Search Results (19)

Background: Genome editing at specific loci is an innovative therapeutic approach; however, it faces many challenges, so optimizing delivery vectors is essential to enhance the safety and efficacy of the CRISPR/Cas9 system. This study investigated whether the hydrodynamic administration of liposomal CRISPR/Cas9 complexes (LCs) in newborn mice induces off-target events or tumors. Methods: Liposomes were obtained through microfluidization. The CRISPR/Cas9 plasmid and a donor plasmid containing the Idua cDNA (alpha-L-iduronidase enzyme) were incorporated by adsorption, and complexes (LCs) were characterized regarding physicochemical properties. C57BL/6 newborn mice were divided in two groups, one received the complexes through hydrodynamic intravenous injection (n = 15) and the other was used as control (n = 15). After 21 months, mice were euthanized and organs were analyzed regarding histological characteristics. Lungs and liver were analyzed by qPCR searching for potential off-target sites in chromosomes 2, 5, 11, and 17 and on-target site in chromosome 6, identified by COSMID. Sequences were analyzed using an ICE tool for indels detection. Results: LCs exhibited 136 nm mean vesicle diameter with PDI below 0.15 and a zeta potential around +43 mV. Immediate biodistribution was predominant in the lungs and liver. There was no significant increase in tumor induction (20% in LCs vs. 33% in control). Molecular analyses indicated 0% off-target effects and around 3% on-target events. Conclusions: We conclude that this set of experiments demonstrates the potential of the chosen gRNA sequence to perform safe gene editing at the murine ROSA26 locus, corroborating the safety of the CRISPR/Cas9 gene editing platform. Full article

One of the major challenges in gene therapy is the efficient and safe introduction of nucleic acids into eukaryotic cells. This process requires overcoming various biological barriers and navigating complex pathways to reach target cells and achieve their biological function. To address this obstacle, numerous transfection methods have been developed, including physical techniques and the use of genetic vectors, both viral and non-viral. However, to date, no transfection method is 100% safe and efficient. Within the spectrum of non-viral genetic vectors, cationic liposomes formed by cationic lipids stand out for their ability to protect and deliver therapeutic NA. These liposomes offer greater biocompatibility and lower immunogenicity compared to viral vectors, although they still do not match the efficiency of viral delivery systems. Consequently, ongoing research focuses on synthesizing a wide variety of cationic lipids in the search for compounds that provide high transfection efficiency with minimal cytotoxicity. This study aimed to design and synthesize a novel cationic lipid (CholCadLys) derived from natural cellular molecules for transferring genetic material to eukaryotic cells. The lipid was synthesized using cholesteryl chloroformate for the hydrophobic region, cadaverine as a linker, and lysine for the polar region, connected by carbamate and amide bonds, respectively. Identification was confirmed through thin-layer chromatography, purification through preparative chromatography, and characterization via infrared spectroscopy and mass spectrometry. The synthesis yielded a 60% success rate, with stable nanoliposomes averaging 76 nm in diameter. Liposomes were formed using this CL and commercial neutral lipids, characterized by transmission electron microscopy and Nanoparticle Tracking Analysis. These liposomes, combined with plasmid DNA, formed lipoplexes used to transfect Hek-293 FT cells, achieving up to 40% transfection efficiency without cytotoxicity in the mixture of CholCadLys and CholCad. This novel CL demonstrates potential as an efficient, safe, and cost-effective gene transfer system, facilitating further development in gene therapy. Full article

Gene therapy is one of the most promising techniques for treating genetic diseases and cancer. The current most important problem in gene therapy is gene delivery. Viral and non-viral vectors like liposomes, used for gene delivery, have many limitations. We have developed new hybrid peptides by combining cell-penetrating peptides (CPPs) with the DNA-binding domain of the human histone H4 protein. These small peptides bind to DNA molecules through their histone domain, leaving the CPP part free and available for binding and penetration into cells, forming complexes that we named “peptosomes”. We evaluated the transfection efficiency of several hybrid peptides by delivering a plasmid carrying the green fluorescent protein gene and following its expression by fluorescent microscopy. Among several hybrid peptides, TM3 achieved a gene delivery efficiency of 76%, compared to 52% for Lipofectamine 2000. TM3 peptosomes may become important gene delivery tools with several advantages over current gene delivery agents. Full article

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR–Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR–Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy. Full article

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Full article

Thermosensitive cationic magnetic liposomes (TCMLs), prepared from dipalmitoylphosphatidylcholine (DPPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000, and didodecyldimethylammonium bromide (DDAB) were used in this study for the controlled release of drug/gene for cancer treatment. After co-entrapping citric-acid-coated magnetic nanoparticles (MNPs) and the chemotherapeutic drug irinotecan (CPT-11) in the core of TCML (TCML@CPT-11), SLP2 shRNA plasmids were complexed with DDAB in the lipid bilayer to prepare TCML@CPT-11/shRNA with a 135.6 ± 2.1 nm diameter. As DPPC has a melting temperature slightly above the physiological temperature, drug release from the liposomes can be triggered by an increase in solution temperature or by magneto-heating induced with an alternating magnetic field (AMF). The MNPs in the liposomes also endow the TCMLs with magnetically targeted drug delivery with guidance by a magnetic field. The successful preparation of drug-loaded liposomes was confirmed by various physical and chemical methods. Enhanced drug release, from 18% to 59%, at pH 7.4 was observed when raising the temperature from 37 to 43 °C, as well as during induction with an AMF. The in vitro cell culture experiments endorse the biocompatibility of TCMLs, whereas TCML@CPT-11 shows some enhancement of cytotoxicity toward U87 human glioblastoma cells when compared with free CPT-11. The U87 cells can be transfected with the SLP2 shRNA plasmids with very high efficiency (~100%), leading to silencing of the SLP2 gene and reducing the migration ability of U87 from 63% to 24% in a wound-healing assay. Finally, an in vivo study, using subcutaneously implanted U87 xenografts in nude mice, demonstrates that the intravenous injection of TCML@CPT11-shRNA, plus magnetic guidance and AMF treatment, can provide a safe and promising therapeutic modality for glioblastoma treatment. Full article

The generation of reactive oxygen species (ROS) can affect cationic liposome-mediated transfection. In this study, we focused on a specific class of antioxidants, flavonoids, to investigate the transfection efficiency using cationic liposome/plasmid DNA complexes (lipoplexes) in 2D and 3D cultures of Colon26 and HepG2 cells, respectively. All tested flavonoids enhanced the transfection efficiency in 2D Colon26 and HepG2 cells. Among the tested flavonoids, 25 µM quercetin showed the highest promotion effect of 8.4- and 7.6-folds in 2D Colon26 and HepG2 cells, respectively. Transfection was also performed in 3D cultures of Colon26 and HepG2 cells using lipoplexes with quercetin. Quercetin (12.5 µM) showed the highest transfection efficiency at all transfection timings in 3D Colon26 and HepG2 cells with increased cell viability. Flow cytometry revealed that quercetin treatment reduced the population of gene expression-negative cells with high ROS levels and increased the number of gene expression-positive cells with low ROS levels in HepG2 cells. Information from this study can be valuable to develop strategies to promote transfection efficiency and attenuate cytotoxicity using lipoplexes. Full article

In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal. Full article

Manganese superoxide dismutase (MnSOD) is a dominant component of the antioxidant defense system in mammalian cells. Since ionizing irradiation induces profound oxidative stress, it was logical to test the effect of overexpression of MnSOD on radioresistance. This task was accomplished by introduction of a transgene for MnSOD into cells in vitro and into organs in vivo, and both paradigms showed clear radioresistance following overexpression. During the course of development and clinical application of using MnSOD as a radioprotector, several prominent observations were made by Larry Oberley, Joel Greenberger, and Michael Epperly which include (1) mitochondrial localization of either manganese superoxide dismutase or copper/zinc SOD was required to provide optimal radiation protection; (2) the time required for optimal expression was 12–18 h, and while acceptable for radiation protection, the time delay was impractical for radiation mitigation; (3) significant increases in intracellular elevation of MnSOD activity were required for effective radioprotection. Lessons learned during the development of MnSOD gene therapy have provided a strategy for delivery of small molecule SOD mimics, which are faster acting and have shown the potential for both radiation protection and mitigation. The purpose of this review is to summarize the current status of using MnSOD-PL and SOD mimetics as radioprotectors and radiomitigators. Full article

The approval of two mRNA vaccines as urgent prophylactic treatments against Covid-19 made them a realistic alternative to conventional vaccination methods. However, naked mRNA is rapidly degraded by the body and cannot effectively penetrate cells. Vectors capable of addressing these issues while allowing endosomal escape are therefore needed. To date, the most widely used vectors for this purpose have been lipid-based vectors. Thus, we have designed an innovative vector called LipoParticles (LP) consisting of poly(lactic) acid (PLA) nanoparticles coated with a 15/85 mol/mol DSPC/DOTAP lipid membrane. An in vitro investigation was carried out to examine whether the incorporation of a solid core offered added value compared to liposomes alone. To that end, a formulation strategy that we have named particulate layer-by-layer (pLbL) was used. This method permitted the adsorption of nucleic acids on the surface of LP (mainly by means of electrostatic interactions through the addition of LAH4-L1 peptide), allowing both cellular penetration and endosomal escape. After a thorough characterization of size, size distribution, and surface charge— and a complexation assessment of each vector—their transfection capacity and cytotoxicity (on antigenic presenting cells, namely DC2.4, and epithelial HeLa cells) were compared. LP have been shown to be significantly better transfecting agents than liposomes through pLbL formulation on both HeLa and DC 2.4 cells. These data illustrate the added value of a solid particulate core inside a lipid membrane, which is expected to rigidify the final assemblies and makes them less prone to early loss of mRNA. In addition, this assembly promoted not only efficient delivery of mRNA, but also of plasmid DNA, making it a versatile nucleic acid carrier that could be used for various vaccine applications. Finally, if the addition of the LAH4-L1 peptide systematically leads to toxicity of the pLbL formulation on DC 2.4 cells, the optimization of the nucleic acid/LAH4-L1 peptide mass ratio becomes an interesting strategy—essentially reducing the peptide intake to limit its cytotoxicity while maintaining a relevant transfection efficiency. Full article

Cationic liposomes have been considered as potential vectors for gene delivery thanks to their ability to transfect cells with high efficiency. Recently, the combination of diagnostic agent and therapeutic agents in the same particle to form a theranostic system has been reported. Magnetic liposomes are one of these examples. Due to the magnetic nanoparticles encapsulated in the liposomes, they can act as a drug delivery system and, at the same time, a magnetic resonance imaging contrast enhancement agent or hyperthermia. In this work, nucleic acid delivery systems based on magnetic cationic liposomes (MCLs) were developed. Two different techniques, reverse phase evaporation and cosolvent sonication, were employed for liposome preparation. Both strategies produced MCLs of less than 200 nm with highly positive charge. Enhancement of their transverse and longitudinal relaxivities r₂ and r₁ was obtained with both kinds of magnetic liposomes compared to free magnetic nanoparticles. Moreover, these MCLs showed high capacity to form complexes and transfect CT-26 cells using the antibiotic-free pFAR4-luc plasmid. The transfection enhancement with magnetofection was also carried out in CT26 cells. These results suggested that our MCLs could be a promising candidate for image-guided gene therapy. Full article

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)₃]²⁺ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between K_app and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide’s condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy. Full article

Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers. Full article

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues. Full article

Gallbladder cancer is associated with a dismal prognosis, and accurate in vivo models will be elemental to improve our understanding of this deadly disease and develop better treatment options. We have generated a transplantation-based murine model for gallbladder cancer that histologically mimics the human disease, including the development of distant metastasis. Murine gallbladder–derived organoids are genetically modified by either retroviral transduction or transfection with CRISPR/Cas9 encoding plasmids, thereby allowing the rapid generation of complex cancer genotypes. We characterize the model in the presence of two of the most frequent oncogenic drivers—Kras and ERBB2—and provide evidence that the tumor histology is highly dependent on the driver oncogene. Further, we demonstrate the utility of the model for the preclinical assessment of novel therapeutic approaches by showing that liposomal Irinotecan (Nal-IRI) is retained in tumor cells and significantly prolongs the survival of gallbladder cancer–bearing mice compared to conventional irinotecan. Full article