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1.
PLoS One ; 19(8): e0308075, 2024.
Article in English | MEDLINE | ID: mdl-39088581

ABSTRACT

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.


Subject(s)
Aorta , Microbubbles , Nitric Oxide Synthase Type III , Nitric Oxide , RNA, Small Interfering , Transfection , Animals , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type III/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Transfection/methods , Aorta/metabolism , Nitric Oxide/metabolism , Mice , Male , Cell Line , Neovascularization, Physiologic/genetics
2.
Nanotechnology ; 35(41)2024 Jul 25.
Article in English | MEDLINE | ID: mdl-39008958

ABSTRACT

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.


Subject(s)
Genetic Vectors , Glutathione , Polyethyleneimine , Transfection , Polyethyleneimine/chemistry , Transfection/methods , Glutathione/metabolism , Glutathione/chemistry , Animals , Humans , Genetic Vectors/chemistry , Genetic Vectors/genetics , Mannitol/chemistry , Mice , Caveolin 1/metabolism , Caveolin 1/genetics , Genetic Therapy/methods , Gene Transfer Techniques , Disulfides/chemistry
3.
Cells ; 13(14)2024 Jul 11.
Article in English | MEDLINE | ID: mdl-39056764

ABSTRACT

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.


Subject(s)
DNA , Extracellular Vesicles , Lipids , Nanoparticles , Plasmids , Transfection , Humans , Plasmids/genetics , Nanoparticles/chemistry , Extracellular Vesicles/metabolism , Hep G2 Cells , Lipids/chemistry , DNA/metabolism , DNA/chemistry , Transfection/methods , Nucleic Acids/metabolism , Nucleic Acids/chemistry , Liposomes
4.
Int J Mol Sci ; 25(13)2024 Jun 24.
Article in English | MEDLINE | ID: mdl-39000028

ABSTRACT

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.


Subject(s)
Cell-Penetrating Peptides , Liposomes , Transfection , Humans , Liposomes/chemistry , Cell-Penetrating Peptides/chemistry , Transfection/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Gene Transfer Techniques , Plasmids/genetics , Genetic Therapy/methods , Histones/metabolism , Histones/chemistry , Histones/genetics , HeLa Cells
5.
Methods Mol Biol ; 2842: 255-265, 2024.
Article in English | MEDLINE | ID: mdl-39012600

ABSTRACT

To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.


Subject(s)
CRISPR-Cas Systems , Epigenesis, Genetic , Gene Editing , Plasmids , Single-Cell Analysis , Transfection , Humans , Plasmids/genetics , Gene Editing/methods , HEK293 Cells , Transfection/methods , Single-Cell Analysis/methods , Epigenomics/methods , Flow Cytometry
6.
J Vis Exp ; (208)2024 Jun 21.
Article in English | MEDLINE | ID: mdl-38975778

ABSTRACT

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).


Subject(s)
DNA, Ribosomal , Naegleria , Transfection , Naegleria/genetics , Transfection/methods , DNA, Ribosomal/genetics , Trophozoites , DNA, Protozoan/genetics , Cloning, Molecular/methods
7.
Methods Mol Biol ; 2829: 49-66, 2024.
Article in English | MEDLINE | ID: mdl-38951326

ABSTRACT

This chapter outlines the workflow using the ExpiSf™ Expression System designed for high-density infection of suspension ExpiSf9™ cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf™ chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf™ Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.


Subject(s)
Baculoviridae , Recombinant Proteins , Workflow , Animals , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Baculoviridae/genetics , Transfection/methods , Culture Media/chemistry , Cell Culture Techniques/methods , Cell Line , Gene Expression
8.
Methods Mol Biol ; 2829: 13-20, 2024.
Article in English | MEDLINE | ID: mdl-38951324

ABSTRACT

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.


Subject(s)
Baculoviridae , Recombinant Proteins , Baculoviridae/genetics , Animals , Recombinant Proteins/genetics , Genetic Vectors/genetics , Transfection/methods , Homologous Recombination , Sf9 Cells , Cell Line , Spodoptera/virology , Insecta/genetics , Insecta/virology
9.
Methods Mol Biol ; 2829: 127-156, 2024.
Article in English | MEDLINE | ID: mdl-38951331

ABSTRACT

The baculovirus expression vector system (BEVS) has now found acceptance in both research laboratories and industry, which can be attributed to many of its key features including the limited host range of the vectors, their non-pathogenicity to humans, and the mammalian-like post-translational modification (PTMs) that can be achieved in insect cells. In fact, this system acts as a middle ground between prokaryotes and higher eukaryotes to produce complex biologics. Still, industrial use of the BEVS lags compared to other platforms. We have postulated that one reason for this has been a lack of genetic tools that can complement the study of baculovirus vectors, while a second reason is the co-production of the baculovirus vector with the desired product. While some genetic enhancements have been made to improve the BEVS as a production platform, the genome remains under-scrutinized. This chapter outlines the methodology for a CRISPR-Cas9-based transfection-infection assay to probe the baculovirus genome for essential/nonessential genes that can potentially maximize foreign gene expression under a promoter of choice.


Subject(s)
Baculoviridae , CRISPR-Cas Systems , Genetic Vectors , Baculoviridae/genetics , Genetic Vectors/genetics , Animals , Genes, Essential , Gene Expression , Transfection/methods , Gene Editing/methods , Sf9 Cells , Humans
10.
Methods Mol Biol ; 2829: 289-300, 2024.
Article in English | MEDLINE | ID: mdl-38951345

ABSTRACT

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.


Subject(s)
Polyethyleneimine , Recombinant Proteins , Transfection , Animals , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection/methods , Polyethyleneimine/chemistry , Plasmids/genetics , Insecta/genetics , Sf9 Cells , Cell Line , Gene Expression , Spodoptera
11.
Methods Mol Biol ; 2829: 329-339, 2024.
Article in English | MEDLINE | ID: mdl-38951347

ABSTRACT

Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of "insect" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.


Subject(s)
Baculoviridae , Recombinant Proteins , Baculoviridae/genetics , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/biosynthesis , HEK293 Cells , Animals , Transfection/methods , Genetic Vectors/genetics , Cell Culture Techniques/methods , Gene Expression , Glycosylation
12.
J Vis Exp ; (208)2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38949388

ABSTRACT

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.


Subject(s)
Neural Stem Cells , Transfection , Animals , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Mice , Transfection/methods , Lateral Ventricles/cytology , Cytological Techniques/methods
13.
ACS Appl Mater Interfaces ; 16(30): 39784-39795, 2024 Jul 31.
Article in English | MEDLINE | ID: mdl-39036892

ABSTRACT

The utilization of hydrogels for DNA/cationic polymer polyplex nanoparticle (polyplex) delivery has significantly advanced gene therapy in tissue regeneration and cancer treatment. However, persistent challenges related to the efficacy and safety of encapsulated polyplexes, stemming from issues such as aggregation, degradation, or difficulties in controlled release during or postintegration with hydrogel scaffolds, necessitate further exploration. Here, we introduce an injectable gene therapy gel achieved by incorporating concentrated polyplexes onto densely packed hydrogel microparticles (HMPs). Polyplexes, when uniformly adhered to the gene therapy gel through reversible electrostatic interactions, can detach from the HMP surface in a controlled manner, contrasting with free polyplexes, and thereby reducing dose-dependent toxicity during transfection. Additionally, the integration of RGD cell adhesion peptides enhances the scaffolding characteristics of the gel, facilitating cell adhesion, migration, and further minimizing toxicity during gene drug administration. Notably, despite the overall transfection efficiency showing average performance, utilizing confocal microscopy to meticulously observe and analyze the cellular states infiltrating into various depths of the gene therapy gel resulted in the groundbreaking discovery of significantly enhanced local transfection efficiency, with primary cell transfection approaching 80%. This phenomenon could be potentially attributed to the granular hydrogel-mediated delivery of polyplex nanoparticles, which revolutionizes the spatial and temporal distribution and thus the "encounter" mode between polyplexes and cells. Moreover, the gene therapy gel's intrinsic injectability and self-healing properties offer ease of administration, making it a highly promising candidate as a novel gene transfection gel dressing with significant potential across various fields, including regenerative medicine and innovative living materials.


Subject(s)
DNA , Genetic Therapy , Hydrogels , Nanoparticles , Nanoparticles/chemistry , Nanoparticles/toxicity , Humans , Hydrogels/chemistry , Hydrogels/pharmacology , DNA/chemistry , Transfection/methods , Animals , Oligopeptides/chemistry , Gene Transfer Techniques
14.
ACS Appl Mater Interfaces ; 16(28): 36095-36105, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-38970470

ABSTRACT

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.


Subject(s)
Saponins , Animals , Humans , Mice , Saponins/chemistry , Saponins/pharmacology , Cell Line, Tumor , Gene Transfer Techniques , Peptides/chemistry , Transfection/methods , Saponaria/chemistry , Saporins/chemistry , Saporins/pharmacology , Genetic Therapy , Cell Survival/drug effects , Cations/chemistry
15.
AAPS PharmSciTech ; 25(5): 131, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38849687

ABSTRACT

Lipid-based vectors are becoming promising alternatives to traditional therapies over the last 2 decades specially for managing life-threatening diseases like cancer. Cationic lipids are the most prevalent non-viral vectors utilized in gene delivery. The increasing number of clinical trials about lipoplex-based gene therapy demonstrates their potential as well-established technology that can provide robust gene transfection. In this regard, this review will summarize this important point. These vectors however have a modest transfection efficiency. This limitation can be partly addressed by using functional lipids that provide a plethora of options for investigating nucleic acid-lipid interactions as well as in vitro and in vivo nucleic acid delivery for biomedical applications. Despite their lower gene transfer efficiency, lipid-based vectors such as lipoplexes have several advantages over viral ones: they are less toxic and immunogenic, can be targeted, and are simple to produce on a large scale. Researchers are actively investigating the parameters that are essential for an effective lipoplex delivery method. These include factors that influence the structure, stability, internalization, and transfection of the lipoplex. Thorough understanding of the design principles will enable synthesis of customized lipoplex formulations for life-saving therapy.


Subject(s)
Gene Transfer Techniques , Genetic Therapy , Lipids , Liposomes , Humans , Lipids/chemistry , Genetic Therapy/methods , Liposomes/chemistry , Animals , Transfection/methods , Genetic Vectors/chemistry , Nucleic Acids/chemistry , Nucleic Acids/administration & dosage
16.
Methods Mol Biol ; 2822: 367-386, 2024.
Article in English | MEDLINE | ID: mdl-38907929

ABSTRACT

Transfection with mRNA has been considered superior to that with plasmids since the mRNA can be translated to a protein in the cytosol without entering the nucleus. One disadvantage of using mRNA is its susceptibility to enzymatic biodegradability, and consequently, significant research has occurred to determine nonviral carriers that will sufficiently stabilize this nucleic acid for cellular transport. Histidine-lysine peptides (HK) are one such class of mRNA carriers, which we think serves as a model for other peptides and polymeric carrier systems. When the HK peptide and mRNA are mixed and interact through ionic and nonionic bonds, mRNA polyplexes are formed, which can transfect cells. In contrast to linear HK peptides, branched HK peptides protected and efficiently transfected mRNA into cells. After describing the preparation and biophysical characterization of these polyplexes, we will provide protocols for in vitro and in vivo transfection for these mRNA polyplexes.


Subject(s)
Histidine , Lysine , Peptides , RNA, Messenger , Transfection , Histidine/chemistry , Histidine/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Lysine/chemistry , Lysine/metabolism , Transfection/methods , Peptides/chemistry , Humans , Animals
17.
Methods Mol Biol ; 2822: 353-365, 2024.
Article in English | MEDLINE | ID: mdl-38907928

ABSTRACT

Polymeric delivery systems could enable the fast- and low-side-effect transport of various RNA classes. Previously, we demonstrated that polyvinylamine (PVAm), a cationic polymer, transfects many kinds of RNAs with high efficiency and low toxicity both in vitro and in vivo. The modification of poly lactic-co-glycolic acid (PLGA) with cartilage-targeting peptide (CAP) enhances its stiffness and tissue-specific delivery of RNA to overcome the avascular nature of articular cartilage. Here we describe the protocol to use PVAm as an RNA carrier, and further, by modifying PVAm with PLGA and CAP, the corresponding co-polymer could be applied for functional RNA delivery for osteoarthritis treatment.


Subject(s)
Polylactic Acid-Polyglycolic Acid Copolymer , Polyvinyls , Polyvinyls/chemistry , Animals , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Humans , Lactic Acid/chemistry , Transfection/methods , Gene Transfer Techniques , Polyglycolic Acid/chemistry , Drug Carriers/chemistry , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Osteoarthritis/drug therapy
18.
PLoS One ; 19(6): e0297817, 2024.
Article in English | MEDLINE | ID: mdl-38833479

ABSTRACT

Lentiviral vectors derived from human immunodeficiency virus type I are widely used to deliver functional gene copies to mammalian cells for research and gene therapies. Post-transcriptional splicing of lentiviral vector transgene in transduced host and transfected producer cells presents barriers to widespread application of lentiviral vector-based therapies. The present study examined effects of indole derivative compound IDC16 on splicing of lentiviral vector transcripts in producer cells and corresponding yield of infectious lentiviral vectors. Indole IDC16 was shown previously to modify alternative splicing in human immunodeficiency virus type I. Human embryonic kidney 293T cells were transiently transfected by 3rd generation backbone and packaging plasmids using polyethyleneimine. Reverse transcription-quantitative polymerase chain reaction of the fraction of unspliced genomes in human embryonic kidney 293T cells increased up to 31% upon the indole's treatment at 2.5 uM. Corresponding yield of infectious lentiviral vectors decreased up to 4.5-fold in a cell transduction assay. Adjusting timing and duration of IDC16 treatment indicated that the indole's disruption of early stages of transfection and cell cycle had a greater effect on exponential time course of lentiviral vector production than its reduction of post-transcriptional splicing. Decrease in transfected human embryonic kidney 293T proliferation by IDC16 became significant at 10 uM. These findings indicated contributions by early-stage transfection, cell proliferation, and post-transcriptional splicing in transient transfection of human embryonic kidney 293T cells for lentiviral vector production.


Subject(s)
Alternative Splicing , Cell Proliferation , Genetic Vectors , Indoles , Lentivirus , Transfection , Humans , Indoles/pharmacology , Cell Proliferation/drug effects , Genetic Vectors/genetics , Lentivirus/genetics , Transfection/methods , HEK293 Cells
19.
Eur J Pharm Biopharm ; 201: 114385, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38945408

ABSTRACT

In the current "era of lipid carriers," numerous strategies have been developed to manufacture lipid nanoparticles (LNPs). Nevertheless, the potential impact of various preparation methods on the characteristics, use, and/or stability of these LNPs remains unclear. In this work, we attempted to compare the effects of three different preparation methods: microfluidics (MF), reverse phase evaporation (RV), and ouzo (OZ) on lipid-peptide NPs (LPNPs) as plasmid DNA delivery carriers. These LPNPs had the same components, namely DOTMA cationic lipid, DSPC, cholesterol, and protamine. Subsequently, we compared the LPNPs in terms of their physicochemical features, functionality as gene delivery vehicles in two distinct cell lines (NT2 and D1-MSCs), and finally, their storage stability over a six-month period. It was clear that all three LPNP formulations worked to deliver EGFP-pDNA while keeping cells alive, and their physicochemical stability was high for 6 months. However, the preparation technique had a significant impact on their physicochemical characteristics. The MF produced LPNPs with a lesser size, polydispersity index, and zeta potential than the other synthesis methods. Additionally, their DNA entrapment efficiency, cell viability, and functional stability profiles were generally superior. These findings provide new insights for comparing different manufacturing methods to create LPNPs with the desired characteristics for effective and safe gene delivery.


Subject(s)
DNA , Gene Transfer Techniques , Lipids , Microfluidics , Nanoparticles , Peptides , Plasmids , Nanoparticles/chemistry , Plasmids/administration & dosage , Humans , Lipids/chemistry , DNA/administration & dosage , DNA/chemistry , Microfluidics/methods , Peptides/chemistry , Cell Line , Transfection/methods , Particle Size , Cell Survival/drug effects
20.
Sci Rep ; 14(1): 14874, 2024 06 27.
Article in English | MEDLINE | ID: mdl-38937523

ABSTRACT

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.


Subject(s)
Norovirus , Plasmids , Rotavirus , Animals , Plasmids/genetics , Rotavirus/genetics , Norovirus/genetics , Enterovirus/genetics , Sf9 Cells , Baculoviridae/genetics , Genetic Vectors/genetics , Transfection/methods , Vaccines, Virus-Like Particle/genetics , Vaccines, Virus-Like Particle/biosynthesis , Insecta , Cell Line
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