branched poly(ß-amino esters) (HPAEs), a type of recently developed gene delivery vector, the high gene transfection efficiency and low cytotoxicity can be achieved simultaneously at high molecular weight (MW). The interactions of HPAE/DNA polyplexes with cell membrane account for the favorable correlation between molecular weight and biocompatibility. In addition to the effect of molecular weight, the molecular configuration of linear and branched segments in HPAEs is also pivotal to endow high transfection efficiency and low cytotoxicity. These findings provide renewed perspective for the further development of clinically viable gene delivery vectors.

The transition from linear to highly branched poly(ß-amino ester)s: Branching matters for gene delivery.

Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(ß-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(ß-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(ß-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(ß-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000. Moreover, we further demonstrate that HPAEs can correct genetic defects in vivo using a recessive dystrophic epidermolysis bullosa graft mouse model. Our findings prove that the A2 + B3 + C2 approach is highly generalizable and flexible for the design and synthesis of HPAEs, which cannot be achieved by the conventional polymerization approach; HPAEs are more efficient vectors in gene transfection than the corresponding LPAEs. This provides valuable insight into the development and applications of nonviral gene delivery and demonstrates great prospect for their translation to a clinical environment.

Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their cationic properties is imperative. Herein, two uncharged functional diblock oligomers P(DMAEL-b-NIPAM) and P(DMAEL-b-Vlm) were prepared from a lactose derivative, N-iso-propyl acrylamide (NIPAM) as well as 1-vinylimidazole (Vlm) and further functionalized with four small ligands folate, glutathione, cysteine and arginine, respectively, aiming to enhance the interactions of complexes with cells, which were quantified utilizing a quartz crystal microbalance (QCM) biosensor, circumventing the tedious material screening process of cell transfection. Upon incorporation with PLL and DNA, the multifunctional oligomers endow the formulated ternary complexes with great properties suitable for transfection, such as anti-aggregation in serum, destabilized endosome membrane, numerous functional sites for promoted endocytosis and therefore robust transfection activity. Furthermore, different from the conventional strategy of decreasing cytotoxicity by reducing the charge density, the multifunctional oligomer incorporation strategy maintains the highly positive charge density, which is essential for efficient cellular uptake. This system develops a new platform to modify natural polycations towards clinical gene therapy.

Tailoring highly branched poly(ß-amino ester)s: a synthetic platform for epidermal gene therapy.

Highly branched poly(ß-amino ester)s (HPAEs) were designed and synthesised for safe and efficient gene delivery to human keratinocytes. HPAEs outperformed commercial transfection reagents: PEI and SuperFect®, for both transfection efficiency and biocompatibility. A 22 and 3.4 fold enhancement of gene transfection was seen coupled with superior biocompatibility.