The use of absorption enhancers to enhance the despersibility of spray-dried powders for pulmonary gene therapy

Background: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. Methods: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler® dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. Results: Spray-dried powder containing dimethyl-β-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. Conclusions: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model. Copyright © 2005 John Wiley & Sons, Ltd.

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein. © 2014 Jiwaji et al.

Gene delivery of the elastase inhibitor elafin protects macrophages from neutrophil elastase-mediated impairment of apoptotic cell recognition

The resolution of inflammation is dependent on recognition and phagocytic removal of apoptotic cells by macrophages. Receptors for apoptotic cells are sensitive to degradation by human neutrophil elastase (HNE). We show in the present study that HNE cleaves macrophage cell surface CD14 and in so doing, reduces phagocytic recognition of apoptotic lymphocytic cells (Mutu 1). Using an improved method of adenovirus-mediated transfection of macrophages with the HNE inbibitor elafin, we demonstrate that elafin overexpression prevents CD14 cleavage and restores apoptotic cell recognition by macrophages. This approach of genetic modification of macrophages could be used to restore apoptotic cell recognition in inflammatory conditions. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Enhanced dispersibility and deposition of spray-dried powders for pulmonary gene therapy

Spray-drying represents a viable alternative to freeze-drying for preparing dry powder dispersions for delivering macromolecules to the lung. The dispersibility of spray-dried powders is limited however, and needs to be enhanced to improve lung deposition and subsequent biological activity. In this study, we investigate the utility of leucine as a dry powder dispersibility enhancer when added prior to spray-drying a model non-viral gene therapy formulation (lipid:polycation:pDNA, LPD). Freeze-dried lactose-LPD, spray-dried lactose-LPD and spray-dried leucine-lactose-LPD powders were prepared. Scanning electron microscopy showed that leucine, increased the surface roughness of spray-dried lactose particles. Particle size analysis revealed that leucine-containing spray-dried powders were unimodally dispersed with a mean particle diameter of 3.12 μm. Both gel electrophoresis and in vitro cell (A549) transfection showed that leucine may compromise the integrity and biological functionality of the gene therapy vector. The deposition of the leucine containing powder was however significantly enhanced as evidenced by an increase in gene expression mediated by dry powder collected at lower stages of a multistage liquid impinger (MSLI). Further studies are required to determine the potential of leucine as a ubiquitous dispersibility enhancer for a variety of pulmonary formulations. © 2003 Taylor & Francis Ltd.

Gene delivery for the treatment of prostate cancer

Prostate cancer is one of the most common cancers diagnosed in men. Whilst treatments for early-stage disease are largely effective, current therapies for metastatic prostate cancer, particularly for bone metastasis, offer only a few months increased lifespan at best. Hence new treatments are urgently required. Small interfering RNA (siRNA) has been investigated for the treatment of prostate cancer where it can ‘silence’ specific cancer-related genes. However the clinical application of siRNA-based gene therapy is limited due to the absence of an optimised gene delivery vector. The optimisation of such gene delivery vectors is routinely undertaken in vitro using 2D cell culture on plastic dishes which does not accurately simulate the in vivo bone cancer metastasis microenvironment. The goal of this thesis was to assess the potential of two different targeted delivery vectors (gold or modified β-cyclodextrin derivatives) to facilitate siRNA receptor-mediated uptake into prostate cancer cells. Furthermore, this project aimed to develop a more physiologically relevant 3D in vitro cell culture model, to mimic prostate cancer bone metastasis, which is suitable for evaluating the delivery of nanoparticulate gene therapeutics. In the first instance, cationic derivatives of gold and β-cyclodextrin were synthesized to complex anionic siRNA. The delivery vectors were targeted to prostate cancer cells using the anisamide ligand which has high affinity for the sigma receptor that is overexpressed by prostate cancer cells. The gold nanoparticle demonstrated high levels of uptake into prostate cancer PC3 cells and efficient gene silencing when transfection was performed in serum-free media. However, due to the absence of a poly(ethylene glycol) (PEG) stabilising group, the formulation was unsuitable for use in serum-containing conditions. Conversely, the modified β-cyclodextrin formulation demonstrated enhanced stability in the presence of serum due to the inclusion of a PEG chain onto which the anisamide ligand was conjugated. However, the maximum level of gene silencing efficacy from three different prostate cancer cell lines (DU145, VCaP and PC3 cells) was 30 %, suggesting that further optimisation of the formulation would be required prior to application in vivo. In order to develop a more physiologically-relevant in vitro model of prostate cancer bone metastasis, prostate cancer cells (PC3 and LNCaP cells) were cultured in 3D on collagenbased scaffolds engineered to mimic the bone microenvironment. While the model was suitable for assessing nanoparticle-mediated gene knockdown, prostate cancer cells demonstrated a phenotype with lower invasive potential when grown on the scaffolds relative to standard 2D cell culture. Hence, prostate cancer cells (PC3 and LNCaP cells) were subsequently co-cultured with bone osteoblast cells (hFOB 1.19 cells) to enhance the physiological relevance of the model. Co-cultures secreted elevated levels of the MMP9 enzyme, a marker of prostate cancer metastasis, relative to prostate cancer cell monocultures (2D and 3D) indicating enhanced physiological relevance of the model. Furthermore, the coculture model proved suitable for investigating nanoparticle-mediated gene silencing. In conclusion, the work outlined in this thesis identified two different sigma receptor-targeted gene delivery vectors with potential for the treatment of prostate cancer. In addition, a more physiologically relevant model of prostate cancer bone metastasis was developed with the capacity to help optimise gene delivery vectors for the treatment of prostate cancer.

The effects of PVP(FE(III)) catalyst and polymer molecular weight on gene delivery via biodegradable crosslinked polyethylenimine

Human gene therapy has faced many setbacks due to the immunogenicity and oncogenity of viruses. Safe and efficient alternative gene delivery vehicles are needed to implement gene therapy in clinical practice. Polymeric vectors are an attractive option due to their availability, simple chemistry, and low toxicity and immunogenicity. Our group has previously reported biodegradable polyethylenimines (PEI) that show high transfection efficiency and low toxicity by cross-linking 800 Da PEI with diacrylate cross-linkers using Michael addition. However, the synthesis was difficult to control, inconsistent, and resulted in polymers with a narrow range of molecular weights. In the present work, we utilized a heterogenous PVP(Fe(III)) catalyst to provide a more controllable PEI crosslinking reaction and wider range of biodegradable PEIs. The biodegradable PEIs reported here have molecular weights ranging from 1.2 kDa to 48 kDa, are nontoxic in MDA-MB-231 cells, and show low toxicity in HeLa cells. At their respective optimal polymer:DNA ratios, these biodegradable PEIs demonstrated about 2-5-fold higher transfection efficiency and 2-7-fold higher cellular uptake, compared unmodified 25 kDa PEI. The biodegradable PEIs show similar DNA condensation properties as unmodified PEI but more readily unpackage DNA, based on ethidium bromide exclusion and heparan sulfate competitive displacement assays, which could contribute to their improved transfection efficiency. Overall, the synthesis reported here provides a more robust, controlled reaction to produce cross-linked biodegradable PEIs that show enhanced gene delivery, low toxicity, and high cellular uptake and can potentially be used for future in vivo studies.

Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Enhancement of non-viral transfection efficiency with nuclear localization signal peptides

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Identification Of Target Genes Using Gene Expression Profile Of Granulocytes From Patients With Chronic Myeloid Leukemia Treated With Tyrosine Kinase Inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

Exposure To Bisphenol-a During Pregnancy Partially Mimics The Effects Of A High-fat Diet Altering Glucose Homeostasis And Gene Expression In Adult Male Mice.

Bisphenol-A (BPA) is one of the most widespread EDCs used as a base compound in the manufacture of polycarbonate plastics. The aim of our research has been to study how the exposure to BPA during pregnancy affects weight, glucose homeostasis, pancreatic β-cell function and gene expression in the major peripheral organs that control energy flux: white adipose tissue (WAT), the liver and skeletal muscle, in male offspring 17 and 28 weeks old. Pregnant mice were treated with a subcutaneous injection of 10 µg/kg/day of BPA or a vehicle from day 9 to 16 of pregnancy. One month old offspring were divided into four different groups: vehicle treated mice that ate a normal chow diet (Control group); BPA treated mice that also ate a normal chow diet (BPA); vehicle treated animals that had a high fat diet (HFD) and BPA treated animals that were fed HFD (HFD-BPA). The BPA group started to gain weight at 18 weeks old and caught up to the HFD group before week 28. The BPA group as well as the HFD and HFD-BPA ones presented fasting hyperglycemia, glucose intolerance and high levels of non-esterified fatty acids (NEFA) in plasma compared with the Control one. Glucose stimulated insulin release was disrupted, particularly in the HFD-BPA group. In WAT, the mRNA expression of the genes involved in fatty acid metabolism, Srebpc1, Pparα and Cpt1β was decreased by BPA to the same extent as with the HFD treatment. BPA treatment upregulated Pparγ and Prkaa1 genes in the liver; yet it diminished the expression of Cd36. Hepatic triglyceride levels were increased in all groups compared to control. In conclusion, male offspring from BPA-treated mothers presented symptoms of diabesity. This term refers to a form of diabetes which typically develops in later life and is associated with obesity.

Pyrimidine-5'-nucleotidase Campinas, A New Mutation (p.r56g) In The Nt5c3 Gene Associated With Pyrimidine-5'-nucleotidase Type I Deficiency And Influence Of Gilbert's Syndrome On Clinical Expression.

Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

Frequency Of The Allelic Variant C.1150t > C In Exon 10 Of The Fibroblast Growth Factor Receptor 3 (fgfr3) Gene Is Not Increased In Patients With Pathogenic Mutations And Related Chondrodysplasia Phenotypes.

Mutations in the FGFR3 gene cause the phenotypic spectrum of FGFR3 chondrodysplasias ranging from lethal forms to the milder phenotype seen in hypochondroplasia (Hch). The p.N540K mutation in the FGFR3 gene occurs in ∼70% of individuals with Hch, and nearly 30% of individuals with the Hch phenotype have no mutations in the FGFR3, which suggests genetic heterogeneity. The identification of a severe case of Hch associated with the typical mutation c.1620C > A and the occurrence of a c.1150T > C change that resulted in a p.F384L in exon 10, together with the suspicion that this second change could be a modulator of the phenotype, prompted us to investigate this hypothesis in a cohort of patients. An analysis of 48 patients with FGFR3 chondrodysplasia phenotypes and 330 healthy (control) individuals revealed no significant difference in the frequency of the C allele at the c.1150 position (p = 0.34). One patient carrying the combination `pathogenic mutation plus the allelic variant c.1150T > C' had a typical achondroplasia (Ach) phenotype. In addition, three other patients with atypical phenotypes showed no association with the allelic variant. Together, these results do not support the hypothesis of a modulatory role for the c.1150T > C change in the FGFR3 gene.

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.