Evaluation Of Transfection Efficiency Research Articles

Combined Effects of Annexin A5 Overexpression, 5-Fluorouracil Treatment, and Irradiation on Cell Viability of Caski Cervical Cancer Cell Line.

Cervical cancer is the fourth leading cause of cancer deaths in women globally. Combining gene therapy with chemo- and radiotherapy may improve cervical cancer treatment outcomes. This study evaluated the effects of Annexin A5(ANXA5) overexpression alongside 5-fluorouracil (5-FU) and irradiation on the viability of CaSki cervical squamous cell carcinoma (SCC) cells. pAdenoVator-CMV-ANXA5-IRES-GFP-plasmid and mock plasmid were transfected into CaSki cells using calcium-phosphate. Seventy-two hours post-transfection, GFP expression was quantified by fluorescence microscopy and flow cytometry to evaluate transfection efficiency. ANXA5 overexpression was confirmed via qPCR. Twenty-four hours post-transfection, cells received a single dose of 8Gy and were treated with 1 and 2µg/ml of 5-FU (IC50 = 2.783µg/ml). Cell viability, apoptosis, cell cycle stage, and Bcl-2 and Bax gene expression were assessed via MTT, annexin V/7-AAD, PI staining, and qPCR assays, respectively. ANXA5 was overexpressed 31.5-fold compared to control (p < 0.0001). MTT assays showed ANXA5 overexpression dose-dependently reduced CaSki cell viability (p < 0.001). IC50 of 5-FU was reduced from 2.783μg/mL to 1.794μg/mL when combined with ANXA5 overexpression. Additive effects on cell death were observed for ANXA5 plus 5-FU or irradiation versus ANXA5 alone. Apoptosis assays indicated combinatorial treatment increased CaSki cell apoptosis over ANXA5 alone. Cell cycle analysis revealed ANXA5 arrested cell cycle at G1/S phases; the percentage of cells in the S phase further rose with combination treatment. Finally, combination therapy significantly decreased Bcl-2 expression and increased Bax versus control (p < 0.001). Altogether, ANXA5 overexpression alongside 5-FU and irradiation may improve cervical squamous cell carcinoma (SCC) treatment efficacy. Further, in vivo investigations are warranted to confirm these in vitro results.

Lactosylated lipid calcium phosphate-based nanoparticles: A promising approach for efficient DNA delivery to hepatocytes.

For safe and effective gene therapy, the ability to deliver the therapeutic nucleic acid to the target sites is crucial. In this study, lactosylated lipid phosphate calcium nanoparticles (lac-LCP) were developed for targeted delivery of pDNA to the hepatocyte cells. The lac-LCP formulation contained lactose-modified cholesterol (CHL), a ligand that binds to the asialoglycoprotein receptor (ASGR) expressed on hepatocytes, and polyethyleneimine (PEI) in the core. Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) were used to monitor the chemical modification, and the physicochemical properties of NPs were studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). To evaluate transfection efficiency, cellular uptake and GFP expression were assessed using fluorescence microscopy and flow cytometry. The results revealed that lactose-targeted particles (lac-LCP) had a significant increase in cellular uptake by hepatocytes. The inclusion of a low molecular weight PEI (1.8 KDa) with a low PEI/pDNA ratio of 1 in the core of LCP, elicited high degrees of GFP protein expression (by 5 and 6-fold), which exhibited significantly higher efficiency than PEI 1.8 KDa and Lipofectamine. The successful functionalization and nuclear delivery of LCP NPs described here indicate its promise as an efficient delivery vector to hepatocyte nuclei.

Cell uptake and intracellular trafficking of bioreducible poly(amidoamine) nanoparticles for efficient mRNA translation in chondrocytes.

Disulfide-containing poly(amidoamine) (PAA) is a cationic and bioreducible polymer, with potential use as a nanocarrier for mRNA delivery in the treatment of several diseases including osteoarthritis (OA). Successful transfection of joint cells with PAA-based nanoparticles (NPs) was shown previously, but cell uptake, endosomal escape and nanoparticle biodegradation were not studied in detail. In this study, C28/I2 human chondrocytes were transfected with NPs co-formulated with a PEG-polymer coating and loaded with EGFP mRNA for confocal imaging of intracellular trafficking and evaluation of transfection efficiency. Compared with uncoated NPs, PEG-coated NPs showed smaller particle size, neutral surface charge, higher colloidal stability and superior transfection efficiency. Furthermore, endosomal entrapment of these PEG-coated NPs decreased over time and mRNA release could be visualized both in vitro and in live cells. Importantly, cell treatment with modulators of the intracellular reducing environment showed that glutathione (GSH) concentrations affect translation of the mRNA payload. Finally, we applied a D-optimal experimental design to test different polymer-to-RNA loading ratios and dosages, thus obtaining an optimal formulation with up to ≈80% of GFP-positive cells and without toxic effects. Together, the biocompatibility and high transfection efficiency of this system may be a promising tool for intra-articular delivery of therapeutical mRNA in OA treatment.

Conjugation of microbial-derived gold nanoparticles to different types of nucleic acids: evaluation of transfection efficiency

In this study, gold nanoparticles produced by eukaryotic cell waste (AuNP), were analyzed as a transfection tool. AuNP were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) were used before and after conjugation with different nucleic acid (NA) types. Graphite furnace atomic absorption spectroscopy (GF-AAS) was used to determine the AuNP concentration. Conjugation was detected by electrophoresis. Confocal microscopy and quantitative real-time PCR (qPCR) were used to assess transfection. TEM, SEM, and EDS showed 25 nm AuNP with round shape. The amount of AuNP was 3.75 ± 0.2 µg/µL and FTIR proved conjugation of all NA types to AuNP. All the samples had a negative charge of − 36 to − 46 mV. Confocal microscopy confirmed internalization of the ssRNA-AuNP into eukaryotic cells and qPCR confirmed release and activity of carried RNA.

Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis.

This study aimed to determine the role of long noncoding RNA (lncRNA) WT1 antisense RNA (WT1-AS) in the occurrence and progression of preeclampsia (PE) and to determine the underlying molecular mechanisms. The associations between WT1-AS and microRNA (miR)-186-5p, and miR-186-5p and cell adhesion molecule 2 (CADM2) were predicted using StarBase software and verified via dual-luciferase assays. To explore the role of the human chorionic trophoblast line HTR-8/SVneo, gene (WT1-AS/miR-186-5p) gain/loss of function experiments were performed. Qualitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to evaluate transfection efficiency. Cell proliferation, apoptosis, cell migration, and invasion were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell analysis, respectively. Moreover, CADM2 protein expression was measured by western blotting. The results indicated that overexpression of WT1-AS inhibited cell viability, migration, and invasion, and induced apoptosis in HTR-8/SVneo cells. We observed that miR-186a-5p directly targeted WT1-AS, and miR-186a-5p knockdown reversed the effects of WT1-AS knockdown in HTR-8/SVneo cells. Binding sites were found between miR-186-5p and CADM2, and CADM2-overexpression reversed the influence of miR-186-5p mimic on HTR-8/SVneo cells. In summary, our findings demonstrated that lncRNA WT1-AS participates in PE by regulating the proliferation and invasion of placental trophoblasts, through the miR-186-5p/CADM2 axis.

Aligned Nanofibrillar Scaffolds as a Platform for the Treatment of Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) injury is the traumatic loss of skeletal muscle fibers, along with associated vascular and neural structures, resulting in long‐term functional disability. To date, several strategies have been developed to target skeletal muscle and vascular regeneration and recovery of muscle function, with only limited improvements reported. Our objective, using a standardized unilateral VML injury to the rat tibialis anterior (TA) muscle, was two‐fold: (1) examine the rate of cell population and distribution of cell phenotypes in a bioengineered aligned collagen scaffold; and (2) evaluate the in vivo transfection efficiency of aligned collagen scaffolds loaded with firefly luciferase (Fluc) using bioluminescence imaging. We hypothesize that a modified mRNA (mmRNA)‐incorporated aligned collagen scaffold could be an ideal platform to stimulate vascular and muscle fiber regeneration following VML injury. In total, 25 adult male Lewis rats were used across studies. In Study 1, following unilateral VML injury to the TA muscle (~82mg removed), rats received implantation of an aligned collagen scaffold (6mm in diameter, 0.5–3.0mm thickness), consisting of two interconnected bundles of BioBridge threads that were cross‐linked, lyophilized, and sterilized. The TA muscle was evaluated histologically at 1, 2, and 4 weeks post‐VML to examine cell population and distribution, fibrotic content, and extent of scaffold degradation within the remaining TA muscle and VML defect area. In Study 2, in vivo bioluminescence imaging quantified localized RNA‐transfected cell expression in rats up to 2 weeks post‐Fluc injection within the TA muscle, with or without VML injury, or implantation of an aligned collagen scaffold loaded with Fluc‐RNA following VML injury to evaluate transfection efficiency for potential mmRNA cargo. Scaffolds exhibited minimal degradation through 4 weeks following implantation. Quantification of cellular distributions within the VML defect area indicated significant macrophage infiltration and inflammation up to 4 weeks post‐VML. Intramuscular Fluc injections indicated peak bioluminescence signal (3.0x105 p/s/cm2/sr) occurs at 7 days post‐injection. These results serve as a reference for our ongoing development of a mmRNA‐loaded scaffold delivery system. Our long‐term goal is to evaluate candidate growth factors to co‐deliver in the mmRNA‐loaded aligned collagen scaffolds to facilitate organized, functional skeletal muscle and vascular regeneration following VML injury.Support or Funding InformationThis work is supported by: US Army Medical Research and Materiel Command under Contract Number W81XWH19C0021; T32AR050938; P217A170051.

Transient transfection of Babesia ovis using heterologous promoters

Babesia species, etiological agents of babesiosis, a recognized emerging tick-borne disease, are a significant animal and human health concern with a worldwide socio-economic impact. The development of genetic manipulation techniques, such as transfection technology, is pivotal to improve knowledge regarding the biology of these poorly studied parasites towards better disease control strategies. For Babesia ovis, responsible for ovine babesiosis, a tick-borne disease of small ruminants, these tools are not yet available. The present study was based on the existence of interchangeable cross-species functional promoters between Babesia species. Herein, we describe for the first time B. ovis transient transfection using two heterologous promoters, the ef-1α-B intergenic regions from B. bovis and B. ovata. Their ability to drive expression of a reporter luciferase in B. ovis supports their cross-species functionality. Also, the ef-1α-B promoter region from B. ovata resulted in statistically significantly higher luminescence values in comparison to the control, thus a possibly suitable promoter for stable gene expression. Evaluation of transfection efficiency using qPCR demonstrated that higher luminescence levels were due to promoter strength rather than a higher transfection efficiency. These findings represent a step forward in the development of methods for B. ovis genetic manipulation, an undoubtedly necessary tool to study this parasite basic biology, including its life cycle, the parasite interactions with host cells and virulence factors.

Non-viral vectors based on cationic niosomes as efficient gene delivery vehicles to central nervous system cells into the brain

Development of safe and efficient non-viral vectors to deliver DNA into the CNS represents a huge challenge to face many neurological disorders. We elaborated niosomes based on DOTMA cationic lipid, lycopene “helper” lipid and polysorbate 60 as non-ionic surfactants for gene delivery to the CNS. Niosomes, and their corresponding nioplexes obtained after the addition of the pCMS-EGFP plasmid, were characterized in terms of size, charge, morphology and capacity to condense, release and protect DNA. In vitro experiments were performed in NT2 cells to evaluate transfection efficiency, viability, cellular uptake and intracellular distribution. Additionally, transfection in primary cortex cells were performed prior to brain administration into rat cerebral cortex. Data obtained showed that nioplexes exhibited not only adequate physicochemical properties for gene delivery applications, but also relevant transfection efficiencies (17%), without hampering viability (90%). Interestingly, In vivo experiments depicted promising protein expression in both cortical glial cells and blood vessels.

Polysorbate 20 non-ionic surfactant enhances retinal gene delivery efficiency of cationic niosomes after intravitreal and subretinal administration

The success of non-viral vectors based on cationic niosomes for retinal gene delivery applications depends on the ability to achieve persistent and high levels of transgene expression, ideally from a single administration. In this work, we studied the effect of the non-ionic surfactant component of niosomes in their transfection efficiency in rat retina. For that purpose, three niosome formulations that only differed in the non-ionic tensioactives were elaborated. Niosomes contained: cationic lipid 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), helper lipid squalene and polysorbate 20, polysorbate 80 or polysorbate 85. Niosomes and corresponding nioplexes were fully characterized in terms of size, polydispersity index, zeta potential, morphology and ability to protect and release DNA. In vitro experiments were carried out to evaluate transfection efficiency, cell viability and intracellular trafficking pathways of the formulations. Nioplexes based on polysorbate 20 niosomes were the most efficient transfecting retinal cells in vitro. Moreover, subretinal and intravitreal administration of those nioplexes in vivo showed also high levels of transgene expression in rat retinas. Our results demonstrate that the incorporation of polysorbate 20 in cationic niosomes enhances retinal gene delivery. Thus, this formulation emerges as a potential non-viral candidate to efficiently transfer specific therapeutic genes into the eye for biomedical purposes.

Viral vector mimicking and nucleus targeted nanoparticles based on dexamethasone polyethylenimine nanoliposomes: Preparation and evaluation of transfection efficiency.

Non-viral vectors such as polymers and liposomes have been used as gene delivery systems to overcome intrinsic problems of viral vectors, but transfection efficiency of these vectors is lower than viral vectors. In the present study, we tried to design non-viral gene delivery vectors that mimic the viral vectors using the benefits of both cationic liposomes and cationic polymer vectors along with targeting glucocorticoid receptors to enhance cellular trafficking of vectors. Cationic liposomes containing DOTAP and cholesterol were prepared by thin-film hydration following extrusion method. Dexamethasone mesylate was synthesized and then conjugated to polyethylenimine through a one-step reaction. A novel gene delivery system, Lipopolyplex was developed by premixing liposome and different molecular weight of bPEI-Dexa as carriers followed by addition of plasmid at three different carrier/pDNA (C/P) weight ratios. The resulted complexes were characterized for their size, zeta potential and ability of DNA condensation. Transfection efficiency of vectors in neuro2A was determined by Luciferase reporter gene assay. Also, the toxicity of gene carriers was investigated in this cell line. Mean particle size of prepared complexes was less than 200 nm and there was no significant difference in their size by increasing the molecular weight of PEIs. All complexes had positive surface charge. Complete condensation of DNA was occurred at C/P ratio of one for all complexes. Lipopolyplexes were more efficient than polyplexes and lipoplexes alone and transfection efficiency was improved by adding dexamethasone. The complexes containing liposome, PEI 10 kDa and dexamethasone (PEI10:Lipo:Dexa(0.05)) had the highest transfection activity about 40-fold and 3.6-fold in comparison with PEI10 and PEI10:Lipo, respectively. Furthermore, the non-viral vectors described in this study showed low cytotoxicity. The results of this study confirmed that PEI in combination with liposome forms lipopolyplex with low toxicity and enhanced transfection efficiency. Moreover, using dexamethasone, in combination with lipopolyplex might be useful to increase the gene delivery potential of these lipopolyplexes.

Synthesis, characterization and evaluation of transfection efficiency of dexamethasone conjugated poly(propyleneimine) nanocarriers for gene delivery#

Context: Polypropylenimine (PPI), a cationic dendrimer with defined structure and positive surface charge, is a potent non-viral vector. Dexamethasone (Dexa) conveys to the nucleus through interaction with its intracellular receptor.Objective: This study develops efficient and non-toxic gene carriers through conjugation of Dexa at various percentages (5, 10 and 20%) to the fourth and the fifth generation PPIs (PPIG4s and PPIG5s).Materials and methods: The 21-OH group of Dexa (0.536 mmol) was modified with methanesulfonyl chloride (0.644 mmol) to activate it (Dexa-mesylate), and then it was conjugated to PPIs using Traut's reagent. After dialysis (48 h) and lyophilization, the physicochemical characteristics of products (PPI-Dexa) including zeta potential, size, buffering capacity and DNA condensing capability were investigated and compared with unmodified PPIs. Moreover, the cytotoxicity and transfection activity of the Dexa-modified PPIs were assessed using Neuro2A cells.Results: Transfection of PPIG4 was close to PEI 25 kDa. Although the addition of Dexa to PPIG4s did not improve their transfection, their cytotoxicity was improved; especially in the carrier to DNA weight ratios (C/P) of one and two. The Dexa conjugation to PPIG5s enhanced their transfection at C/P ratio of one in both 5% (1.3-fold) and 10% (1.6-fold) Dexa grafting, of which the best result was observed in PPIG5-Dexa 10% at C/P ratio of one.Discussion and conclusions: The modification of PPIs with Dexa is a promising approach to improve their cytotoxicity and transfection. The higher optimization of physicochemical characteristics, the better cell transfection and toxicity will be achieved.

N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride: Synthesis, interaction with DNA and evaluation of transfection efficiency

А novel cationic chitosan derivative, N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), with different degrees of substitution (DS) was synthesized by a chemoselective interaction of 4-formyl-N,N,N-trimethylanilinium iodide with chitosan amino groups using a reductive amination method. Several factors (pH, reactant ratio, reaction time, and chitosan structure) were studied for their effects on the DS of the resulting TMAB-CS. The obtained derivatives were characterized by 1H NMR and FTIR spectroscopy. Turbidimetric titration showed enhanced solubility over a wide pH range even for low-substituted TMAB-CS. TMAB-CS provided strong DS-dependent binding of plasmid DNA. Dynamic light scattering measurements revealed the formation of stable polyplexes with hydrodynamic diameters of 200–300nm and ζ-potential of 20–30mV. TMAB-CS with relatively low DS (25%) demonstrated more pronounced transfection efficiency (up to 2000 cell/cm2) of plasmid DNA into the HEK293 cell line promoted by free TMAB-CS. The positive effects of lower DS can be related to a better polyplex dissociation within the cell. The cytotoxicity of TMAB-CS was comparable to that of the initial chitosan at concentrations up to 300ng/μL, even at high DS.

Hyperbranched–dendrimer architectural copolymer gene delivery using hyperbranched PEI conjugated to poly(propyleneimine) dendrimers: synthesis, characterization, and evaluation of transfection efficiency

The applications of dendrimer-based vectors seem to be promising in non-viral gene delivery because of their potential for addressing the problems with viral vectors. In this study, generation 3 poly(propyleneimine) (G3-PPI) dendrimers with 1, 4-diaminobutane as a core initiator was synthesized using a divergent growth approach. To increase the hydrophobicity and reduce toxicity, 10% of primary amines of G3-PPI dendrimers were replaced with bromoalkylcarboxylates with different chain lengths (6-bromohexanoic and 10-bromodecanoic). Then, to retain the overall buffering capacity and enhance transfection, the alkylcarboxylate–PPIs were conjugated to 10 kDa branched polyethylenimine (PEI). The results showed that the modified PPI was able to form complexes with the diameter of less than 60 nm with net-positive surface charge around 20 mV. No significant toxicity was observed in modified PPIs; however, the hexanoate conjugated PPI–PEI (PPI-HEX-10% PEI) and the decanoate conjugated PPI–PEI (PPI-DEC-10%-PEI) showed the best transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, even PPI-HEX-10%-PEI showed transfection efficiency equal to standard PEI 25 kDa with reduced toxicity. This study suggested a new series of hyperbranched (PEI)–dendrimer (PPI) architectural copolymers as non-viral gene delivery vectors with high transfection efficiency and low toxicity.

The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells

In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

The influence of the polar head-group of synthetic cationic lipids on the transfection efficiency mediated by niosomes in rat retina and brain

The development of novel non-viral delivery vehicles is essential in the search of more efficient strategies for retina and brain diseases. Herein, optimized niosome formulations prepared by oil-in water (o/w) and film-hydration techniques were characterized in terms of size, PDI, zeta potential, morphology and stability. Three ionizable glycerol-based cationic lipids containing a primary amine group (lipid 1), a triglycine group (lipid 2) and a dimethylamino ethyl pendent group (lipid 3) as polar head-groups were part of such niosomes. Upon the addition of pCMS-EGFP plasmid, nioplexes were obtained at different cationic lipid/DNA ratios (w/w). The resultant nioplexes were further physicochemically characterized and evaluated to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed to evaluate transfection efficiency and cell viability in HEK-293, ARPE-19 and PECC cells. Interestingly, niosome formulations based on lipid 3 showed better transfection efficiencies in ARPE-19 and PECC cells than the rest of cationic lipids showed in this study. In vivo experiments in rat retina after intravitreal and subretinal injections together with in rat brain after cerebral cortex administration showed promising transfection efficiencies when niosome formulations based on lipid 3 were used. These results provide new insights for the development of non-viral vectors based on cationic lipids and their applications for efficient delivery of genetic material to the retina and brain.

Synthesis of efficient gene delivery systems by grafting pegylated alkylcarboxylate chains to PAMAM dendrimers: Evaluation of transfection efficiency and cytotoxicity in cancerous and mesenchymal stem cells.

The applications of polyamidoamine (PAMAM) dendrimers have attracted much attention in biomedicine specially non-viral gene delivery because of thier unique characteristics including hyperbranching, multivalency, and well-defined uniform globular three-dimensional structures. In the current study, in order to enhance the transfection efficiency and reduce the cytotoxicity of PAMAMs, bromoalkylcarboxylates with different chain length (2-bromoacetic, 6-bromohexanoic, 10-bromodecanoic and 16-bromohexadecanoic acids) were covalently conjugated with 10% and 30% of primary amines of generation 4 and 5 (G4 and G5) of PAMAM dendrimers to increase the hydrophobicity of the carrier. At the next stage, the alkylcarboxylate-PAMAMs were pegylaed to further modify the PAMAM structures for biological applications. Obtained results demonstrated that the prepared PAMAM derivatives had particle size around 140 nm with net-positive surface charge. None of the prepared PAMAM-based non-viral vactors exhibited significant hemolytic activity and also cytotoxicity. Meanwhile decahexanoate-PAMAM G4 [G4(16C-10%)] and decanoate-PAMAM G4 conjugated to polyethylene glycol (PEG) (G4[(10C-30%)(10C-PEG)]) showed highest transfection efficiency in murine neuroblastoma (Neuro-2a) cell line, interestingly only the latter had improved transfection efficiency in mesenchymal stem cells (MSCs). This study proved the potential utility of alkylcarboxylate-grafted PAMAM dendrimers (G4 and G5) with or without PEG modification for efficient gene transfer into cancerous cells as well as MSCs.

Anticancer activity of stoppin based on a novel peptide delivery system.

Stoppin (L1) is a newly identified anticancer peptide, which is a potent p53‑MDM2/MDMX inhibitor. Due to its limitation in cell delivery efficiency, a new peptide delivery system was developed based on a nucleic acid‑polypeptide‑liposome complex and its stability and effectiveness in vitro was investigated. The nucleic acid‑stoppin‑liposome complex was prepared and characterization of the complex was conducted. The stability of the complex was evaluated by enzyme digestion. Following transfection of the A549 cells with the complex, detection of green fluorescent protein (GFP) and luciferase activity was conducted to evaluate transfection efficiency. In addition, the anticancer activity of the complex was determined by 3‑(4,5‑dimethyl‑thiazolyl‑2)‑2,5 diphenyltetrazolium bromide assay and apoptosis was detected by flow cytometry. The results indicated that the particle size of the complex was 102±10 nm and the encapsulation rate was ~100% when the ratio of liposome, L1 and plasmid was: 4 µl:1 µg:2 µg. The enzyme digestion experiment demonstrated that the complex was resistant to pancreatic and DNA enzyme degradation, indicating that the complex had biological stability. Cell transfection demonstrated that it had a mutual promotion effect on delivery, which could be confirmed by GFP fluorescence and luciferase assay. The cell‑killing efficiency of this novel delivery system was three times higher than with stoppin alone at a low concentration. In conclusion, this novel stoppin peptide delivery system was stable. The nucleic acid‑peptide‑liposome complex can protect the internal component from the degradation of enzymes, promote entry of the peptide into the cells and enhance the anti‑tumor activity of stoppin. Therefore, it is a promising approach for peptide delivery, which can be characterized and visualized using plasmids with GFP or luciferase.

Use of ultrasound to enhance PEI-mediated gene delivery in vivo on diabetic model

Background and aims Gene therapy has strong therapeutic potential for the personalized treatment of type 1 diabetes. Local gene delivery methods for gene therapy are a promising strategy to increase the therapeutic genes concentration at the target location. Transient ultrasound can cause the local increase of cell membrane permeability thus promoting the penetration of transfection particles into the cell. The objective of this work is to evaluate transfection efficiency and safety for intrahepatic polyplex-mediated gene delivery by sonoporation. Methods Polyplexes of expression vector DNAs complexed to galactose-bearing polyethylenimine (L-PEI) have been used. Expression vectors are two plasmids containing expression cassette for full-size human preproinsulin gene controlled with CMV promoter and flanked by inverted terminal repeats of AAV (pTRhins) and the same cassette for marker gfp gene (pTRegfp). Animals were injected under US guidance with polyplexes in a dose of 40 µg pTRegfp /0.7 ml (rat) and of 15 µg pTRhins /0.15 ml (diabetic mouse) into the liver parenchyma of subdiaphragmal segments using 31 G needles. Afterwards injection locus in depth of 1 cm during 180 sec was insonated by 130 Db ultrasound using multifrequency 3-5 MHz probe. Results Sonoporation is able to increase polyplex gene transfer to liver cells in vivo. Flow cytometry analysis of primary hepatocytes isolated from the liver of experimental rats showed that ultrasound -enhanced polyplex gene transfer was highly localized, and was superior to all controls. At least 42% of the liver cells in vivo can be transfected in this way with ultrasound exposure versus 1.2% without it. Hypoglycemic effect of the insulin gene delivery followed by 3m in US exposure was observed on the third day: glucose level of diabetic mice (hyperglycemic 6- week) decreased in average by 30%. Nevertheless sonoporation does not increase naked plasmid DNA (without L-PEI ) transfection efficiency. A week and a month after the procedure serial sections from rat liver injected with polyplexes containing 40 µg marker plasmid or saline solution alone exposed to US and without it was analyzed for the presence of inflammation. Pathomorphological and histological analysis of experimental livers revealed no inflammatory processes in tissues, and any detectable side effects of US-enhanced gene delivery were seen. Experimental mice and rat liver DNAs were positive in transgene PCR for 3 months after gene delivery. Conclusions

Autologous Hematopoietic Stem/Progenitor Cell (HSPC) Therapy For Monogenic Blood Disorders: Scalable, cGMP-Compliant Process For Generating Highly Efficient Genome Edited HSPC

BackgroundAllogeneic bone marrow transplant (BMT) is the only curative method for a number of monogenic blood disorders, including various forms of hemoglobinopathies and severe combined immunodeficiencies. Aside from the significant hurdle of finding an identical HLA-matched related donor, allogeneic BMT recipients require chronic immunosuppression to mitigate the significant risk of GVHD and are at greater risk for graft failure. Autologous gene modified HSPC potentially provide a much safer alternative to allogeneic BMT and abrogate the need for finding HLA-matched donors. Here, we report the development of a highly efficient process for generating gene modified human HSPC at clinical-scale (>150 x 10^6 cells for 2x10^6/kg) using clinical-grade equipment. MethodsHealthy donors were administered Neupogen® (10mg/kg/day) for 4-5 consecutive days and then apheresed. Enrichment of CD34+ cells was performed with the Miltenyi CliniMACS® system. Genome editing was achieved via the introduction of mRNA encoding two engineered zinc finger nucleases (ZFN) using a scalable electroporation device. Cells were harvested and cryo-preserved with a controlled-rate freezer. Cell recovery and viability, gene modification efficiency, stem cell pluripotency and engraftment potential were evaluated by in vitro assays and in a humanized NSG mouse model. ResultsEnrichment of CD34+ cells from mobilized leukopak products was highly efficient with the Miltenyi CliniMACS® system (median recovery = 327 million CD34+ cells per 10L mobilized leukopak). The positively selected fractions were >98% CD34+ by FACS analysis. Two large scale electroporation devices (BTX AgilePulse Max® and MaxCyte GT®) were evaluated. Each device is capable of electroporating up to 300 million cells. The optimal transfection conditions for both devices were first identified by using a GFP mRNA to evaluate transfection efficiency by flow cytometry, which resulted in the identification of conditions (voltage and duration) that yielded gene transfer efficiencies of >90%. Compatibility of this protocol with driving endogenous gene modification was evaluated using mRNA encoding ZFNs that target various endogenous gene loci. Highly efficient levels of genome editing were observed in CD34+ HSPC each transfected with a different pair of ZFNs (median = 53%, 43%, 45% and 42% modified alleles at four distinct disease-relevant loci). At the optimal mRNA dose for each ZFN pair, cell viability post electroporation was >80%, comparable to untransfected controls. Process suitability was evaluated by in vitro colony forming cell assay. No significant differences in colony formation were observed between gene modified and untransfected control samples. The capacity of electroporated HSPC to engraft and support multi-lineage development of human hematopoietic cells was evaluated in NSG mice, and no differences were observed between the ZFN-treated and untransfected control cells. In addition, high levels of gene modification (19-28%) were detected in bulk human cells from the blood and tissues of engrafted mice, and in various sorted cell types (bone marrow CD34 and differentiated B and T cells). ConclusionWe have developed a scalable process capable of deriving >300 million gene-modified CD34+ HSPC. This process supports high levels of ZFN-driven genome editing, is well tolerated, and causes no discernable defect in the hematopoietic potential of these cells to develop into multiple cell lineages, with high gene editing levels maintained in the differentiated progeny of the HSPC. These results support the use of gene modified autologus HSPC for the treatment of monogenic blood disorders. Disclosures:Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Ya-Li:Sangamo BioSciences: Employment. Kim:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Wang:Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Ando:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment.

A novel cationic niosome formulation for gene delivery to the retina

Niosomes represent a recent promising approach for gene delivery purposes. We elaborated on a novel niosome formulation based on the 2,3-di(tetradecyloxy)propan-1-amine cationic lipid, combined with squalene and polysorbate 80 to evaluate the transfection efficiency in rat retinas. Niosomes prepared by the solvent emulsification–evaporation technique were mixed with the pCMSEGFP plasmid to form lipoplexes which were characterized in terms of morphology, size, surface charge, and DNA condensation, protection and release. In vitro studies were conducted to evaluate transfection efficiency, viability and internalization mechanism in HEK-293 and ARPE-19 cells. The efficacy of the most promising formulation was evaluated in rat eyes by monitoring the expression of the EGFP after intravitreal and subretinal injections. Lipoplexes at 15/1 ratio were 200nm in size, 25mV in zeta potential and exhibited spherical morphology. At this ratio, niosomes condensed and protected the DNA from enzymatic digestion. Lipoplexes successfully transfected HEK-293 and specially ARPE-19 cells, without affecting the viability. Whereas lipoplexes entered mainly retinal cells by clathrin-mediated endocytosis, HEK-293 cells showed a higher caveolae-dependent entry. After ocular administration, the expression of EGFP was detected in different cells of the retina depending on the administration route. This novel niosome formulation represents a promising approach to deliver genetic material into the retina to treat inherited retinal diseases.