Alameh, Mohamad-Gabriel

Biniecki, Kristof

Buschmann, Michael D.

Lavertu, Marc

Merzouki, Abderrazzak

PolyPublie

Titre:Title:Chitosan/siRNA nanoparticles demonstrate superior silencing efficiency and lower toxicity compared to lipid nanoparticlesAuteurs:Authors:Mohamad-Gabriel Alameh, Marc Lavertu, Abderrazzak Merzouki, Kristof Biniecki, & Michael D. Buschmann Date: 2014Type: Communication de conférence / Conference or Workshop ItemRéférence:Citation:Alameh, M.-G., Lavertu, M., Merzouki, A., Biniecki, K., & Buschmann, M. D. (mai 2014). Chitosan/siRNA nanoparticles demonstrate superior silencing efficiency and lower toxicity compared to lipid nanoparticles [Résumé]. 17th Annual Meetingof the American Society of Gene & Cell Therapy, Washington, DC. Publié dans Molecular Therapy, 22(S1). https://doi.org/10.1016/s1525-0016(16)35588-5Document en libre accès dans PolyPublieOpen Access document in PolyPublieURL de PolyPublie:PolyPublie URL:https://publications.polymtl.ca/4741/Version: Version officielle de l'éditeur / Published version Révisé par les pairs / Refereed Conditions d’utilisation:Terms of Use: CC BY Document publié chez l’éditeur officielDocument issued by the official publisherNom de la conférence:Conference Name:17th Annual Meeting of the American Society of Gene & Cell TherapyDate et lieu:Date and Location:2014-05-21 - 2014-05-24, Washington, DC Maison d’édition:Publisher:ElsevierURL officiel:Official URL:https://doi.org/10.1016/s1525-0016(16)35588-5Mention légale:Legal notice:Ce fichier a été téléchargé à partir de PolyPublie, le dépôt institutionnel de Polytechnique MontréalThis file has been downloaded from PolyPublie, the institutional repository of Polytechnique Montréalhttps://publications.polymtl.caMolecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of  Gene & Cell Therapy S223CHEMICAL/MOLECULAR CONJUGATESand gene therapies. Here, to explore a potential of pH-sensitive polymer-liposome complexes for the tumor-specifi c combinatorial delivery of anticancer agents and siRNA, conventional liposomes (ConL), polymer-liposome complexes (PLC) and polymer-cationic liposome complexes (PCLC) were prepared. Pluronic® P104-based multiblock copolymer (MBCP-2) was included as pH-sensitive polymer. Physicochemical properties, release under different pH, cytotoxicity and in vitro cellular uptake of DOX-loaded liposomes were investigated. From the release test, an acidic pH was determined to be an important factor for release from the PLC vehicles. The novel PLC vehicle itself showed low cytotoxicity demonstrating suitable viability. Observing cellular uptake of DOX by confocal microscopy imaging, a greater amount of DOX was delivered to cells with the pH-sensitive polymer-anchored vehicles than that with free DOX and ConL. It was verifi ed that the novel vehicles could effectively deliver both DOX and GFP-siRNA. Novel pH-sensitive PCLC have a potential for targeted therapy of anticancer agents and gene therapy under acidic tumor microenvironment.575. Chitosan/siRNA Nanoparticles Demonstrate Superior Silencing Effi ciency and Lower Toxicity Compared To Lipid Nanoparticles Mohamad-Gabriel Alameh,1 Marc Lavertu,1 Abderrazzak Merzouki,1 Kristof Biniecki,2 Michael D. Buschmann.11Biomedical and Chemical Engineering, Ecole Polytechnique de Montreal, Montreal, QC, Canada; 2ANRis Pharmaceuticals Inc, Kirkland, QC, Canada.Purpose: Nanoparticles (NP) using specifi c chitosans (CS) have been developed for effi cient delivery of siRNA, in vitro and in vivo. Safety of such systems as defi ned by the lack of both systemic and chronic toxicity is the most critical aspect as per FDA, EMEA and ICH guidelines. Development of CS NPs for clinical use requires comparative toxicity profi les. A fractional factorial design (FFD) was used to screen factors affecting toxicity to select non-toxic formulations with optimal biological activity for in vivo studies.Methods: A combinatorial approach was adopted to evaluate the in vitro toxicity of NP using the H1299 cell line. A 25-1 FFD was used to screen for the effects of chitosan molecular weight (Mn), amine to phosphate ratio (N:P), presence of serum at time of transfection and siRNA concentration ([NA]) on NP toxicity. Post transfection metabolic impairment induced by NP delivering anti-eGFP siRNA was assessed after 4 and 48 hours using alamarBlue assay. Quantifi cation of NP genotoxicity and evaluation of silencing effi ciency (SE) were performed using the alkaline comet assay and fl ow cytometry 48 hours post-transfection respectively. Results relative to non transfected cells were compared to DharmaFECT (DF) and Doxorubicin.Results: CS-NP induced signifi cantly less toxicity (0-25% vs 60-70% cell death) than DF 48 hours post-transfection. Low molecular weight chitosans (LMWCS 10kDa) induced a slightly higher toxic effect than their high molecular weight counterparts (HMWCS 150kDa). Serum proteins showed no infl uence on cytotoxicity. NP prepared in diluted versus concentrated regimes (0.05 vs 0.2μg/L) showed signifi cantly different sizes but no infl uence on toxicity. Hence NP size did not correlate with toxicity. For a fi nal siRNA concentration of 100nM/well, the increase in the quantity of chitosan (0.33-3.3μg/well, i.e. N:P of 5 and 60) did not affect the toxicity profi le observed either with LMWCS or HMWCS. [NA] up to 500 nM showed no increase in toxicity regardless of the N:P ratio. These results suggest that cells readily tolerate siRNA and chitosan quantities near 7μg/mL and 165μg/mL, respectively. Silencing effi ciency reached ~50-70%. Interestingly, an increase in chitosan charge density (DDA from 92 to 98%) resulted in higher toxicity for LMWCS, reminiscent of the mitochondrial induced toxicity of Polyethylenimine but only for chitosan of the highest DDA. Genotoxicity assessment using the comet assay corroborated the results obtained with alamarBlue. DF, a lipid formulation used as positive control for SE, demonstrated high toxicity compared to chitosan in all tests.Conclusion: Chitosan is a superior choice for preparation of NP compared to lipids owing to its high effi ciency (>60%) combined with very low toxicity. Assessment of apoptotic pathway activation for LMWCS by pathway focus qPCR gene profi ling arrays is ongoing. Finally, cell cycle and DNA damage response pathway profi ling will reveal mechanistic insights into toxicity pathways.576. Formulation Optimization of PEI-DNA Complexes for the Effi cient Gene Delivery To Cultured HeLa Cells Cho-A. Lee,1 Jeong-Sook Park,1 Cheong-Weon Cho.11College of Pharmacy, Chungnam National Univeristy, Daejeon, Korea.The purpose of this study is to examine the optimized formulation of the luciferase-expressing plasmid DNA complexed with the synthetic cationic polymer polyethyleneimine (PEI) that shows the highest transfection activity in HeLa cell. The PEI/DNA ratio that produced the highest luciferase activity upon transfection into HeLa cells was found to be around 5. Assuming that the size and shape of the PEI-DNA complex is one of the most determinative variables for the high transfection effi ciency and that the mixing dynamics of the two macromolecules can have a profound effect in this regard, we examined the effect of the concentrations of PEI and DNA and the size of PEI on the transfection effi ciency at N/P ratio=5. The highest transfection effi ciency was observed when DNA (2 ug/ul) was mixed with 10mM PEI (25kD). Upon electron microscopic examination, the PEI-DNA complex assumed regular spherical shapes with 40-60 nm in size. We also examined the effect of serum and antibiotics and found that the best results were obtained at 0.5% FBS without any antibiotics. At optimal conditions the PEI-DNA complexes yielded 2-3 fold higher transfection effi ciency than commercial liposomal agents. Our results should prove informative in formulating PEI-DNA complexes for optimal gene delivery to cultured cell lines and the optimized formulation of PEI-DNA complexes may be further improved to be adopted for effi cient in vivo gene delivery.577. DOTAP and MPEG-PCL Hybrid Nanoparticles for Cancer Gene Therapy Ke Men,1 Xingmei Duan,1 Pan Wang,1 Maling Gou,1 Yuquan Wei.11State Key Laboratory of Biotherapy, West China Hospital and West China Medical School, Sichuan Univesity, Chengdu, Sichuan, China.Cationic nanoparticles show promise for applications in gene delivery. Commercial liposomal gene carriers have high transfection efficiency but strong cytotoxicity which is caused by the high content of cationic DOTAP in the liposome. Here, DOTAP is used to modify degradable monomethoxy poly(ethylene glycol)–poly(ε-caprolactone) (MPEG–PCL, MP) micelles in one step, creating the cationic self-assembled DOTAP and MPEG–PCL hybrid micelles (DMP). These micelles have a mean particle size of 46 nm and a zeta potential of +41 mV, and are able to bind plasmid DNA. The content of DOTAP in DMP micelles is as low as 10%. Compared with PEI25K (the gold standard), DMP micelles had higher transfection effi ciency and lower cytotoxicity. DMP delivered survivin-T34A gene could inhibit the growth of C-26 colon cancer cells through inducing apoptosis in vitro. Intraperitoneal administration of DMP delivered Survivin-T34A gene effi ciently inhibited the growth of abdominal metastatic C-26 colon cancer and the malignant ascites. These data suggest that DMP micelles are a novel gene carrier, and its delivery of survivin-T34A gene may have promising applications in colon cancer treatment.

Chitosan/siRNA nanoparticles demonstrate superior silencing efficiency and lower toxicity compared to lipid nanoparticles

Veilleux, Daniel

Titre:Title:Lyophilized chitosan nanoparticles for pDNA and siRNA delivery: Physico-chemical properties, transfection efficiency, and cytotoxicityAuteurs:Authors:Daniel Veilleux, Marc Lavertu, Kristof Biniecki, & Michael D. Buschmann Date: 2014Type: Communication de conférence / Conference or Workshop ItemRéférence:Citation:Veilleux, D., Lavertu, M., Biniecki, K., & Buschmann, M. D. (mai 2014). Lyophilized chitosan nanoparticles for pDNA and siRNA delivery: Physico-chemical properties, transfection efficiency, and cytotoxicity [Affiche]. American Society of Gene & CellTherapy 17th Annual Meeting, Washington, DC. Publié dans Molecular Therapy, 22(S1). https://doi.org/10.1016/s1525-0016(16)35365-5Document en libre accès dans PolyPublieOpen Access document in PolyPublieURL de PolyPublie:PolyPublie URL:https://publications.polymtl.ca/5159/Version: Version officielle de l'éditeur / Published version Révisé par les pairs / Refereed Conditions d’utilisation:Terms of Use:CC BY-NC-ND Document publié chez l’éditeur officielDocument issued by the official publisherNom de la conférence:Conference Name:American Society of Gene & Cell Therapy 17th Annual MeetingDate et lieu:Date and Location:2014-05-21 - 2014-05-24, Washington, DC Maison d’édition:Publisher:ElsevierURL officiel:Official URL:https://doi.org/10.1016/s1525-0016(16)35365-5Mention légale:Legal notice:Ce fichier a été téléchargé à partir de PolyPublie, le dépôt institutionnel de Polytechnique MontréalThis file has been downloaded from PolyPublie, the institutional repository of Polytechnique Montréalhttps://publications.polymtl.ca Molecular Therapy Volume 22, Supplement 1, May 2014  Copyright © The American Society of  Gene & Cell TherapyS134PHYSICAL/NON-PHYSICAL METHODS OF DELIVERYJACS 2008). Time-correlated single photon counting allowed the determination of the amount of free and bound EB in solution based on the ratio of the areas under the decay associated spectra, which were used in the Hill equation to determine K (Bishop, JACS 2013). Polyplex diameter and x were quantified using NanoSight and dynamic light scattering. Transfection and cytotoxicity were assessed using fl ow cytometry and CellTiter 96®. Results As molecular weight decreased and as the number of carbons increased in the backbone and sidechain, K decreased. There was not a signifi cant trend between K and either particle size, x, or cytotoxicity. There was a biphasic response when comparing transfection against K in both cell lines with optimal Ks ranging from (1-6) x 104 M-1 (Figure 1; GB319).Conclusions A K in the range of (1-6) x 104 M-1 was necessary but not suffi cient for optimal transfection. The binding affi nity can be modifi ed to be in the optimal range via GPC fractionation or through the addition of carbons in the backbone or sidechain. Investigating subtle structural differences revealed new quantitative and mechanistic relationships for gene delivery.352. Lyophilized Chitosan Nanoparticles for pDNA and siRNA Delivery: Physico-Chemical Properties, Transfection Effi ciency, and Cytotoxicity Daniel Veilleux,1 Marc Lavertu,1 Kristof Biniecki,2 Michael D. Buschmann.11Biomedical and Chemical Engineering, Ecole Polytechnique de Montreal, Montreal, QC, Canada; 2ANRis Pharmaceuticals Inc, Kirkland, QC, Canada.Purpose: Polymer/nucleic acid nanoparticles (NP) using the natural polysaccharide chitosan (CS) have been optimised for effi cient and safe delivery of plasmid DNA and siRNA, in vitro and in vivo. Further development of this technology for clinical and commercial applications requires stability and storage through lyophilisation. Specifi c compositions and methods were identifi ed in this study to retain structural properties and biological activity of lyophylized chitosan nucleic acid nanoparticles and to permit concentration by rehydration in reduced volumes to achieve high doses.Methods: CS/DNA NP were prepared using a 10kDa CS and the plasmid eGFPLuc, either formulated with or without 0.5% (w/V) lyoprotectant (sucrose, dextran 5kDa, or trehalose) and/or buffer (3.44 mM histidine at pH 6.5). NP formulations were then subjected or not to FD and rehydration at various concentrations. For each formulation, NP size and polydispersity index (PDI) were assessed by Dynamic Light Scattering (DLS); zeta potential, by Laser Doppler Velocimetry; and morphology, by Scanning Electron Microscopy (SEM). Transfection effi ciency of formulations was quantifi ed in vitro using a HEK293 cells, measuring the expression of reporter genes eGFP and Luciferase by fl ow cytometry and luminometry respectively. Cytotoxicity of formulations was measured in vitro using the lactate dehydrogenase (LDH) assay, the alamar blue assay, and the annexin V/propidium iodide apoptosis assay.Results: Data showed that both lyoprotectant and buffer were required to prevent complex aggregation following FD and rehydration: hydrodynamic diameters ranged from 125 to 235nm, with PDI values from 0.15 to 0.25 and zeta potentials from +19 to +25mV. Optimal formulations could be concentrated up to 20-fold upon rehydration in lower volumes (Rh20X), while achieving near physiological pH and tonicity. Complexes obtained with these formulations were mainly spherical. Following FD and rehydrated to initial volume (Rh1X), these formulations had similar transfection effi ciencies as fresh complexes without excipient, whereas after Rh20X, formulations with trehalose and histidine had the best effi ciency.Luciferase expression levels of formulations with histidine and sucrose or trehalose were not affected by FD and rehydration. All formulations had negligible cytotoxicity. Similar outcomes for CS siRNA systems for gene silencing were also found.Conclusion: These novel biocompatible DNA and RNA delivery systems permit broad applications, increased doses, and improved storage properties. Characterization of these systems during accelerated and long term stability studies is ongoing.353. Approaches for Improved Polymeric Gene Delivery To Umbilical Cord Blood-Derived Mesenchymal Stromal Cells Laura Rose,1 Cezary Kucharski,1 Dulan Jayasooriya,1 Leah A. Marquez-Curtis,2 Hilal Gul-Uludag,1 Anna Janowska-Wieczorek,2 Hasan Uludag.11Chemical Engineering, University of Alberta, Edmonton, Canada; 2Canadian Blood Services, Edmonton, Canada.Introduction: Mesenchymal stromal cells harvested from umbilical cord blood (CB) offer vast potential for cell-based therapies, but their modifi cation with plasmid DNA (pDNA) remains ineffi cient. Here, we investigated various approaches for improving transfection using small molecular weight (2 kDa) polyethylenimine modifi ed with linoleic acid (PEI-LA) [1] as a low toxicity alternative to lipofection. Approaches pursued included centrifugation of polymer/pDNA complexes after addition to cells, modifying complexes with hyaluronic acid (HA) to target CD44 receptors, and spiking complexes with non-silencing RNAs. Materials & Methods: CB-derived cells [2] were transfected in 24-well plates with pDNA expressing GFP. PEI-LA or Lipofectamime™ 2000 was mixed with pDNA at controlled polymer/pDNA weight ratios for 30 minutes at room temperature before addition to cell cultures. After the complexes’ addition, plates were centrifuged for 5 minutes at 210 g. Media was replaced after 4 hours. For coatings, gelatin or HA was added to complexes after formation. As a control, gelatin or HA was mixed with pDNA prior to PEI-LA. Finally, either control non-silencing small interfering RNA (siRNA) or micro RNA (miRNA) was mixed with 

Lyophilized chitosan nanoparticles for pDNA and siRNA delivery: Physico-chemical properties, transfection efficiency, and cytotoxicity

Tavakoli Naeini, Ashkan

Titre:Title:Large scale preparation of chitosan/nucleic acid nanoparticles usingan automated inline mixing systemAuteurs:Authors:Ashkan Tavakoli Naeini, Marc Lavertu, Michael D. Buschmann, & Kristof Biniecki Date: 2014Type: Communication de conférence / Conference or Workshop ItemRéférence:Citation:Tavakoli Naeini, A., Lavertu, M., Buschmann, M. D., & Biniecki, K. (mai 2014). Large scale preparation of chitosan/nucleic acid nanoparticles using an automated inline mixing system [Résumé]. American Society of Gene & Cell Therapy 17th Annual Meeting, Washington, DC. Publié dans Molecular Therapy, 22. https://doi.org/10.1016/s1525-0016%2816%2935591-5Document en libre accès dans PolyPublieOpen Access document in PolyPublieURL de PolyPublie:PolyPublie URL:https://publications.polymtl.ca/5149/Version: Version officielle de l'éditeur / Published version Révisé par les pairs / Refereed Conditions d’utilisation:Terms of Use: CC BY-NC-ND Document publié chez l’éditeur officielDocument issued by the official publisherNom de la conférence:Conference Name:American Society of Gene & Cell Therapy 17th Annual MeetingDate et lieu:Date and Location:2014-05-21 - 2014-05-24, Washington, DC Maison d’édition:Publisher:ElsevierURL officiel:Official URL:https://doi.org/10.1016/s1525-0016%2816%2935591-5Mention légale:Legal notice:Ce fichier a été téléchargé à partir de PolyPublie, le dépôt institutionnel de Polytechnique MontréalThis file has been downloaded from PolyPublie, the institutional repository of Polytechnique Montréalhttps://publications.polymtl.ca Molecular Therapy Volume 22, Supplement 1, May 2014  Copyright © The American Society of  Gene & Cell TherapyS224DIABETES, METABOLIC AND GENETIC DISEASES IIThis work was supported by the National Natural Science Foundation (81201785) and the National “863” Program (SQ2014SF02D01077).578. Large Scale Preparation of Chitosan/Nucleic Acid Nanoparticles Using an Automated Inline Mixing System Ashkan Tavakoli Naeini,1 Marc Lavertu,1 Michael Buschmann,1 Kristof Biniecki.21Biomedical and Chemical Engineering, Ecole Polytechnique de Montreal, Montreal, QC, Canada; 2ANRis Pharmaceuticals Inc, Kirkland, QC, Canada.Purpose: Polymer/nucleic acid (NA) nanoparticles (NPs) using the natural polysaccharide chitosan (CS) have been developed for effi cient and safe delivery of plasmid DNA and siRNA (NAs), in vitro and in vivo. Further optimisation of this technology for clinical and commercial applications requires the implementation of large scale preparation methods. The rapid kinetics of NP formation (< 5 μs) has practical limitations on scalability, reproducibility and homogeneity of their production by manual mixing. In this study, an automated in-line mixing system was developed for large scale mixing of chitosan and NA solutions. This system was used to investigate the infl uence of mixing parameters on NP physicochemical properties.Methods: A fully automated in-line mixing system consisting of two computer-controlled peristaltic pumps was developed to simultaneously drive large volumes of CS and NA solutions through a mixing connector, using equal volumetric ratios. NPs were prepared using a 10 kDa CS, and either plasmid DNA or siRNA. The infl uence of fl ow rate (Reynolds number, Re, ranging from 20 to 2000) and concentrations of CS (27 to 0.81 μg/ml of chitosan) and NAs (10 to 300 μg/ml) on the physicochemical properties of NPs was examined. Manually mixed NPs were prepared for comparison purposes. Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Environmental Scanning Electron Microscopy (ESEM) were used to assess the size, polydispersity (PDI), and morphology of NPs.Results: The in-line mixing system allowed for the reproducible preparation of homogeneous NP batches of 1 L or more. In comparison, manual mixing resulted in broader particle size distribution and showed limited reproducibility. Mixing concentration had a very signifi cant infl uence on NPs size. Indeed, a decrease in concentration of pDNA from 0.3 to 0.01 mg/mL resulted in a marked decrease of NPs hydrodynamic diameter and PDI (250 to 80 nm and 0.4 to 0.1, respectively). For an equivalent decrease in siRNA concentration, NP size and PDI decreased from 130 to 50 nm and from 0.21 to 0.18, respectively Aggregation occurred above a threshold NA concentration of 0.1 and 0.2 mg/mL for pDNA and siRNA, respectively. In contrast, the mixing fl ow rate or Re (in laminar fl ow regime) had no signifi cant effect on NP size distribution. Below the aggregation concentration thresholds, ESEM observations of NPs prepared with either pDNA or siRNA revealed spherical or globular morphologies, independent of the mixing parameters.Conclusion: The developed automated in-line mixing system is a promising method for the large scale and reproducible production of NPs. The significant influence of mixing concentration on chitosan NPs size revealed in this study will allow the preparation of homogeneous NPs of various sizes, which will be useful to provide NPs at the volumes and doses required in preclinical and clinical studies of these therapeutics.Diabetes, Metabolic and Genetic Diseases II579. Long-Term Follow-Up of Lentiviral Integration Profi les from Patients Undergoing Clinical Gene Therapy for X-Adrenoleukodystrophy Cynthia C. Bartholomae,1 Nathalie Cartier,2,3 Salima Hacein-Bey-Abina,2,4 Ina Kutschera,1 Alain Fischer,2,4 Marina Cavazzana-Calvo,2,4 Patrick Aubourg,2,3 Manfred Schmidt,1 Christof von Kalle.11German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany; 2INSERM U986, University Paris-Descartes, Paris, France; 3Hopital Bicêtre-Paris Sud, Le Kremlin-Bicêtre, France; 4Hopital Necker-Enfants Malades, Paris, France.HIV-1 based lentiviral vectors with self-inactivating (SIN) confi guration are showing a reduced genotoxic risk in preclinical testing’s compared to retroviral vectors harboring full LTRs. X-Adrenoleukodystrophy (X-ALD) has been the fi rst monogenetic disease to be treated with gene therapy using HIV-1 based SIN vectors. In total, four patients have been enrolled in this clinical gene therapy trial. The fi rst patient (P1) has been treated already more than six years ago, P2 5 years ago, P3 4.5 years and P4 two years ago. So far no severe adverse events have been reported from this and other clinical trials using HIV-1 based SIN vectors.In the present study, we report an in depth integration site (IS) analysis of samples from 4 patients from this fi rst clinical trial to treat a monogenetic cerebral disease using autologous hematopoietic stem cell transplantation with a HIV-1 based lentiviral SIN-vector. The correction of hematopoietic stem cells has not been accompanied by signs of clonal outgrowth or even premalignant disproportional of cellular contributions in the 4 treated patients. Linear amplifi cation mediated PCR (LAM-PCR) performed on ex vivo transduced cells prior to reinfusion and on engrafted cells revealed a polyclonal hematopoietic reconstitution in all patients. High-throughput sequencing of LAM-PCR amplicons by 454 pyrosequecing and Illumina MiSeq sequencing identified a highly diverse clonal repertoire. In total, more than 33,000 unique IS have been retrieved from distinct time points and sorted cell fractions (P1: 6-74 months (M); P2: M6 – M64; P3: M2 – M54; P4: M1 – M24). Downstream bioinformatics analysis confi rmed the characteristic insertion profi le reported for lentiviral vectors. More than 70% of the detected IS are located within gene coding regions, which are known to be targeted preferentially by lentiviral vectors. Moreover, a favored integration on chromosomes 17 and 19 harboring gene dense regions has been observed. The affected genomic regions, close to KDM2A, PACS1 and HLA genes, are described for lentiviral clustering. The landscape of ALD vector integration was very similar in all four patients investigated. A successful ex vivo transduction of early hematopoietic progenitors is indicated by the detection of identical IS identifi ed in myeloid and lymphoid lineages as well as in CD34+ cells. Lentiviral gene therapy shows to be safe and effective as the cerebral disease has been stabilized in 3 out of 4 patients.

Large scale preparation of chitosan/nucleic acid nanoparticles using an automated inline mixing system

https://publications.polymtl.ca/4741/1/2014_Alameh_Chitosan_siRNA_nanoparticles_demonstrate_superior.pdf

Chitosan/siRNA nanoparticles demonstrate superior silencing efficiency and lower toxicity compared to lipid nanoparticles

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