Non-viral Vectors Research Articles

PEI functionalized cell membrane for tumor targeted and glutathione responsive gene delivery

Polyethylenimine (PEI) is a broadly exploited cationic polymer due to its remarkable gene-loading capacity. However, the high cytotoxicity caused by its high surface charge density has been reported in many cell lines, limiting its application significantly. In this study, two different molecular weights of PEI (PEI10k and PEI25k) were crosslinked with red blood cell membranes (RBCm) via disulfide bonds to form PEI derivatives (RMPs) with lower charge density. Furthermore, the targeting molecule folic acid (FA) molecules were further grafted onto the polymers to obtain FA-modified PEI-RBCm copolymers (FA-RMP25k) with tumor cell targeting and glutathione response. In vitro experiments showed that the FA-RMP25k/DNA complex had satisfactory uptake efficiency in both HeLa and 293T cells, and did not cause significant cytotoxicity. Furthermore, the uptake and transfection efficiency of the FA-RMP25k/DNA complex was significantly higher than that of the PEI25k/DNA complex, indicating that FA grafting can increase transfection efficiency by 15 %. These results suggest that FA-RMP25k may be a promising non-viral gene vector with potential applications in gene therapy.

Non-viral and viral delivery systems for hemophilia A therapy: recent development and prospects.

Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy.

Influence of the Charge Ratio of Guanine-Quadruplex Structure-Based CpG Oligodeoxynucleotides and Cationic DOTAP Liposomes on Cytokine Induction Profiles.

Cationic liposomes, specifically 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) liposomes, serve as successful carriers for guanine-quadruplex (G4) structure-based cytosine-guanine oligodeoxynucleotides (CpG ODNs). The combined benefits of CpG ODNs forming a G4 structure and a non-viral vector carrier endow the ensuing complex with promising adjuvant properties. Although G4-CpG ODN-DOTAP complexes show a higher immunostimulatory effect than naked G4-CpG ODNs, the effects of the complex composition, especially charge ratios, on the production of the pro-inflammatory cytokines interleukin (IL)-6 and interferon (IFN)-α remain unclear. Here, we examined whether charge ratios drive the bifurcation of cytokine inductions in human peripheral blood mononuclear cells. Linear CpG ODN-DOTAP liposome complexes formed micrometer-sized positively charged agglomerates; G4-CpG ODN-DOTAP liposome complexes with low charge ratios (0.5 and 1.5) formed ~250 nm-sized negatively charged complexes. Notably, low-charge-ratio (0.5 and 1.5) complexes induced significantly higher IL-6 and IFN-α levels simultaneously than high-charge-ratio (2 and 2.5) complexes. Moreover, confocal microscopy indicated a positive correlation between the cellular uptake of the complex and amount of cytokine induced. The observed effects of charge ratios on complex size, surface charge, and affinity for factors that modify cellular-uptake, intracellular-activity, and cytokine-production efficiency highlight the importance of a rational complex design for delivering and controlling G4-CpG ODN activity.

Sarcomere disassembly and transfection efficiency in proliferating human iPSC-derived cardiomyocytes

Abstract Introduction Proper contraction of cardiomyocytes (CMs) is dictated by the architecture of the sarcomeres and appears together with polyploidy inversely correlated with limited self-renewal and regeneration of the adult heart. This limited regenerative potential correlates with the decrease in mononuclear diploid cells in adult versus neonatal myocardium. Previous animal studies have shown that the sarcomeres are dissembled during proliferation of neonatal CMs. However, little is known about sarcomere assemblage during human CMs nuclear and/or cell division, which would be relevant information for strategies to boost endogenous heart repair. Purpose In this study we used the human induced pluripotent stem cell (hiPSC) model to investigate sarcomere assemblage during mitosis, followed by cytokinesis, multinucleation and/or self-duplication in massively expanding hiPSC-CMs. Furthermore, we examined whether CMs undergoing nuclear and/or cellular duplication would be more susceptible for genomic editing than non-proliferative hiPSC-CMs. Methods hiPSC-CMs were massively expanded using our previously described CHIR99021 small molecular treatment and cell-cell contact removal. We then performed immunofluorescence staining with cardiac Troponin T (cTnT) and Ki67 to follow sarcomere organization during cell cycle activity. Next, we performed Time-Lapse imaging in a hiPSC-CM culture system to follow the sequence of cellular and nuclear divisions in hiPSC-CMs. RNA sequencing lookup was performed for proliferation, sarcomere and molecular pathway-related genes. Efficiency of non-viral vector-based fluorescence (mCherry) expression and transfection efficiency was done by flow cytometry analysis and Time-Lapse imaging. Results We observed that both mononuclear and binuclear hiPSC-CMs give rise to mononuclear daughter cells or binuclear progeny. Within this source of highly proliferative hiPSC-CMs, treated with CHIR99021 small molecule, we found that Wnt and Hippo signaling were more present when compared to metabolic matured and non-proliferative hiPSC-CMs and adult human heart tissue. Moreover, we found that CHIR99021 treatment increased the efficiency of non-viral vector incorporation in high-proliferative hiPSC-CMs, in which fluorescent transgene expression became present after completion of chromosomal segregation (M-phase). Conclusion This study demonstrates that there is a complex landscape in proliferation, multinucleation and self-duplication of human CMs. Moreover, enhanced incorporation of alien non-viral vectors is related to Wnt activation and cell cycle activity of hiPSC-CMs. Altogether, our findings provide insights in duplication of human CMs and tools for molecular gene studies.

Iron oxide-coupled CRISPR-nCas9-based genome editing assessment in mucopolysaccharidosis IVA mice

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by mutations in the GALNS gene that leads to the lysosomal accumulation of keratan sulfate (KS) and chondroitin 6-sulfate, causing skeletal dysplasia and cardiopulmonary complications. Current enzyme replacement therapy does not impact the bone manifestation of the disease, supporting that new therapeutic alternatives are required. We previously demonstrated the suitability of the CRISPR/nCas9 system to rescue the phenotype of human MPS IVA fibroblasts using iron oxide nanoparticles (IONPs) as non-viral vectors. Here, we have extended this strategy to an MPS IVA mouse model by inserting the human GALNS cDNA into the ROSA26 locus. The results showed increased GALNS activity, mono-KS reduction, partial recovery of the bone pathology, and non-IONPs-related toxicity or antibodies-mediated immune response activation. This study provides, for the first time, in vivo evidence of the potential of a CRISPR/nCas9-based gene therapy strategy for treating MPS IVA using non-viral vectors as carriers.

Development of a trilipid-based liposome system as a delivery vector for plasmid DNA in an MCF-7 cell line: Preparation, optimization, physical characterization and In Vitro cytotoxicity evaluation

Breast cancer cases have recorded an increase for the past decade globally. Currently, available treatments affect patients both physically and mentally, prompting the development of a safer alternative treatment, such as gene therapy. Clinical trials mainly utilise viruses to deliver genes though it has adverse immunological issues. Thus, non-viral vectors such as liposomes, an alternative delivery system without immunological problems, are extensively considered. Liposomes, consisting of lipid bilayers made into nanoparticles as a form of the delivery system, encompass a therapeutic gene cargo to protect and efficiently traverse through the biological barriers for effective gene delivery. Various liposome formulations involving DPPC, OCTA and CHOL lipids were investigated. The optimum method was developed for formulating liposomes which involved several methods and techniques producing particles of below ∼300 nm in size and was confirmed via TEM imaging forming spherical agglomeration. The cytotoxicity of the liposome and nucleic acid complexes was determined using MTT cytotoxicity assay with ∼65% cell viability at 2 µg/µl (w/v) concentration, a higher concentration used compared to those published in the literature (µg/ml). Through this work, a formulation of liposome consisting of DPPC:OCTA:CHOL at 18:72:10 ratio with a reporter gene (pEGFP) was developed and has shown promising size properties, zeta potential, encapsulation efficiency with a capacity to use at a higher concentration as a potential non-viral gene therapy carrier for utilization in MCF-7 breast cancer cell line.

Nucleic acid delivery to retinal cells using lipopeptides as a potential tool towards ocular gene therapies

We evaluated the use of lipopeptides capable to bind to nucleic acids towards plasmid DNA (pDNA) delivery. The investigations were particularly focused on arising retinal pigment epithelial cells (ARPE-19) as motivated by the considerable number of ocular disorders linked to gene aberrations. The lipopeptides comprised the artificial oligoamino acid succinyl-tetraethylene pentamine (Stp) as well as incorporated lysines, histidines, cysteines, fatty acids, and tyrosine trimers. Regardless of the structural differences, the lipopeptides demonstrated to efficiently condense pDNA at nitrogen-to-phosphate molar ratio (N/P) ≥ 6. Spheric nanoparticles were observed by cryo-TEM and dynamic light scattering determined hydrodynamic sizes ranging from 50 to 130 nm. The biological assays evidenced highly efficient pDNA delivery with a lower degree of cytotoxicity compared to the well-known transfecting agent linear polyethylenimine (LPEI). Although more efficient than LPEI, cysteine-containing carriers were demonstrated to be less efficient than the other counterparts possibly due to exceeding polyplex stabilization via disulfide cross links, which could hamper pDNA unpacking at the target site. Therefore, clearly a balance between complex stability and cargo release should be taken into account to optimize the transfection efficiency of the non-viral vectors. The gene transfer activity in ARPE-19 cells suggests the applicability of this kind of carrier for ocular treatments based on retinal gene delivery.

Recombinant VAR2CSA malaria protein-targeted exosome-mediated DACT2 gene therapy for effective glioma treatment

A good non-viral vector is the key to the success of gene delivery and therapy; hence, modified exosomes may overcome the physiological barrier in the delivery in vivo. This study aims to design a novel non-viral vector and verify its gene delivery function in vitro and in vivo for dapper homolog 2 (DACT2) genes. In this study, amphipathicity cationic exosomes with receptor targeted function and DACT2 gene loading function were constructed by exosomes, PEG, glycidyl hexadecyl dimethylammonium chloride, and recombinant VAR2CSA malaria protein (RVP) antibody. Characterizations of RVP antibody and PEG modified cationic lipid exosome (RVP-CL) nanocomplexes were evaluated by dynamic light scattering, atomic force microscopy, FTIR, and so on. The DNA condensation ability and stability were assessed by agarose gel electrophoresis. Cellular uptake efficiency and cytotoxicity in glioma cells were investigated. Furthermore, the tumor suppressive effect was evaluated in vitro and in vivo. The results indicated that RVP-CL had a uniform size of 100–200 nm and positive zeta potential. With high uptake efficiency, RVP-CL can rapidly target, recognize, and enter the glioma cell (KNS-42 and U118 mg) and release the gene. Moreover, RVP-CL/DACT2 can effectively inhibit the growth of glioma cells in vitro and in vivo. The modified cationic exosomes may offer a promising strategy for gene delivery in the treatment of RVP-positive gliomas or other tumors.

Polymer-Initiating Caveolae-Mediated Endocytosis and GSH-Responsive MiR-34a Gene Delivery System for Enhanced Orthotopic Triple Negative Breast Cancer Therapy.

Gene therapy based on miRNAs has broad application prospects in the treatment of tumors. However, due to degradation and ineffective release during intracellular transport, current gene delivery vectors used for miRNAs limited their actual transfection efficiency. This study develops a novel nonviral vector PEI-SPDP-Man (PSM) that can simultaneously target cellular uptake pathways and intracellular responsive release for miR-34a. PSM is synthesized by connected mannitol (Man) to branched polyethylenimine (PEI) using a disulfide bond. The prepared PSM/miR-34a gene delivery system can induce and enter to tumor cells through caveolae-mediated endocytosis to reduce the degradation of miR-34a in lysosomes. The disulfide bond is sensed at high concentration of glutathione (GSH) in the tumor cells and miR-34a is released, thereby reducing the expression of Bcl-2 and CD44 to suppress the proliferation and invasion of tumor cells. In vitro and in vivo experiments show that through the targeted cellular uptake and the efficient release of miR-34a, an effective antitumor and antimetastasis profiles for the treatment of orthotopic triple negative breast cancer (TNBC) are achieved. This strategy of controlling intracellular transport pathways by targeting cellular uptake pathways in the gene therapy is an approach that could be developed for highly effective cancer therapy.

Crystal structure simulation of cationic cholesteric Liquid-Crystal polyesters. Non-Viral vectors

The crystal structure of six cationic cholesteric liquid crystal polyesters with choline, ammonium, and amide end groups are reported: PTOBEE-Choline [(C26H20O8)n-C5H13N]; PTOBDME-Choline [(C34H36O8)n-C5H13N]; PTOBEE-Ammonium [(C26H20O8)n-C5H13N]; PTOBDME-Ammonium [(C34H36O8)n-C5H13N]; PTOBEE-Amide (C26H19O9N)n and PTOBUME-Amide (C33H33O9N)n, have demonstrated non-viral vector properties in gene therapy by delivering DNA to the cell nucleus. They were synthesized by functionalization of precursor racemic polyesters via a condensation reaction between 4,4′-(terephthaloyl-di-oxydibenzoic chloride) (TOBC) and the racemic mixture of glycol (R-S-1,2-butanediol) or (R-S-1,2-dodecanediol), respectively. The resulting helical macromolecular structures exhibit unexpected optical activity and chiral morphology, previously attributed to the kinetic resolution of one preferable helical conformer of the possible four observed by NMR. Conformational analysis and energy minimization are performed on the four helical conformer monomeric models which are then polymerized to obtain helical chains. Three levels of chirality were observed in these polymer structures. Crystal models are built in triclinic primitive P1cells, with their molecular chains oriented parallel to the Z-axis (c lattice parameter equal to the helical pitch length), the cell parameters are obtained by performing geometry optimization tasks. A raw estimation of the relative weight of the four possible helical conformers on each synthetic cationic polymer has been performed on ideal mixtures considering their approximate known crystal structure with their experimental X-ray powder diffraction by Powder Quantitative Phase Analysis. The results would confirm the theory of the preferable recrystallization of a diastereoisomer, among the four possible, during the synthetic procedures. These findings shed light on the observed optical activity and provide insights into the crystal structure of these cationic polymers. The morphology of the cationic polymers is studied by ESEM in wet samples and crystals after drying the solvent.

Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia.

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR. Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells. LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro. Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

Chitosan Modification-Enhanced Silencing Effect of Ad5-shPDGF-D Vector in Breast Cancer Cell Line MDA-MB-231.

Gene therapeutics are being developed to treat metastatic breast tumors, which are mostly resistant to conventional therapies. Targeting platelet-derived growth factor-D (PDGF-D) is a viable approach because it is known to play roles in angiogenesis and tumor growth. The success of gene therapy is largely dependent on delivery vectors, but both viral and nonviral delivery vectors have their disadvantages. Evolving hybrid vectors are being used to overcome those disadvantages. In this study, we aimed to prepare a recombinant adenovirus type-5 (Ad5)/chitosan hybrid vector to deliver shPDGF-D in a breast cancer cell line by the noncovalent coating of the Ad5 surface with chitosan, a natural polymer. The Ad5/chitosan hybrid vector was prepared by the noncovalent coating of the Ad5 surface with different molecular weights (low and high) and different amounts of chitosan (12.5, 25, and 50 μg), and the effect of silencing PDGF-D was investigated in the MDA-MB-231 cell line. In vitro characterization studies showed that the noncovalent chitosan coating increased the size of the Ad5 particle and changed the surface charge from -16.53 mV to slightly neutral. In vitro cell culture studies also showed that the addition of chitosan with both low (73.61%) and high (65.86%) molecular weight increased the PDGF-D silencing efficiency of the Ad5 vector (42.44%) at 48 hours. While low-molecular-weight chitosan had faster effects, high-molecular-weight chitosan provided a more sustained effect in PDGF-D silencing. The results indicate that noncovalent chitosan modification may improve the therapeutic effects of the Ad5 vector, offering the potential for further in vitro and in vivo experiments.

Ocular Gene Therapy: Literature Review

This literature offers complete information about the advances in gene treatment in the of the eye, inclusive of cornea, conjunctiva, lacrimal gland, and trabecular meshwork. We talked about gene transport systems, collectively with viral and non-viral vectors as exact as gene modifying techniques, generally CRISPR-Cas9, and epigenetic treatments, consisting of antisense and siRNA therapeutics. We moreover furnish a specific assessment of gene treatment has been examined with corresponding outcomes. Disease stipulations embody corneal and conjunctival fibrosis and scarring, corneal epithelial wound healing, corneal graft survival, corneal neovascularization, genetic corneal dystrophies, herpetic keratitis, glaucoma, dry eye disease, and specific ocular surface diseases. Although most of the analyzed consequences on the use and validity of gene treatment at the ocular surface have been offered in vitro or the utilization of animal models, we moreover talked about the on hand human studies. Gene treatment methods are nowadays viewed very promising as rising future remedies of a variety of diseases, and this area is unexpectedly expanding.

Updates on Emerging Interventions for Autosomal Recessive ABCA4-Associated Stargardt Disease.

Autosomal recessive Stargardt disease (STGD1) is an inherited retinal degenerative disease associated with a mutated ATP-binding cassette, subfamily A, member 4 (ABCA4) gene. STGD1 is the most common form of juvenile macular degeneration with onset in late childhood to early or middle adulthood and causes progressive, irreversible visual impairment and blindness. No effective treatment is currently available. In the present article, we review the most recent updates in clinical trials targeting the management of STGD1, including gene therapy, small molecule therapy, and stem cell therapy. In gene therapy, dual adeno-associated virus and non-viral vectors have been successful in delivering the human ABCA4 gene in preclinical studies. For pharmaceutical therapies ALK-001, deuterated vitamin A shows promise with preliminary data for phase 2 trial, demonstrating a decreased atrophy growth rate after two years. Stem cell therapy using human pluripotent stem cell-derived retinal pigment epithelium cells demonstrated long-term safety three years after implantation and visual acuity improvements in the first two years after initiation of therapy. Many other treatment options have ongoing investigations and clinical trials. While multiple potential interventions have shown promise in attenuating disease progression, further exploration is necessary to demonstrate treatment safety and efficacy.

Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Gene-activated hyaluronic acid-based cryogels for cartilage tissue engineering

Articular cartilage injuries are very frequent lesions that if left untreated may degenerate into osteoarthritis. Gene transfer to mesenchymal stem cells (MSCs) provides a powerful approach to treat these lesions by promoting their chondrogenic differentiation into the appropriate cartilage phenotype. Non-viral vectors constitute the safest gene transfer tools, as they avoid important concerns of viral systems including immunogenicity and insertional mutagenesis. However, non-viral gene transfer usually led to lower transfection efficiencies when compared with their viral counterparts. Biomaterial-guided gene delivery has emerged as a promising alternative to increase non-viral gene transfer efficiency by achieving sustained delivery of the candidate gene into cellular microenvironment. In the present study, we designed hyaluronic acid-based gene-activated cryogels (HACGs) encapsulating a novel formulation of non-viral vectors based on niosomes (P80PX) to promote MSCs in situ transfection. The developed HACG P80PX systems showed suitable physicochemical properties to promote MSCs in situ transfection with very low cytotoxicity. Incorporation of a plasmid encoding for the transcription factor SOX9 (psox9) into HACG P80PX systems led to an effective MSCs chondrogenic differentiation with reduced expression of fibrocartilage and hypertrophic markers. The capacity of the developed systems to restore cartilage extracellular matrix was further confirmed in an ex vivo model of chondral defect.

Biomaterial‐Based Gene Delivery to Central Nervous System Cells for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a devastating traumatic injury often causing permanent loss of function. The challenge of treating SCI stems from the development of a complex pathophysiology at the site of injury, involving multiple biochemical cascades, widespread inflammation, blood supply interruption, inhibitory scar formation, and poor regrowth of injured axons. Clinical options are limited to surgical stabilization and attempt to ameliorate secondary damage following injury. Gene therapy has significant potential to tackle multiple aspects of SCI and improve functional outcomes. The emergence of a diverse array of biomaterial‐based nonviral nanoparticle vectors capable of delivering gene‐modifying nucleic acids offers the potential to improve the efficiency and specificity of genetic cargos for spinal cord regeneration. In this review, the progress that has been made in the field of SCI repair and the different types of nanoparticles and nucleic acid cargoes that have been used are outlined, placing a particular focus on the different cell types and pathways targeted. While many challenges remain, a perspective on the future of the field of nanoparticle‐mediated gene delivery for SCI is provided, including using biomaterial scaffolds engineered specifically for SCI to deliver gene therapeutics and the exciting opportunities that exist in the post‐COVID landscape.

PH-Responsive polymer boosts cytosolic siRNA release for retinal neovascularization therapy

Small interfering RNA (siRNA) has a promising future in the treatment of ocular diseases due to its high efficiency, specificity, and low toxicity in inhibiting the expression of target genes and proteins. However, due to the unique anatomical structure of the eye and various barriers, delivering nucleic acids to the retina remains a significant challenge. In this study, we rationally design PACD, an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block (A), a siRNA binding block (B) and a pH-responsive block (C). PACDs can self-assemble into nanosized polymeric micelles that compact siRNAs into polyplexes through simple mixing. By evaluating its pH-responsive activity, gene silencing efficiency in retinal cells, intraocular distribution, and anti-angiogenesis therapy in a mouse model of hypoxia-induced angiogenesis, we demonstrate the efficiency and safety of PACD in delivering siRNA in the retina. We are surprised to discover that, the PACD/siRNA polyplexes exhibit remarkable intracellular endosomal escape efficiency, excellent gene silencing, and inhibit retinal angiogenesis. Our study provides design guidance for developing efficient nonviral ocular nucleic acid delivery systems.

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias.