Non-viral Vectors Research Articles

Hypoxia-sensitive miRNA regulation via CRISPR/dCas9 loaded in hybrid exosomes: A novel strategy to improve embryo implantation and prevent placental insufficiency during pregnancy.

Assisted reproductive techniques as a new regenerative medicine approach have significantly contributed to solving infertility problems that affect approximately 15% of couples worldwide. However, the success rate of an in vitro fertilization (IVF) cycle remains only about 20%-30%, and 75% of these losses are due to implantation failure (the crucial rate-limiting step of gestation). Implantation failure and abnormal placenta formation are mainly caused by defective adhesion, invasion, and angiogenesis. Placental insufficiency endangers both the mother's and the fetus's health. Therefore, we suggested a novel treatment strategy to improve endometrial receptivity and implantation success rate. In this strategy, regulating mir-30d expression as an upstream transcriptomic modifier of the embryo implantation results in modified expression of the involved genes in embryonic adhesion, invasion, and angiogenesis and consequently impedes implantation failure. For this purpose, "scaffold/matrix attachment regions (S/MARs)" are employed as non-viral episomal vectors, transfecting into trophoblasts by exosome-liposome hybrid carriers. These vectors comprise CRISPR/dCas9 with a guide RNA to exclusively induce miR-30d gene expression in hypoxic stress conditions. In order to avoid concerns about the fetus's genetic manipulation, our vector would be transfected specifically into the trophoblast layer of the blastocyst via binding to trophoblast Erb-B4 receptors without entering the inner cell mass. Additionally, S/MAR episomal vectors do not integrate with the original cell DNA. As an on/off regulatory switch, a hypoxia-sensitive promoter (HRE) is localized upstream of dCas9. The miR-30d expression increases before and during the implantation and placental insufficiency conditions and is extinguished after hypoxia elimination. This hypothesis emphasizes that improving the adhesion, invasion, and angiogenesis in the uterine microenvironment during pregnancy will result in increased implantation success and reduced placental insufficiency, as a new insight in translational medicine.

Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways.

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes' cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

CRISPR medicine for blood disorders: Progress and challenges in delivery.

Blood disorders are a group of diseases including hematological neoplasms, clotting disorders and orphan immune deficiency diseases that affects human health. Current improvements in genome editing based therapeutics demonstrated preclinical and clinical proof to treat different blood disorders. Genome editing components such as Cas nucleases, guide RNAs and base editors are supplied in the form of either a plasmid, an mRNA, or a ribonucleoprotein complex. The most common delivery vehicles for such components include viral vectors (e.g., AAVs and RV), non-viral vectors (e.g., LNPs and polymers) and physical delivery methods (e.g., electroporation and microinjection). Each of the delivery vehicles specified above has its own advantages and disadvantages and the development of a safe transferring method for ex vivo and in vivo application of genome editing components is still a big challenge. Moreover, the delivery of genome editing payload to the target blood cells possess key challenges to provide a possible cure for patients with inherited monogenic blood diseases and hematological neoplastic tumors. Here, we critically review and summarize the progress and challenges related to the delivery of genome editing elements to relevant blood cells in an ex vivo or in vivo setting. In addition, we have attempted to provide a future clinical perspective of genome editing to treat blood disorders with possible clinical grade improvements in delivery methods.

Developing non-viral or viral vectors for efficient and targeted delivery of genetic material, such as DNA or RNA, for gene therapy applications

Gene therapy has emerged as a promising approach for treating a wide range of genetic and acquired diseases by introducing or modifying genetic material within cells. To achieve successful gene therapy, efficient and precise delivery of genetic material, such as DNA or RNA, to target cells is essential. This abstract explores the development of both non-viral and viral vectors for the delivery of genetic material in gene therapy applications. Non-viral vectors, including lipid nanoparticles (LNPs), polymer-based carriers, and cell-penetrating peptides, have gained significant attention due to their safety profile and ease of production. These vectors are designed to protect genetic material from degradation, facilitate cellular uptake, and release the cargo at the desired location. Recent advancements in nanotechnology have enabled the design of customizable non-viral vectors with enhanced delivery efficiency and reduced offtarget effects. Viral vectors, on the other hand, harness the natural infectivity of viruses to transport genetic material into target cells. Retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) are commonly used viral vectors in gene therapy. Viral vectors offer high transduction efficiency but may trigger immune responses or pose risks of insertional mutagenesis. Efforts in vector engineering have led to the development of safer viral vectors with improved targeting capabilities and reduced immunogenicity. AAVs, in particular, have gained prominence due to their ability to achieve long-lasting gene expression with minimal adverse effects. Targeted delivery strategies aim to enhance vector specificity, ensuring that genetic material reaches the intended cell type or tissue. These strategies include modifying vector surface proteins, employing tissue-specific promoters, or utilizing ligand-receptor interactions. In conclusion, the successful application of gene therapy relies on the development of efficient and targeted delivery systems for genetic material. Non-viral and viral vectors offer distinct advantages and continue to evolve to meet the demands of gene therapy applications. Advances in vector design, safety, and targeting strategies hold promise for the continued progress of gene therapy as a transformative medical intervention.

Gene therapy and its applications

Gene therapy is the treatment of abnormal or mutated genes present in cells through the addition of healthy genes or replacement/deletion/site-specific modification of faulty genes. Deoxyribonucleic acid, messenger ribonucleic acid (RNA), small interference RNA, microsomal RNA and antisense oligonucleotides are the genetic materials implicated in gene therapy. They are inserted into the diseased cells using viral or non-viral vectors through an in vivo or ex vivo transduction. Gamma retrovirus, lentivirus, herpesvirus, adenovirus and adeno-associated virus are common viral vectors, while transposons, cationic polymers, dendrimers and cell-penetrating peptides or liposomes are common non-viral vectors. Allologous or autologous T cells, haematopoietic stem cells and chimeric antigen receptor T cells are used for ex vivo gene transduction. Conventional gene therapy of inserting new genetic material shows toxicity such as off-target effects, altered immune responses, inflammatory reactions and possible oncogenic transformation in the recipient. Newer gene editing techniques such as zinc-finger nuclease, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats allow the site-specific correction or control of expression of mutated genes present in cells. Until August 2020, 23 gene-based medicines received approval from drug regulatory agencies in various countries and 362 were in development. Single-gene disorders have shown encouraging results, but evidence of using gene therapy in polygenic and common age-related diseases is still required. Recently, the horizon of gene therapy widened to include COVID vaccines and as an adjunct to chemotherapy. If we could overcome its limitations such as immunogenicity, mutagenicity and high costs, gene therapy can be the medicine of the next generation.

Chimeric antigen receptor (CAR) T cells targeting CD19 for acute lymphoblastic leukemia

Chimeric antigen receptor transgenic T cell (CAR-T) therapy targeting the CD19 antigen was approved for relapsed/refractory acute lymphocytic leukemia in the United States in 2017 and in Japan in 2019. Despite the excellent efficacy of CAR-T therapy, the relapse rate is about 50%. To reduce this rate, it will be important to examine predictive factors for relapse and which patients should receive hematopoietic cell transplantation. In addition, as the high cost of CAR-T cells has become a financial toxicity that threatens the health insurance system in many countries, development of less expensive CAR-T products using non-viral vectors is also underway.

The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model.

Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α-smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC-treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.

The nanostructure of polyelectrolyte complexes of QPDMAEMA-b-POEGMA copolymers and oppositely charged polyelectrolytes, and their stability in the presence of serum albumin.

Quaternized poly(2-(dimethylamino) ethyl methacrylate)-b-poly(oligo(ethyleneglycol) methyl ether methacrylate) (QPDMAEMA-b-POEGMA) is a copolymer of a positively charged block and a non-ionic hydrophilic block. The positively charged block, QPDMAEMA, electrostatically interacts with oppositely charged polymers, e.g., poly(acrylic acid) (PAA) and DNA, to form a complex. This complex is stable in aqueous solution due to the hydrophilic block, POEGMA, which provides colloidal stability and biocompatibility. Polyplexes can be used as non-viral vectors in gene therapy. Polyplexes are essential for delivering genetic materials into cells because they protect the genetic material from degradation before reaching the target cells, thus increasing the transfection efficiency. However, currently used polyplexes show a low transfection efficiency in vivo, probably because the polyplexes are exposed to blood proteins, such as serum albumin, which cause their dissociation. The main goal of this research is the morphology characterization of QPDMAEMA-b-POEGMA complexes with the sodium salt of polyacrylic acid (NaPAA), and with DNA by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). These methods give qualitative and quantitative data about the morphology of the complexes. The morphology of the complexes was examined at different charge ratios (CRs). Complexes with NaPAA form core-corona spherical micelles and vesicular structures, whereas complexes with DNA form lamellar and hexagonal structures. The QPDMAEMA-b-POEGMA and DNA complexes were also examined after exposing them to bovine serum albumin (BSA). We found that BSA does not affect the complexes for seven days. This morphology characterization is essential for better design and formulation of vectors for gene therapy and polyelectrolyte complexes for biomedical applications.

Comparison of chemically mediated CRISPR/Cas9 gene editing systems using different nonviral vectors in porcine embryos.

The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.

EVALUATION AND COMPREHENSIVE COMPARISON OF OCTADECYLAMINE AND STEARAMIDE-BASED SOLID LIPID NANOPARTICLES FOR EFFICIENT GENE DELIVERY

In recent years various nonviral vector systems have been developed to overcome the disadvantages of viral vectors. Thus, researchers have turned to more secure and easy to reach vector systems. In this study, we have evaluated the gene transfer efficiency of two different nonviral vectors prepared using two lipids: octadecylamine and stearamide. The solid lipid nanoparticles were produced with the emulsion-solvent evaporation method. Particles' mean size, polydispersity index (PDI), and zeta potential values for octadecylamine nanoparticle (ONP) are, respectively, 382.7 nm (&amp;#177; 20), 0.4, and 28 mV (&amp;#177; 2.03) and for stearamide nanoparticle (SNP) are 276.9 nm (&amp;#177; 10.45), 0.3, and 20.4 mV (&amp;#177; 4.03), as determined by using Zetasizer via the dynamic light scattering (DLS) method. Morphological characterization of nanoparticles was performed with scanning electron microscope (SEM) and atomic force microscope (AFM). The chemical characterization was performed by Fourier-transform infrared spectroscopy (FTIR). The cytotoxicity of nanoparticles were determined via 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay with an MCF-7 human breast cancer cell line. The cytotoxicity for both ONP and SNP were calculated over 55&amp;#37;. Also nanoparticles/plasmid DNA conjugates cytotoxicity were determined with no significant toxicity obtained. The transfection efficiency was evaluated by observing green fluorescent protein (GFP) expression in the MCF-7 human breast cancer cell line. The transfection efficiency was 67.5&amp;#37; (&amp;#177; 2.3) for ONP, 77.6&amp;#37; (&amp;#177; 1.2) for SNP, and 70.7&amp;#37; (&amp;#177; 1.4) for commercially available transfection agent.

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.

Recent advances in stimuli-responsive polymeric carriers for controllable CRISPR/Cas9 gene editing system delivery.

Non-viral polymeric vectors with good biocompatibility have been recently explored as delivery systems for clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nucleases. In this review, based on current limitations and critical barriers, we summarize the advantages of stimulus-responsive polymeric delivery vectors (i.e., pH, redox, or enzymes) towards controllable CRISPR/Cas9 genome editing system delivery as well as the advances in using stimulus-responsive CRISPR/Cas9 polymeric carriers towards cancer treatment. Last but not least, the key challenges and promising development strategies of stimulus-responsive polymeric vector designs for CRISPR/Cas9 systems will also be discussed.

Gene Therapy for Intervertebral Disc Degeneration: An Overview of Current Strategies and Applications

Intervertebral disc degeneration is a pervasive condition contributing to chronic back pain, affecting up to a third of the population, with risk further increasing with age. It is a significant driver of disability for millions of Americans and others worldwide. The standard of care today is reliant on symptomatic treatment rather than addressing the root cause of disease. Surgical interventions alter the structural integrity and biomechanics of the spine, often leading to loss of function and motion, and post-operative complications. This is the basis for innovation in novel biologic treatments, including gene therapy, which aims to reestablish the optimal balance between matrix catabolism and anabolism within pathologically degenerating disc cells. This review will cover the significant advances that have led to identification of target therapeutic genes combined with regulated expression of the therapeutic transgene and successful systems for gene delivery into cells. Recent advances in viral and non-viral vectors for gene transfer, silencing of genes by RNA interference, editing of genes by clustered regularly interspaced short palindromic repeats, and modifying mammalian target of rapamycin signaling pathwayS offer promising treatment avenues. Clinical translation of these approaches, however, will require further investigation of the pathological basis of disc degeneration in addition to systematic safety measures for the adoption of gene therapy.

Superior gene transfection efficiency in triple negative breast cancer by RAFT-mediated amino acid-based cationic diblock copolymers.

To date, the synthesis of efficient and safe gene carriers with low toxicity and appreciable gene transfection efficiency has been the major hurdle associated with non-viral gene carriers. Herein, we synthesized three amino acid-based diblock copolymers comprising glycine-leucine, leucine-phenyl alanine and glycine-phenyl alanine group containing blocks. The synthesis of all the diblock copolymers was confirmed by FTIR, 1H NMR, DLS and GPC techniques. All the polymers showed a high positive zeta potential value that varies from 45 ± 1 mV to 56 ± 1 mV, and the hydrodynamic size of the polymers varies from 250 ± 8 to 303 ± 14 nm. The three polymers showed negligible cytotoxicity compared with PEI (25 kDa) for MDA-MB-231 and NKE cells. Among all other polymers, P(HGN)n-b-P(HPN)m exhibited the highest biocompatibility with ∼70% cell viability at a concentration of 200 μg mL-1. Hemolysis data revealed that among all three polymers, P(HGN)n-b-P(HPN)m exhibited the highest blood compatibility, while up to a high concentration of 200 μg mL-1, it showed a very negligible amount (∼18%) of hemolysis. Most importantly, excellent gene complexation capability and good protection of pDNA against enzymatic degradation were observed with all three diblock copolymers. Interestingly, P(HGN)n-b-P(HPN)m/pDNA complex showed the smallest particle size (∼15 nm) and highest positive zeta potential as observed from TEM micrographs and DLS analysis, which probably results significantly higher level of cellular uptake and hence the highest transfection efficiency (∼85%) against MDA-MB-231 cells. Therefore, the diblock copolymer P(HGN)n-b-P(HPN)m with superior gene transfection efficiency in triple negative breast cancer may be an efficient non-viral vector for successful TNBC therapy in the future.

In vitro transfection efficiencies of T-shaped spermine-based cationic lipids with identical and nonidentical tails under high serum conditions.

T-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic tails having variable carbon lengths (from C10 to C18) were designed and synthesized. These lipids were characterized, and their structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. These liposomes were then applied as non-viral vectors to transfect HEK293T, HeLa, PC3, H460, HepG2, and Calu'3 cell lines with plasmid DNA encoding the green fluorescent protein. ST9, ST12 and ST13 with nonidentical tails could deliver DNA into HEK293T cells up to 60% under serum-free conditions. The lipid ST15 bearing nonidentical tails was found to be a potent gene transfer agent under 40% serum conditions in HEK293T and HeLa cells. Besides their low cytotoxicity, these lipoplexes also exhibited greater transfection efficiency than the commercially available transfection agent, Lipofectamine 3000.

In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector

Biological applications deriving from the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 site-specific nuclease system continue to impact and accelerate gene therapy strategies. Safe and effective in vivo co-delivery of the CRISPR/Cas9 system to target somatic cells is essential in the clinical therapeutic context. Both non-viral and viral vector systems have been applied for this delivery matter. Despite elegant proof-of-principle studies, available vector technologies still face challenges that restrict the application of CRISPR/Cas9-facilitated gene therapy. Of note, the mandated co-delivery of the gene-editing components must be accomplished in the potential presence of pre-formed anti-vector immunity. Additionally, methods must be sought to limit the potential of off-target editing. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the key requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of a simian (chimpanzee) adenovirus isolate 36 (SAd36) to achieve targeted modifications of vector tropism. The SAd36 vector with the myeloid cell-binding peptide (MBP) incorporated in the capsid has allowed selective in vivo modifications of the vascular endothelium. Importantly, vascular endothelium can serve as an effective non-hepatic cellular source of deficient serum factors relevant to several inherited genetic disorders. In addition to allowing for re-directed tropism, capsid engineering of nonhuman primate Ads provide the means to circumvent pre-formed vector immunity. Herein we have generated a SAd36. MBP vector that can serve as a single intravenously administered agent allowing effective and selective in vivo editing for endothelial target cells of the mouse spleen, brain and kidney. Data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

Efficient gene transfection of suspension cells by highly branched poly(β-amino ester)

Suspension cells play a crucial role in many biological processes. However, compared to adherent cells, it is particularly challenging to introduce exogenous genes into suspension cells to regulate their biological functions with non-viral gene vectors, mainly due to the low cellular uptake and endosomal escape of polyplexes. Herein, to improve the interactions of polyplexes with cellular membranes, we design and synthesize highly branched poly(β-amino ester) (HPAE) via an “A2 + B4 + C2” Michael addition strategy. Results show that branching significantly increases DNA condensation of HPAE, cellular uptake and endosomal escape of HPAE/DNA polyplexes. In mast cells (MCs), HPAE exhibits up to 80-fold higher gene transfection efficiency compared to the corresponding linear poly(β-amino ester) (LPAE) and the leading commercial gene transfection reagents PEI25k, jetPEI, and Lipofectamine 3000, without causing obvious cytotoxicity. Our study establishes a reliable non-viral platform for efficient gene transfection of suspension cells.

Antioxidant Stress of Transdermal Gene Delivery by Non-Viral Gene Vectors Based on Chitosan-Oligosaccharide.

Oxidative stress initiated by reactive oxygen species (ROS) is the cause of many acquired or congenital skin diseases. Oral antioxidants or using topical antioxidants preparations may bring the nonspecific distribution of drugs or anaphylaxis due to repeated use. In this study, a biocompatible gene vector by cross-linking of chitosan-oligosaccharide (CSO) and N,N'-cystamine-bis-acrylamide (CBA) was synthesized (CSO-CBA), which could carry therapeutic genes into the skin, express functional proteins in epidermal cells, and play an efficient antioxidant effect. Infrared and 1H NMR spectrum data showed that CSO-CBA was successfully synthesized. Gel electrophoresis results showed that the gene could be successfully compressed by the carrier and can be released in a reducing environment. Hemolysis experiments showed that the carrier had good biocompatibility. Transdermal gene delivery experiments proved that the vector can bring genes into the skin, express functional proteins, and protect the skin from reactive oxygen species damage after 7 days of administration. Skin compatibility experiments show that our therapy is biocompatible. Our study provides a minimally invasive and painless, high-biocompatibility, and long-term effective treatment for skin damage caused by reactive oxygen species, which has a potential application.

Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle.

RNA interference is a promising therapeutical approach presently hindered by delivery concerns such as rapid RNA degradation and targeting of individual tissues. Injectable hydrogels are one potentially simple and direct route towards overcoming these barriers. Here we report on the utility of a combination of a mildly modified form of the clinically utilised fibrin hydrogel with Invivofectamine® 3.0, a lipid nonviral transfection vector, for local and sustained release. PEGylation of fibrin allowed for controlled release of small interfering RNA (siRNA)-lipopolyplexes for at least 10 days and greatly increased the stability of fibrin in vitro and in vivo. A 3D cell culture model and a release study showed transfection efficacy of siRNA-lipopolyplexes was retained for a minimum of 7 days. Injection in conjunction with PEGylated-fibrinogen significantly increased retention of siRNA-lipopolyplexes in mouse skeletal muscle and enhanced knockdown of myostatin mRNA that correlated with muscle growth. Thus, the increased efficacy observed here for the combination of a lipid nanoparticle, the only type of nonviral vector approved for the clinic, with fibrin, might allow for more rapid translation of injectable hydrogel-based RNA interference.

Plasmid DNA cationic non-viral vector complexes induce cytotoxicity-associated PD-L1 expression up-regulation in cancer cells in vitro

Non-viral vectors are promising nucleic acid carriers which have been utilized in gene-based cancer immunotherapy. The aim of this study is to compare the transfection efficiency and cytotoxicity of three cationic non-viral vectors namely Polyethylenimine (PEI), Lipofectamine 2000 (LPF) and stable nucleic acid lipid particles (SNALPs) of different lipid compositions, for the delivery of plasmid DNA (pDNA) expressing immunostimulatory molecules, OX40L or 4-1BBL, to cancer cells in vitro. The results indicate that PEI and LPF are efficient vectors for pDNA delivery with high transfection efficiency obtained. However, pDNA-PEI and pDNA-LPF complexes up-regulated the expression of programmed death ligand-1 (PD-L1) and induced significant cytotoxicity in both B16F10 and CT26 cell lines. The up-regulation of PD-L1 expression induced by pDNA-PEI and pDNA-LPF complexes was independent of cancer cell line, nor was it linked to the presence of GpC motifs in the pDNA. In contrast, the use of biocompatible SNALPs (MC3 and KC2 types) resulted in lower pDNA transfection efficiency, however no significant up-regulation of PD-L1 or cytotoxicity was observed. A strong correlation was found between up-regulation of PD-L1 expression and cytotoxicity. Up-regulation of PD-L1 expression could be mitigated with RNAi, maintaining expression at basal levels. Due to the improved biocompatibility and the absence of PD-L1 up-regulation, SNALPs represent a viable non-viral nucleic acid vector for delivery of pDNA encoding immunostimulatory molecules. The results of this study suggest that PD-L1 expression should be monitored when selecting commercial transfection reagents as pDNA vectors for cancer immunotherapy in vitro.