Can painting human cells with exogenous maltoporin enable efficient therapeutic gene transfer by bacteriophage lambda vectors?

Published versio

Hypothesis: Inserting bacterial natural transformation protein complexes into human cells for efficient gene therapy using naked DNA

Naked DNA is a non-toxic vector for therapeutic gene delivery. However, current methods of transfection with naked DNA reach a limited range of susceptible tissues and have a low efficiency. The transfection of clinically important post-mitotic cells is particularly challenging because in these cells DNA need to pass the nuclear barrier. Thus, new principles for the transfer of naked DNA into human cells are required and can be found among the genetic exchange mechanisms in bacteria, where gene entry into cells via pick-up and transfer of naked DNA is known as “transformation”. In a number of bacteria, dedicated molecular machinery facilitates cell entry of free DNA by the process of “natural transformation”. In transformation-competent bacterial cells, specialised protein complexes mediate the binding of free double-stranded DNA, its fragmentation, cell entry and conversion to single-stranded DNA. I propose to exploit bacterial natural transformation machinery for a two-step transfection of human cells with therapeutic naked DNA. Firstly, the bacterial transformation protein complexes are inserted into the plasma membranes or nuclear envelopes of the target human cells and, secondly, the double-stranded vector DNA is supplied for the processing by the installed DNA transfer apparatus. I hypothesize that non-toxic bacterial transformation complexes residing in their new human milieu can promote the ultra-efficient transfer of exogenous therapeutic naked DNA. As the introduction of DNA into mammalian cells by non-viral means is called “transfection”, I propose to name the bacterial transformation complexes functioning in their new eukaryotic surroundings as “transfectosomes”. The initial step of the gene delivery should exploit the modern methods of extraneous protein insertion into mammalian cells, such as cell painting, engineering of cell permeable proteins with targeted intracellular localization, physical techniques of protein transfer like electroinsertion and electroporation. Sequence-selective natural transformation systems are known and can be taken advantage of to exclude undesired (e.g. gene silencing) portions of vector DNA from entering human nucleoplasm. Improved transfectosomes can possibly be engineered for better establishment and performance in human membranes. The hypothesis can be tested by comparing the naked DNA transfer efficiency into the transfectosome-bearing and the naive human cells in ex-vivo and in-vivo gene therapy settings. Immunogenicity of the transfectosomes can be modulated by protein engineering. As the delivered fragments of single-stranded DNA are highly recombinogenic, the confirmation of the hypothesis can lead to a breakthrough in gene repair therapy of dominantly inherited familial hypercholesterolemia, polycystic kidney disease and trinucleotide repeat disorders.Published versio

Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro.

BACKGROUND: Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro. METHODS: To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin and the polycation transduction enhancer Transfectam. The EGFP-positive transduced cells were then enriched by flow cytometry. RESULTS: More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes. CONCLUSION: The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients own skeletal myoblasts

Синтез та деякі перетворення 5-ізоксазолілсульфонілхлоридів

The effect of the structure of 5-(benzylthio)isoxazoles on selectivity of the synthesis of 5-(chlorosulfonyl)isoxazoles has been determined. The chemical behavior in relation to amines has been described.Aim. To develop the methods for the synthesis of 5-(chlorosulfonyl)- isoxazoles and 4-chloro-5-(chlorosulfonyl)isoxazoles as promising reagents for construction of prospective bioactive compounds.Results and discussion. The number of 5-(benzylthio)isoxazoles was obtained by cyclocondensation of N-hydroxyimidoyl chlorides or 2-chloro-2-(hydroxyimino) acetates with benzylethynylsulfide. Their oxidative chlorination with gaseous chlorine led to formation of the mixture of isoxazole-5-sulfonyl chlorides and 4-chloroisoxazole-5-sulfonyl chlorides. The ratio between these products in the mixture depended on the nature of the substitution group in position 3 of the isoxazole ring. For the synthesis of 4-chloro-5-(chlorosulfonyl)isoxazoles with acceptable yields the approach of an advance chlorination of 5-benzylthioisoxazoles by N-chlorosuccinimide with further oxidative chlorination was used.Experimental part. The synthesis of the starting and target compounds was performed in classic preparative conditions; flesh-chromatography; elemental analysis; LCMS; 1H and 13C NMR-spectroscopy were used.Conclusions. The reaction of oxidative chlorination of 5-(benzylthio)-3-isoxazoles has been studied. The synthetic approach for the previously unknown representatives of isoxazole-5-sulfonylchlorides has been developed.Определено влияние структуры 5-бензилтиоизоксазолов на селективность образования 5-изоксазолилсульфонилхлоридов и выявлено химическое поведение последних по отношению к аминам.Цель  работы – создание методов синтеза 5-изоксазолил- и 4-хлор-5-изоксазолилсульфонилхлоридов как перспективных реагентов для конструирования потенциально биоактивных веществ.Результаты и их обсуждение. Циклоконденсацией N-гидроксиимидоилхлоридов или 2-хлоро-2-(гидроксиимино)ацетатов из бензилтиоацетиленом синтезировано ряд 5-бензилтиоизоксазолов. Их окислительное хлорирование приводит к образованию смеси 5-изоксазолилсульфонилхлоридов и 4-хлор-5-изоксазолилсульфонилхлоридов, соотношение между которыми зависит от характера заместителей в положении 3 изоксазольного цикла. Для синтеза 4-хлор-5-изоксазолилсульфонилхлоридов с удовлетворительными выходами использован вариант предварительного хлорирования ядра 5-бензилтиоизоксазолов N-хлорсукцинимидом с дальнейшим окислительным хлорированием.Экспериментальная часть. Синтез исходных и целевых соединений в классических препаративных условиях; методы флеш-хроматографии, элементного анализа, хроматомасс-спектрометрии, ЯМР 1Н и 13С-спектроскопии. Выводы. Исследована реакция окислительного хлорирования 5-бензилтиоизоксазолов и разработан синтетический подход к ранее неизвестным представителям 5-изоксазолилсульфонилхлоридов.Встановлено вплив структури 5-бензилтіоізоксазолів на селективність утворення 5-ізоксазолілсульфонілхлоридів та з’ясована хімічна поведінка останніх по відношенню до амінів.Мета роботи – створення методів синтезу 5-ізоксазоліл- та 4-хлоро-5-ізоксазолілсульфонілхлоридів як перспективних реагентів для конструювання потенційно біоактивних речовин.Результати та їх обговорення. Циклоконденсацією N-гідроксіімідоїлхлоридів або 2-хлоро-2-(гідроксііміно)ацетатів із бензилтіоацетиленом синтезовано низку 5-бензилтіоізоксазолів. Їх окиснювальне хлорування приводить до утворення суміші 5-ізоксазолілсульфонілхлоридів та 4-хлоро-5-ізоксазолілсульфонілхлоридів, співвідношення між якими залежить від характеру замісників у положенні 3 ізоксазольного циклу. Для синтезу 4-хлоро-5-ізоксазолілсульфонілхлоридів із задовільними виходами використано варіант попереднього хлорування ядра 5-бензилтіоізоксазолів N-хлоросукцинімідом із подальшим окиснювальним хлоруванням.Експериментальна частина. Синтез вихідних та цільових сполук у класичних препаративних умовах; методи флеш-хроматографії, елементного аналізу, хроматомас-спектрометрії, ЯМР 1Н та 13С-спектроскопії.Висновки. Досліджена реакція окиснювального хлорування 5-бензилтіоізоксазолів та розроблено синтетичний підхід до раніше невідомих представників 5-ізоксазолілсульфонілхлоридів

Нейровизуализационные методики оценки головного мозга при сахарном диабете (литературный обзор)

Diabetes mellitus (DM) is associated with changes in the structure of the brain and deterioration of cognitive functions from mild to moderate according to neuropsychological testing. With the growing DM epidemic and the increasing number of people living to old age, cognitive dysfunctions associated with DM can have serious consequences for the future of public and practical health. Chronic hyperglycemia, severe episodes of hypoglycemia, and microvascular complications are important risk factors common for type 1 and type 2 diabetes. DM is also associated with structural and functional changes in the brain, which can be diagnosed by various types of magnetic resonance imaging (MRI) of the brain. In this review, we investigate studies conducted over the past two decades to improve the understanding of how DM effects the brain function and structure. We also describe the changes characteristic of type 1 and type 2 diabetes during standard MRI, functional MRI and proton magnetic-resonance spectroscopy (proton MRS) as well as their features.Сахарный диабет (СД) связан с изменениями в структуре головного мозга и ухудшением когнитивных функций от легкой до умеренной степени по данным нейропсихологического тестирования. В условиях растущей эпидемии СД и увеличения числа людей, доживающих до старости, когнитивная дисфункция, ассоциированная с СД, может иметь серьезные последствия для будущего общественного и практического здравоохранения. Хроническая гипергликемия, тяжелые эпизоды гипогликемии и микрососудистые осложнения являются важными факторами риска, общими для СД 1-го и 2-го типа. Также СД связан со структурными и функциональными изменениями в головном мозге, которые возможно диагностировать посредством различных вариантов магнитно-резонансной томографии (МРТ) головного мозга. В представленном обзоре рассмотрены исследования, проведенные за последние два десятилетия, чтобы улучшить понимание того, как СД влияет на функцию и структуру головного мозга. Также описаны изменения, характерные для СД 1-го и 2-го типа при проведении стандартной,  функциональной МРТ и протонной магнитно-резонансной спектроскопии, и их особенности

Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo

Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials. © 2013 The Author(s)

Silencing of Transgene Expression: A Gene Therapy Perspective

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Development of a new S/MAR containing Minicircle DNA vector: a new promise for Gene Therapy.

A barrier limiting the use of nonviral vectors for gene therapy is related to the short duration of transgene expression in vivo. Development, evaluation, and optimisation of a long term transgene expression using a non viral vector is currently the primary aim in our research field. Recently, we have demonstrate that a nonviral episomal plasmid (pDNA) vector combined with a scaffold/matrix attachment region (S/MAR) is able to sustain long-term expression in murine liver for at least six months following hydrodynamic injection. However, plasmids contain sequences, which are essential for propagation in bacteria but are unnecessary for expression in mammalian cells. These bacterial components are responsible for the epigenetic silencing of the vector and toxicity when applied in vivo

DEVELOPMENT OF S/MAR MINICIRLES VECTOR FOR PERSISTENT EXPRESSION IN VIVO.

An ideal vector for gene therapy must fulfil the following requirements: non-toxicity, mitotic stability and persistent therapeutic levels of transgene expression. Viral vectors are widely used due to their ability to sustain prolonged expression. Their potentially tumorigenic effects are a limiting factor for in vivo applications. Non-viral vectors, which can be designed to be free from viral sequences, are a promising alternative for gene transfer although they often produce transient transgene expression. This limitation of this vector type is primarily due to bacterial sequences they contain and these have proven to be toxic for the mammalian cells as they contain a high number of unmethylated CpG motifs. We have developed a new prototype gene expression vector devoid of any bacterial sequence. This minicircle vector comprises entirely mammalian sequences. Following hydrodynamic delivery into murine liver we have shown that this construct is able to sustain persistent expression of the luciferase marker gene in vivo for more than 25 days. In contrast, expression from an original expression plasmid (from which the minicircle is derived) which harbours these extraneous bacterial sequences dropped significantly to 10% of its initial expression after 25 days. The inclusion of an S/MAR element into the minicircle sequence not only produced increasing levels of expression following administration but after 25 days showed sustained levels which were twice as high as those found immediately following administration. These promising results demonstrate the utility of minimally sized non-viral vectors in combination with S/MAR motifs for persistent, non toxic therapeutic gene transfer