Fig 1 - uploaded by George Dickson
Content may be subject to copyright.
Source publication
![Table 2. Constituents of Escherichia coli Lysates [70]](profile/George-Dickson/publication/47698259/figure/tbl2/AS:394179382398987@1470991096723/Constituents-of-Escherichia-coli-Lysates-70_Q320.jpg)
After some decades of research, development and first clinical approaches to use DNA vectors in gene therapy, cell therapy and DNA vaccination, the requirements for the pharmaceutical manufacturing of gene vectors has improved significantly step by step. Even the expression level and specificity of non viral DNA vectors were significantly modified...
Similar publications
Due to their unique biological characteristics, mesenchymal stem cells (MSCs) are useful in developing cell-based therapies, tissue regeneration, and many other applications of cellular and molecular genetics. Use of lentiviral system allows the integration of specific nucleic acid sequence into a target cell genome near an active transcription sit...
Citations
... Doğrudan doku tarafından üretildiği için antijenler doğal formda ve istikrarlı bir şekilde sentezlenir. Tek başına DNA enjeksiyonu ile elektroporasyonun ardından enjeksiyon arasındaki ilişki araştırıldığında elektrik alanları uygulandıktan sonra hem hücresel hem de humoral yanıtta artış olduğu gösterilmiştir (109,110). ...
... GET esas olarak enfeksiyon hastalıkları, kanser, artrit, multipl skleroz ve organ naklini takiben iltihaplanmaya karşı DNA aşılaması ve rejeneratif tıp uygulamaları için hücreleri genetik olarak modifiye etmek için de kullanılmıştır (56,82,(110)(111)(112)(113). ...
... Faz I klinik çalışmalar melanom tedavisinde plazmid kodlama antianjiogenik metargit peptid (AMEP) ile elektroporasyon değerlendirmek için yapılmaktadır (101). Kanser tedavisinde, GET preklinik çalışmalarının çoğu, IL-12, IL-2, IL-15 ve GM-CSF gibi immünomodülatör genlerin iletilmesine odaklanmıştır (110,111). Yine vazostatin, endostatin ve vasküler endotelyal büyüme faktörü (VEGF) (121-124) gibi tümör damar yapısını hedefleyen genlerin kullanımını belgeleyen birkaç çalışma da yapılmıştır. İntegrinleri hedefleyen bir antianjiyojenik metargidin peptidinin (AMEP) GET'i ve endoglini hedefleyen siRNA'nın GET'inin melanom tümörleri (AMEP için) ve meme karsinom tümörleri (siRNA için) üzerinde antiproliferatif ve antianjiyojenik etkiler uyguladığı gösterilmiştir (125,126). ...
Sağlık sistemlerinin güçlendirilmesi; sağlık hizmetlerinin ve teknolojilerinin iyileştirilmesi, sunulması ve sürekli ardışık inovatif süreçleri içerir. İhtiyaç temelli inovasyon bu süreci hızlandırmaktadır. Sağlık ürün ve hizmetlerinin inovatif yönü ile geliştirilerek sunulması, sağlık yeniliğinde bütüncül bir yaklaşımın ayrılmaz bileşenleridir. Sağlık inovasyonunu, finansal olarak sürdürülebilir çözümlerle geliştirip büyütmek için de kritik öneme sahiptir. Sağlık inovasyonuna yaklaşımımız bu nedenle bütünseldir. Sağlıktaki tıkanan düğüme ve karmaşık zorluklara çözümler geliştirmek için bilimsel / teknolojik, sosyal ve ticari inovasyonun koordineli uygulaması ile yeni nesil inovatif hekimlik anlayışının dijital bir güncelleme ile yakın zamanda karşımıza gelmesi kaçınılmaz olacaktır.
... Viral vectors (e.g., adenoviruses, poxviruses), recombinant bacteria, plasmid DNA and RNA, are different kinds of these vaccines [170]. RNA or DNA-based vaccines are expressed in the host cell instead of direct injection of the antigen or complete virus particles [171]. ...
Almost 80% of people confronting COVID-19 recover from COVID-19 disease without any particular treatments. They experience heterogeneous symptoms; a wide range of respiratory symptoms, cough, dyspnea, fever, and viral pneumonia. However, some others need urgent intervention and special treatment to get rid of this widespread disease. So far, there isn't any unique drug for the potential treatment of COVID 19. However, some available therapeutic drugs used for other diseases seem beneficial for the COVID-19 treatment. On the other hand, there is a robust global concern for developing an efficient COVID-19 vaccine to control the COVID-19 pandemic sustainably. According to the WHO report, since 8 October 2021, 320 vaccines have been in progress. 194 vaccines are in the pre-clinical development stage that 126 of them are in clinical progression. Here, in this paper, we have comprehensively reviewed the most recent and updated information about coronavirus and its mutations, all the potential therapeutic approaches for treating COVID-19, developed diagnostic systems for COVID- 19 and the available COVID-19 vaccines and their mechanism of action.
... Regarding their outcome in MC production, the main difference between these two families is related with the underlying mechanism of recombination. Due to their unidirectional recombination mechanism, serine recombinases greatly reduce the formation of MC multimeric forms [15]. Current systems for MC production typically rely on recombinases from the serine family due to this better performance. ...
Minicircles (MCs) are DNA molecules that are produced in Escherichia coli by replicating a parental plasmid (PP) and inducing its site-specific intramolecular recombination into miniplasmid (MP; containing the prokaryotic backbone) and MC molecules (comprised by the eukaryotic cassette). The determination of the recombination efficiency and the monitoring of PP, MC and MP species during processing and in the final product are critical aspects of MC manufacturing. This work describes a real-time PCR method for the specific identification of PP, MP or MC that uses sets of primers specific for each species. The method was evaluated using artificial mixtures of (i) PP and MP, (ii) PP and MC and (iii) MP and MC that were probed for all three DNA molecules. The ratio of molecules of each DNA species in these mixtures were determined with differences lower than 10% relatively to the expected ratio of the species in 90% of the mixtures. Next, the recombination efficiency was successfully estimated by analysing pre-purified DNA samples obtained from cell cultures. A standard deviation <2% was obtained between replicas and results closely correlated with those obtained by densitometry analysis of agarose gels. Further optimization is required to determine recombination efficiency directly from whole cells.
... Although this can be valuable for DNA vaccination, these sequences are a disadvantage if the vector is intended to be used for delivery in gene therapy (Mairhofer and Grabherr 2008;Hardee et al., 2017). Altogether, the drawbacks of the typical pDNA vector triggered the development of safer and more efficient non-viral delivery vectors, a topic that has been the focus of several reviews [e.g., (Mairhofer and Grabherr 2008;Schleef et al., 2010;Hardee et al., 2017;Shankar et al., 2017)]. ...
... The implementation of a minicircle purification strategy is highly dependent on the DNA mixture obtained after the lysis of cells collected at the end of bacterial growth. Unlike the typical pDNA production, in which two topological isoforms of the product are obtained (open circular and supercoiled), in minicircle production additional DNA species are obtained, which include the miniplasmid and residual un-recombined parental plasmid, with the corresponding topoisomers (Schleef et al., 2010;Mayrhofer and Iro 2012). This complicates minicircle purification since these other molecules display similar physicochemical properties. ...
... Suggested specifications for pDNA-based vaccines and possible analytical methods (adapted from FDA -Center for BiologicsEvaluation and Research, 2007;Schleef et al., 2010;Urthaler et al., 2012). ...
Minicircles are non-viral delivery vectors with promising features for biopharmaceutical applications. These vectors are plasmid-derived circular DNA molecules that are obtained in vivo in Escherichia coli by the intramolecular recombination of a parental plasmid, which generates a minicircle containing the eukaryotic therapeutic cassette of interest and a miniplasmid containing the prokaryotic backbone. The production process results thus in a complex mixture, which hinders the isolation of minicircle molecules from other DNA molecules. Several strategies have been proposed over the years to meet the challenge of purifying and obtaining high quality minicircles in compliance with the regulatory guidelines for therapeutic use. In minicircle purification, the characteristics of the strain and parental plasmid used have a high impact and strongly affect the purification strategy that can be applied. This review summarizes the different methods developed so far, focusing not only on the purification method itself but also on its dependence on the upstream production strategy used.
... While choosing the vector, it should be considered that different tissues/cells require vectors with different properties and based on the application, vector choice can also be different [3]. Tools for gene delivery can be classified into two major groups as viral and non-viral vectors [4][5][6]. For what concerns viral vectors, parts of viral genome were deleted and replaced by genetic elements [7]. ...
Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.
... 7 To date, we have prepared all MC-DNA vectors for injection from bacterial cultures using commercially available endotoxin free DNA purification kits that utilize a classic alkaline lysis approach followed by anion-exchange resin column (see the Supplemental Materials and Methods). Although we had no indication for inflammatory or immune-stimulatory effects in MC-treated mice whatsoever, 7 we examined whether more highly purified 18 MC-DNA, prepared using PlasmidFactory's (PF) affinity chromatography-based MC production technology (see the Supplemental Materials and Methods), would exhibit better therapeutic performance. As displayed in Figures S3A and S3B, standard kit grade MC.PKU28 DNA demonstrated that most of the MC DNA was present as a monomeric covalently closed circular (ccc) supercoiled molecule, but a significant amount was relaxed open circular monomer, dimer, trimmer, tetra-, and larger concatemers. ...
... To date, we have prepared all MC-DNA vectors for injection from bacterial cultures using commercially available endotoxin free DNA purification kits that utilize a classic alkaline lysis approach followed by anion-exchange resin column (see Supplementary Materials and Methods). Although we had no indication for inflammatory or immune-stimulatory effects in MC-treated mice whatsoever [7], we examined whether more highly purified [18] Table 2). ...
Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of <1 vector genome per diploid hepatocyte genome and was achieved at a vector dose that was significantly lowered. Partial hepatectomy and subsequent liver regeneration was associated with >95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.
... MC DNA was recovered from RP using affinity chromatography based on interaction of lactose operon (LacO) with repressor of lactose operon (LacI) as previously described. 52 In-Process-Control showed that just the MC binds to the chromatography matrix, while the undesired MP is not binding. The resuting MC fraction was precipitated and resuspended in water for injection at 1 mg/ml and the product QC was performed (see Supplementary Figure S2). ...
Adeno-associated viral (AAV) vectors are considered as one of the most promising delivery systems in human gene therapy. In addition, AAV vectors are frequently applied tools in preclinical and basic research. Despite this success, manufacturing pure AAV vector preparations remains a difficult task. While empty capsids can be removed from vector preparations owing to their lower density, state-of-the-art purification strategies as of yet failed to remove antibiotic resistance genes or other plasmid backbone sequences. Here, we report the development of minicircle (MC) constructs to replace AAV vector and helper plasmids for production of both, single-stranded (ss) and self-complementary (sc) AAV vectors. As bacterial backbone sequences are removed during MC production, encapsidation of prokaryotic plasmid backbone sequences is avoided. This is of particular importance for scAAV vector preparations, which contained an unproportionally high amount of plasmid backbone sequences (up to 26.1% versus up to 2.9% (ssAAV)). Replacing standard packaging plasmids by MC constructs not only allowed to reduce these contaminations below quantification limit, but in addition improved transduction efficiencies of scAAV preparations up to 30-fold. Thus, MC technology offers an easy to implement modification of standard AAV packaging protocols that significantly improves the quality of AAV vector preparations. © 2016 Official journal of the American Society of Gene & Cell Therapy
... Cette section décrit les éléments à prendre en compte pour la construction d'un vecteur plasmidique ainsi que les pistes d'optimisation ayant été proposées ces dernières années. La topologie encadrée en jaune correspond à la forme dite surenroulée monomérique considérée comme la plus efficace pour le transfert de gène (Schleef et al. 2010). ...
... Après divers tests de contrôle qualité du produit final, il peut être concentré, formulé, conditionné et stocké en fonction de sa future application (d). D'après (Schleef et al. 2010;Schmeer & Schleef 2014). ...
L’un des principaux défis de la thérapie génique est d’identifier un vecteur sûr capable d’assurer un transfert efficace et une expression soutenue d’un gène d’intérêt thérapeutique dans les cellules cibles. L’émergence de vecteurs plasmidiques de nouvelles générations a permis d’atteindre ces objectifs et de considérer la thérapie génique non virale comme une alternative prometteuse aux vecteurs viraux pour le traitement de maladies génétiques ou acquises. Appartenant à ces nouvelles générations, les dérivés du vecteur pFAR4 sont des miniplasmides dépourvus de gène de résistance à un antibiotique. Leur propagation dans les cellules d’Escherichia coli est basée sur la suppression d’une mutation non-sens de type ambre introduite dans un gène essentiel de la souche productrice, permettant ainsi d’éliminer les risques associés à l’utilisation de gène de résistance à un antibiotique tout en diminuant la taille du vecteur. Le but de cette thèse est d’étudier le potentiel de ces vecteurs dans deux contextes de thérapie génique non virale : Dans une première approche, le potentiel du vecteur pFAR4 a été évalué pour l’expression d’un gène thérapeutique dans le foie de souris. Pour ce faire, un dérivé de ce vecteur exprimant le gène Sgsh à partir d’un promoteur spécifique des hépatocytes et codant la protéine sulfamidase, protéine défectueuse chez les patients souffrant de la maladie de Sanfilippo de type A, a été administré par injection hydrodynamique à des souris. Nous avons montré que le vecteur pFAR4 promeut dans le foie une expression élevée et soutenue de la sulfamidase, qui décline rapidement lorsque le gène Sgsh est administré par un vecteur contenant un gène de résistance à la kanamycine. Dans le cadre de cette étude, il a été établi que le profil d’expression obtenu avec le vecteur pFAR4 n’est pas lié à son insertion dans le génome des hépatocytes mais résulte, de par sa taille réduite, d’une protection contre les phénomènes d’extinction de transgène couramment observés in vivo avec les vecteurs conventionnels. Dans une seconde approche, le vecteur pFAR4 a été combiné à la technologie Sleeping Beauty (SB), dont l’un des constituants majeurs est la transposase hyperactive SB100X qui promeut la transposition d’un transgène, en l’excisant du plasmide qui le porte et en l’insérant dans le génome des cellules hôtes. Cette combinaison a été étudiée in vitro dans des cellules HeLa, en utilisant un transposon contenant soit le gène de résistance à la néomycine soit le gène codant la protéine fluorescente Vénus. Nous avons ainsi montré que le plasmide pFAR4 constituait un vecteur efficace pour les composants du système SB et que la combinaison pFAR4/SB conduisait à un taux de transgénèse augmenté par rapport à une association avec des plasmides conventionnels. Cette efficacité élevée résulte d’un niveau de transfection et d’un taux d’excision augmentés, tous deux favorisés par la taille réduite du plasmide. La combinaison pFAR4/SB devrait prochainement être utilisée pour transférer le gène codant le facteur anti-angiogénique PEDF (Pigment Epithelium-Derived Factor) à des cellules primaires de l’épithélium pigmentaire de la rétine ou de l’iris dans deux essais cliniques (Phase I/II) de thérapie génique ex vivo pour le traitement de la dégénérescence maculaire liée à l’âge (DMLA).
... DNA is easy to produce compared to proteins or antigens (i.e. conventional vaccine material) and it is a stable molecule that can be stored for relatively long periods in normal conditions [63]. In addition, naked DNA is the only vector that does not generate anti-vector immune response, meaning that this approach is safer than the others in term of infection. ...
Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.
