Non-viral Gene Delivery Research Articles

On the mechanism of tissue-selective gene delivery by lipid nanoparticles

The era of nucleic acid nanomedicine has arrived, as evidenced by Patisiran, a small interfering RNA (siRNA) encapsulated lipid nanoparticle (LNP), and mRNA-loaded LNPs used in COVID-19 vaccines. The diversity of nano-designs for delivering nucleic acid molecules tested in Phase II/III clinical trials reflects the potential of these technologies. These breakthroughs in non-viral gene delivery, including the use of LNPs, have attracted substantial interest worldwide for developing more effective drugs. A next step in this field is to target tissues other than the liver, which requires significant research efforts and material development. However, mechanistic studies in this area are lacking. This study compares two types of LNPs with different tissue-selectivity for delivering plasmid DNA (pDNA), one being liver-selective and the other spleen-selective, in an effort to understand the mechanisms responsible for differences in gene expression of delivered genes. We observed little difference in the biodistribution of these two LNPs despite the 100–1000-fold differences in gene expression. We then quantified the amount of delivered pDNA and mRNA expression in each tissue by quantitative real-time PCR (qPCR) to evaluate various intracellular processes, such as nuclear delivery, transcription and translation. The results showed a >100-fold difference in the translation step but there were little differences in amount of pDNA delivered to the nucleus or the amount of mRNA expression for the two LNP deliveries. Our findings suggest that endogenous factors affect gene expression efficiency not the extent of biodistribution.

Targeting telomerase utilizing zeolitic imidazole frameworks as non-viral gene delivery agents across different cancer cell types

Telomerase, a ribonucleoprotein coded by the hTERT gene, plays an important role in cellular immortalization and carcinogenesis. hTERT is a suitable target for cancer therapeutics as its activity is highly upregulated in most of cancer cells but absent in normal somatic cells. Here, by employing the two Metal-Organic Frameworks (MOFs), viz. ZIF-C and ZIF-8, based biomineralization we encapsulate Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 plasmid system that targets hTERT gene (CrhTERT) in cancer cells. When comparing the two biocomposites, ZIF-C shows the better loading capacity and cell viability. The loaded plasmid in ZIF-C is highly protected against enzymatic degradation. CrhTERT@ZIF-C is efficiently endocytosed by cancer cells and the subcellular release of CrhTERT leads to telomerase knockdown. The resultant inhibition of hTERT expression decreases cellular proliferation and causing cancer cell death. Furthermore, hTERT knockdown shows a significant reduction in tumour metastasis and alters protein expression. Collectively we show the high potential of ZIF-C-based biocomposites as a promising general tool for gene therapy of different types of cancers.

Abstract 1791: Next generation UltraCAR-T® cells with intrinsic checkpoint inhibition and overnight manufacturing overcome suppressive tumor microenvironment leading to sustained antitumor activity

Abstract The UltraCAR-T® platform, with decentralized overnight manufacturing, has shifted the autologous CAR-T manufacturing paradigm. This disruptive approach has been validated in clinical and pre-clinical studies for both hematologic and solid tumors. UltraCAR-T cells, manufactured using an overnight process with minimal ex vivo manipulation of autologous T cells. UltraCAR-T cells are engineered to co-express CAR, membrane bound IL-15, and kill switch genes using non-viral gene transfer via high-throughput UltraPorator™ system. Multigenic design and overnight manufacturing renders UltraCAR-T cells with enhanced in vivo expansion and persistence for durable antitumor activity. Chronic antigen stimulation from the tumor can lead to CAR T having an exhausted phenotype contributing to treatment failures. Next generation UltraCAR-T cells also incorporate a novel cell intrinsic blockade of PD-1, superseding the need for combination therapy with checkpoint inhibitors and mitigate classic T cell exhaustion that occurs from chronic stimulation, thereby expanding the therapeutic window for efficacy. Using mesothelin (MSLN) as an exemplar from the CAR-target library, we developed a robust in vitro and in vivo model of continuous tumor antigen exposure to evaluate antitumor activity of the UltraCAR-T cells. MSLN UltraCAR-T cells were generated with and without intrinsic PD-1 blockade. In vitro, UltraCAR-T cells with intrinsic PD-1 blockade demonstrated enhanced inflammatory cytokine expression and cytotoxicity at low effector:target ratios. In long-term co-culture assays with recurring challenge of MSLN+ PD-L1+ tumor cells every 2-3 days, UltraCAR-T cells with intrinsic PD-1 blockade showed sustained potent cytotoxicity which was superior to CAR-T cells lacking PD-1 blockade and in combination with αPD-1 antibody. Intrinsic PD-1 blockade markedly enhanced polyfunctionality of UltraCAR-T cells in the presence of MSLN+ PD-L1+ tumor. In xenograft solid tumor model, a single administration of MSLN UltraCAR-T cells manufactured using UltraPorator demonstrated significant antitumor efficacy. Blood analysis showed sustained downregulation of PD-1, robust antigen specific expansion and durable persistence with central memory/stem-cell memory as the dominant phenotype of UltraCAR-T in vivo. Rechallenging the mice, who became tumor-free after a single UltraCAR-T infusion, with tumor for a second time to simulate tumor relapse led to the significant reduction in tumor burden without additional UltraCAR-T treatment. Collectively, these pre-clinical data highlight the improved efficacy of intrinsic PD-1 blockade in next generation UltraCAR-T cells using non-viral gene delivery and an established rapid, decentralized manufacturing process. Citation Format: Giorgio Zenere, Carol Poortman, Afreen Sayed, Devi Gunasekera, Cheryl Bolinger, Pooja Chauhan, Jacques Plummer, Shamim Ahmad, Rutul R. Shah, Helen Sabzevari, Douglas E. Brough, Tim Chan. Next generation UltraCAR-T® cells with intrinsic checkpoint inhibition and overnight manufacturing overcome suppressive tumor microenvironment leading to sustained antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1791.

A New Optimization Strategy of Highly Branched Poly(β-Amino Ester) for Enhanced Gene Delivery: Removal of Small Molecular Weight Components

Highly branched poly(β-amino ester) (HPAE) has become one of the most promising non-viral gene delivery vector candidates. When compared to other gene delivery vectors, HPAE has a broad molecular weight distribution (MWD). Despite significant efforts to optimize HPAE targeting enhanced gene delivery, the effect of different molecular weight (MW) components on transfection has rarely been studied. In this work, a new structural optimization strategy was proposed targeting enhanced HPAE gene transfection. A series of HPAE with different MW components was obtained through a stepwise precipitation approach and applied to plasmid DNA delivery. It was demonstrated that the removal of small MW components from the original HPAE structure could significantly enhance its transfection performance (e.g., GFP expression increased 7 folds at w/w of 10/1). The universality of this strategy was proven by extending it to varying HPAE systems with different MWs and different branching degrees, where the transfection performance exhibited an even magnitude enhancement after removing small MW portions. This work opened a new avenue for developing high-efficiency HPAE gene delivery vectors and provided new insights into the understanding of the HPAE structure-property relationship, which would facilitate the translation of HPAEs in gene therapy clinical applications.

The steep road to nonviral nanomedicines: Frequent challenges and culprits in designing nanoparticles for gene therapy.

The potential of therapeutically loaded nanoparticles (NPs) has been successfully demonstrated during the last decade, with NP-mediated nonviral gene delivery gathering significant attention as highlighted by the broad clinical acceptance of mRNA-based COVID-19 vaccines. A significant barrier to progress in this emerging area is the wild variability of approaches reported in published literature regarding nanoparticle characterizations. Here, we provide a brief overview of the current status and outline important concerns regarding the need for standardized protocols to evaluate NP uptake, NP transfection efficacy, drug dose determination, and variability of nonviral gene delivery systems. Based on these concerns, we propose wide adherence to multimodal, multiparameter, and multistudy analysis of NP systems. Adoption of these proposed approaches will ensure improved transparency, provide a better basis for interlaboratory comparisons, and will simplify judging the significance of new findings in a broader context, all critical requirements for advancing the field of nonviral gene delivery.

Nanite debuts AI platform for polymer design

Nanite has netted $6 million in seed funding to launch its artificial intelligence platform for designing polymer nanoparticles for nonviral gene delivery. A drug’s delivery mechanism can be as important as the drug itself. For gene therapies, adeno-associated viruses (AAVs) are the most established delivery vehicle. But AAVs have a limited payload and can trigger an immune response. Lipid nanoparticles, which are used in COVID-19 messenger RNA vaccines , are an alternative delivery vehicle. But they tend to get stuck in the liver so aren’t suited for tissue-specific delivery. Polymer nanoparticles are the latest nonviral delivery alternatives. Their main advantage is their chemical diversity, says Sean Kevlahan, Nanite’s CEO and cofounder. Their resulting versatility allows scientists to tailor polymers for different cargo and tissue targets. The polymer capsule involves many design considerations, including the monomer chemistry, polymer architecture, and surface charge distribution. Nanite employs AI to predict the design for

Elastin-Derived VGVAPG Fragment Decorated Cell-Penetrating Peptide with Improved Gene Delivery Efficacy.

Cell-penetrating peptides (CPPs) are attractive non-viral gene delivery vectors due to their high transfection capacity and safety. Previously, we have shown that cell-penetrating peptide RALA can be a promising gene delivery vector for chronic wound regeneration application. In this study, we engineered a novel peptide called RALA-E by introducing elastin-derived VGVAPG fragment into RALA, in order to target the elastin-binding protein on the cell surface and thus improve delivery efficacy of RALA. The transfection efficiency of RALA-E was evaluated by transfecting the HEK-293T and HeLa cell lines cells with RALA-E/pDNA complexes and the flow-cytometry results showed that RALA-E significantly increased the transfection efficiency by nearly 20% in both cell lines compared to RALA. Inhibition of pDNA transfection on HEK-293T cells via chlorpromazine, genistein and mβCD showed that the inhibition extent in transfection efficiency was much less for RALA-E group compared to RALA group. In addition, RALA-E/miR-146a complexes showed up to 90% uptake efficiency in macrophages, and can escape from the endosome and enter the nucleus to inhibit the expression of inflammation genes. Therefore, the developed RALA-E peptide has high potential as a safe and efficient vector for gene therapy application.

An Overview of Gene Editing Modalities and Related Non-clinical Testing Considerations.

Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.

GSH-Activatable Aggregation-Induced Emission Cationic Lipid for Efficient Gene Delivery.

The key to gene therapy is the design of biocompatible and efficient delivery systems. In this work, a glutathione (GSH)-activated aggregation-induced-emission (AIE) cationic amphiphilic lipid, termed QM-SS-KK, was prepared for nonviral gene delivery. QM-SS-KK was composed of a hydrophilic biocompatible lysine tripeptide headgroup, a GSH-triggered disulfide linkage, and a hydrophobic AIE fluorophore QM-OH (QM: quinoline-malononitrile) tail. The peptide moiety could not only efficiently compact DNA but also well modulate the dispersion properties of QM-SS-KK, leading to the fluorescence-off state before GSH treatment. The cleavage of disulfide in QM-SS-KK by GSH generated AIE signals in situ with a tracking ability. The liposomes consisted of QM-SS-KK, and 1,2-dioleoylphosphatidylethanolamine (DOPE) (QM-SS-KK/DOPE) delivered plasmid DNAs (pDNAs) into cells with high efficiency. In particular, QM-SS-KK/DOPE had an enhanced transfection efficiency (TE) in the presence of 10% serum, which was two times higher than that of the commercial transfection agent PEI25K. These results highlighted the great potential of peptide and QM-based fluorescence AIE lipids for gene delivery applications.

Increased PRL-1 in BM-derived MSCs triggers anaerobic metabolism via mitochondria in a cholestatic rat model

Mesenchymal stem cell (MSC) therapy in chronic liver disease is associated with mitochondrial anaerobic metabolism. Phosphatase of regenerating liver-1 (PRL-1), known as protein tyrosine phosphatase type 4A, member 1 (PTP4A1), plays a critical role in liver regeneration. However, its therapeutic mechanism remains obscure. The aim of this study was to establish genetically modified bone marrow (BM)-MSCs overexpressing PRL-1 (BM-MSCsPRL-1) and to investigate their therapeutic effects on mitochondrial anaerobic metabolism in a bile duct ligation (BDL)-injured cholestatic rat model. BM-MSCsPRL-1 were generated with lentiviral and nonviral gene delivery systems and characterized. Compared with naive cells, BM-MSCsPRL-1 showed an improved antioxidant capacity and mitochondrial dynamics and decreased cellular senescence. In particular, mitochondrial respiration in BM-MSCsPRL-1 generated using the nonviral system was significantly increased as well as mtDNA copy number and total ATP production. Moreover, transplantation of BM-MSCsPRL-1 generated using the nonviral system had predominantly antifibrotic effects and restored hepatic function in a BDL rat model. Decreased cytoplasmic lactate and increased mitochondrial lactate upon the administration of BM-MSCsPRL-1 indicated significant alterations in mtDNA copy number and ATP production, activating anaerobic metabolism. In conclusion, BM-MSCsPRL-1 generated by a nonviral gene delivery system enhanced anaerobic mitochondrial metabolism in a cholestatic rat model, improving hepatic function.

Non-viral gene coating modified IOL delivering PDGFR-α shRNA interferes with the fibrogenic process to prevent posterior capsular opacification.

Posterior capsule opacification (PCO), the most common complication after cataract surgery, is caused by the proliferation, migration and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells in the capsule bag. Although the surface modification and drug loading of intraocular lens (IOLs) have been effective in preventing PCO to some extent, the intraocular safety of anti-proliferative drug application is still a major limitation in clinical application. In this study, we used non-viral gene delivery systems in combination with layer-by-layer (LBL) self-assembly technology, and the modified IOL could effectively prevent the development of PCO by interfering with the EMT process mediated by the platelet-derived growth factor receptor-α (PDGFR-α). Herein, the gene fragments were wrapped by electrostatic conjugation using polyethyleneimine-graft-poly(ethylene glycol) to form gene complexes. Gene complexes were characterized by dynamic light scattering, transmission electron microscopy (TEM) and agarose gel electrophoresis, and evaluated for storage and serum stability. The layer assembly behavior of the IOL surface, changes in optical properties and the release behavior of the gene complexes were characterized using quartz crystal microbalance, UV-vis, contact angle and TEM. In vitro experiments showed that the IOL coating has good bio-compatibility and can achieve the corresponding transfection effect, and the released gene complexes exhibited excellent cell internalization and lysosomal escape behaviors, as well as effective inhibition of PDGFR-α expression and its mediated EMT process. The early PCO prevention effect and bio-compatibility evaluation of the modified IOL in vivo were evaluated by implantation into animal eyes. This study provides a new strategy for the development of surface modifications of small nucleic acid drugs and non-toxic EMT interference therapies for PCO.

Balancing gene transfection and cytotoxicity of nucleic acid carriers with focus on ocular and hepatic disorders: evaluation of hydrophobic and hydrophilic polyethyleneimine derivatives.

Polyethyleneimine (PEI) derivatives substituted by lactose, succinic acid or alkyl domains were evaluated as nonviral gene delivery vectors towards balancing gene transfection and cytotoxicity. The investigations were focused on pDNA transfection into arising retinal pigment epithelia (ARPE-19) and human hepatocellular carcinoma (HepG2) cell lines. The first mentioned cell line was chosen as motivated by the non-negligible number of ocular disorders linked to gene aberrations, whereas the second one is a cell line overexpressing the asialoglycoprotein receptor (ASGP-R), which can bind to galactose residues. The presence of short alkyl domains (C4 and C6), and particularly the succinylation of the PEI chains, improved the biological outputs of the gene vectors. The presence of hydrophobic units possibly enhances lytic activity, whereas the incorporation of succinic acid slightly reduces polymer-DNA interaction strength, thereby enabling more efficient intracellular unpacking and cargo release. Succinylation is also supposed to decrease cytotoxicity and avoid protein adsorption to the polyplexes. The presence of long carbon chains (for instance, C12) nevertheless, results in higher levels of cytotoxicity and respective lower transfection rates. The sugar-decorated polyplexes are overall less cytotoxic, but the presence of lactose moieties also leads to larger polyplexes and notably weak polymer-DNA binding, which compromise the transfection efficiency. Yet, along with the presence of short lytic alkyl domains, the double-substitution of PEI synergistically boosts gene transfection probably due to the uptake of higher DNA and polymer amounts without cell damage. Overall, the experimental data suggest that ocular and hepatic gene therapies may be potentialized by fine-tuning the hydrophobic-to-hydrophilic balance, and succinic acid is a favorable motif for the modification of PEI.

Analysis of PEG-lipid anchor length on lipid nanoparticle pharmacokinetics and activity in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) affects millions of people worldwide, yet there are currently no therapeutics that address the long-term impairments that develop in a large portion of survivors. Lipid nanoparticles (LNPs) are a promising therapeutic strategy that may address the molecular basis of TBI pathophysiology. LNPs are the only non-viral gene delivery platform to achieve clinical success, but systemically administered formulations have only been established for targets in the liver. In this work, we evaluated the pharmacokinetics and activity of LNPs formulated with polyethylene glycol (PEG)-lipids of different anchor lengths when systemically administered to a mouse model of TBI. We observed an increase in LNP accumulation and activity in the injured brain hemisphere compared to the uninjured contralateral brain hemisphere. Interestingly, transgene expression mediated by LNPs was more durable in injured brain tissue compared to off-target organs when compared between 4 and 24 hours. The PEG-lipid is an important component of LNP formulation necessary for the stable formation and storage of LNPs, but the PEG-lipid structure and content also has an impact on LNP function. LNP formulations containing various ratios of PEG-lipid with C18 (DSPE-PEG) and C14 (DMG-PEG) anchors displayed similar physicochemical properties, independent of the PEG-lipid compositions. As the proportion of DSPE-PEG was increased in formulations, blood circulation times of LNPs increased and the duration of expression increased. We also evaluated diffusion of LNPs after convection enhanced delivery (CED) in healthy brains and found LNPs distributed >1 mm away from the injection site. Understanding LNP pharmacokinetics and activity in TBI models and the impact of PEG-lipid anchor length informs the design of LNP-based therapies for TBI after systemic administration.

A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes.

Transfection describes the delivery of exogenous nucleic acids (NAs) to cells utilizing non-viral means. In the last few decades, scientists have been doing their utmost to design ever more effective transfection reagents. These are eventually mixed with NAs to give rise to gene delivery complexes, which must undergo characterization, testing, and further refinement through the sequential reiteration of these steps. Unfortunately, although microfluidics offers distinct advantages over the canonical approaches to preparing particles, the systems available do not address the most frequent and practical quest for the simultaneous generation of multiple polymer-to-NA ratios (N/Ps). Herein, we developed a user-friendly microfluidic cartridge to repeatably prepare non-viral gene delivery particles and screen across a range of seven N/Ps at once or significant volumes of polyplexes at a given N/P. The microchip is equipped with a chaotic serial dilution generator for the automatic linear dilution of the polymer to the downstream area, which encompasses the NA divider to dispense equal amounts of DNA to the mixing area, enabling the formation of particles at seven N/Ps eventually collected in individual built-in tanks. This is the first example of a stand-alone microfluidic cartridge for the fast and repeatable preparation of non-viral gene delivery complexes at different N/Ps and their storage.

Covalent functionalization of graphene sheets for plasmid DNA delivery: experimental and theoretical study.

Several approaches, including plasmid transfection and viral vectors, were used to deliver genes into cells for therapeutic and experimental purposes. However, due to the limited efficacy and questionable safety issues, researchers are looking for better new approaches. Over the past decade, graphene has attracted tremendous attention in versatile medical applications, including gene delivery, which could be safer than the traditional viral vectors. This work aims to covalently functionalize pristine graphene sheets with a polyamine to allow the loading of plasmid DNA (pDNA) and enhance its delivery into cells. Graphene sheets were successfully covalently functionalized with a derivative of tetraethylene glycol connected to polyamine groups to improve their water dispersibility and capacity to interact with the pDNA. The improved dispersibility of the graphene sheets was demonstrated visually and by transmission electron microscopy. Also, it was shown by thermogravimetric analysis that the degree of functionalization was about 58%. Moreover, the surface charge of the functionalized graphene was +29 mV as confirmed by zeta potential analysis. The complexion of f-graphene with pDNA was achieved at a relatively low mass ratio (10 : 1). The incubation of HeLa cells with f-graphene loaded with pDNA that encodes enhanced green fluorescence protein (eGFP) resulted in the detection of fluorescence signal in the cells within one hour. f-Graphene showed no toxic effect in vitro. Density functional theory (DFT) and quantum theory of atoms in molecules (QTAIM) calculations revealed strong binding with ΔH 298 = 74.9 kJ mol-1. QTAIM between the f-graphene and a simplified model of pDNA. Taken together, the developed functionalized graphene could be used for the development of a new non-viral gene delivery system.

Synthesis of nanoparticles of Poly (ethyleneimine) and their characterization by transmission electron microscopy, thin layer chromatography, and infrared spectroscopy

Intracellular gene delivery alters the expression of a gene and corrects a defective gene that may be the cause of a disease or a disorder. Nonviral gene delivery is more appropriate than viral-mediated due to their low cytotoxicity and immunogenicity. Amongst these, polycationic nanoparticles i.e. Polyethyleneimine (PEI) were used most successfully. The PEGylation of such cationic polymer reduces its cytotoxicity. Different molecular weight poly (ethylene glycol) was used for the PEGylation of such cationic nanoparticles which have an individual effect. For this purpose, the PEG is esterified, which was then reacted with a cationic polymer. Four different molecular weights of PEG were used. The size of nanoparticles so formed depends upon the molecular weight of PEG. So formed nanoparticles were dialyzed, lyophilized, and then characterized by IR and TEM. The nanoparticles so formed are directly affected by the different molecular weights of PEG. Higher the molecular weights of PEG smaller size of nanoparticles so formed but only up to a limited extent. The decreasing order of nanoparticles as an increment of molecular weight of PEG was found as a -0.85 coefficient of correlation. The smaller-sized nanoparticles have higher transfection efficiency than larger-sized nanoparticles. So, the higher the molecular weight of PEG higher will be the transfection efficiency.

Preparation and characterization of a gemini surfactant-based biomimetic complex for gene delivery

Gemini surfactants (GS) have been explored as non-viral gene delivery systems. Nevertheless, their cytotoxicity and the limitations in the in vivo studies have impeded their development. To attenuate toxicity and further explore their possibilities in gene delivery, a series of GS (18-7-18)-based gene delivery systems complexed with red blood cell membranes (RBCM) or/and DOPE-PEG2000 (DP) were prepared and evaluated. EGFP-encoding plasmids were delivered via GS-based complexes and the efficiency of gene transfection was evaluated by imaging of the major organs after intravenous administration in mice and qPCR quantification in hepatocytes. In order to assess the safety of GS-based complexes, the hemolysis test, serum biochemical indices, H&E staining and CCK-8 test were examined. The results revealed that EGFP was primarily expressed in livers, and all complexes showed minimal acute toxicity to major organs. Moreover, we found that the dual incorporation of RBCM and DP could significantly elevate the transfection efficiency and cell viability in hepatocytes. Overall, the results indicated that GS-based complexes possessed great potential as vectors for gene delivery both in vivo and in vitro and the dual incorporation of RBCM and DP could be a promising gene delivery approach with high transfection efficacy and low toxicity.

Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells.

In this article, we used monolayer two dimensional (2D) and 3D multicellular spheroid models to improve our understanding of the gene delivery process of a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly)-loaded plasmid encoding Enhanced Green Fluorescent Protein (EGFP). A comparison between the cytotoxicity effect and transfection efficiency of the plasmid DNA (pDNA)-loaded Ppoly system in 2D and 3D spheroid cells determined that the transfection efficiency and cytotoxicity of Ppoly-pDNA nanocomplexes were lower in 3D spheroids than in 2D monolayer cells. Furthermore, histopathology visualization of Ppoly-pDNA complex cellular uptake in 3D spheroids demonstrated that Ppoly penetrated into the inner layers. This study indicated that the Ppoly, as a non-viral gene delivery system in complex with pDNA, is hemocompatible, non-toxic, high in encapsulation efficiency, and has good transfection efficiency in both 2D and 3D cell cultures compared to free pDNA and lipofectamine (as the control).

A new nano hyperbranched β-pinene polymer: Controlled synthesis and nonviral gene delivery

Recently, an extensive research effort has been directed toward the improvement of nonviral transfection vectors, such as polymeric materials. The macromolecular structure of polymers has a substantial effect on their transfection efficacy. In this context, the modern advances in polymer production techniques, such as the deactivation-enhanced radical atom transfer polymerization (DE-ATRP), have been fundamental for the synthesis of controlled architecture nanomaterials. In this study, hyperbranched poly(β-pinene)-PDMAEMA-PEGDMA nanometric copolymers were synthesised at high conversion via DE-ATRP using different concentrations of β-pinene for gene delivery applications. The structural characterization and the biological performance of the materials were investigated. The copolymers’ molar mass (10,434–16,438 mol l-1), dispersity, and conversion (90–95%) varied significantly with β-pinene proportion on the polymerizations. The polymer-gene complexes generated (280–110 nm) presented excellent solution stability due to the β-pinene segment on the copolymers. Gene delivery and transfection were highly dependent on the copolymer composition. The copolymers containing the highest β-pinene proportions exhibited the best results with high transfection effectivity. In conclusion, the incorporation of β-pinene in DMAEMA-PEGMA copolymer formulations is a renewable option to improve the materials’ branching ratio, polyplex stability, and gene delivery performance without causing cytotoxic effects.

Recent advances in gene therapy for bone tissue engineering.

Autografting, a major treatment for bone fractures, has potential risks related to the required surgery and disease transmission. Bone morphogenetic proteins (BMPs) are the most common osteogenic factors used for bone-healing applications. However, BMP delivery can have shortcomings such as a short half-life and the high cost of manufacturing the recombinant proteins. Gene delivery methods have demonstrated promising alternative strategies for producing BMPs or other osteogenic factors using engineered cells. These approaches can also enable temporal overexpression and local production of the therapeutic genes in the target tissues. This review addresses recent progress on engineered viral, non-viral, and RNA-mediated gene delivery systems that are being used for bone repair and regeneration. Advances in clustered regularly interspaced short palindromic repeats/Cas9 genome engineering for bone tissue regeneration also is discussed.