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Oleg Tolmachov

Publications and source records attributed to Oleg Tolmachov.

8 recordsLinked to original sources
PubMed2006

Covalent attachment of multifunctional chimeric terminal proteins to 5' DNA ends: A potential new strategy for assembly of synthetic therapeutic gene vectors.

The generation of synthetic therapeutic gene vectors requires the coupling of DNA to transfer-promoting peptides including cellular receptor ligands, protein transduction domains, hydrophobic peptides for attachment to lipid membranes, nuclear localisation signals, cytoskeleton attachment motifs, nuclear matrix association elements and immune evasion moieties. Existing methods of peptide-DNA joining often interfere with transgene expression and, therefore, are inadequate for production of effective therapeutic vector complexes, particularly destined for gene delivery in the challenging environment in vivo. However, there is a natural mechanism for rigid coupling of polypeptides with DNA. Some bacterial and eukaryotic linear plasmids, adenoviruses and a number of bacteriophages including phi29 of Bacillus subtilis and PRD1 of Escherichia coli use terminal proteins covalently bound to 5' DNA ends to prime replication. Inverted terminal DNA repeats, normally short DNA sequences, contain all the sequences required in cis for the covalent coupling reaction. The complex of the terminal protein, DNA polymerase and some known auxiliary proteins supplies sufficient trans-functions, thus enabling simple linking of the terminal proteins to DNA in vitro. We hypothesise that chimeric fusion proteins, constructed on the basis of terminal proteins of adenoviruses, linear plasmids or bacteriophages with protein-primed replication, can on the one hand retain the ability to bind covalently 5' DNA termini in conditions established previously for protein-primed replication in vitro, and on the other hand confer gene transfer facilitating properties and enhanced longevity of efficient transgene expression. Terminal localisation of the chimeric proteins can ensure that they do not interfere with transgene transcription. At the same time a covalent bond between polypeptide and DNA can provide rigid coupling ensuring their stable association en route to nuclei. Bound to 5'-ends of the delivered DNA, terminal protein-based chimeras could also protect the vector DNA from 5'-3' and possibly 3'-5' exonuclease attack, thus limiting its intracellular degradation and increasing longevity of transgene expression. Our hypothesis can be tested by measuring the gene transfer efficiency of the novel complexes containing linear DNA fragments with covalently linked multifunctional chimeric terminal proteins, using previously described synthetic gene vectors as standards.

5' Untranslated Regions↗
PubMed2006

A heterologous system for assembly of retroviral gene vectors: intracellular budding in yeast?

Mammalian retroviral gene vectors are of particular importance in gene therapy because of their efficient chromosomal integration and resulting stable maintenance of transgenes. These vectors are currently produced in mammalian cells, with low yield and at substantial expense. Therefore, a more efficient heterologous production system for retroviral vectors would be desirable. With their impressive track record in biotechnology, various yeast species appear like ideal organisms to generate retroviral vectors. Typically, retroviral vector particles emerge from mammalian cells after budding at the plasma membrane. However, in yeast, viral budding at the plasma membrane is blocked by the cell wall. At the same time, mass production of enveloped viral vectors in yeast protoplasts is technically challenging. Recent reports indicated the generation of infectious virions via intracellular budding for some combinations of retroviruses and mammalian cells. Relying on these data I hypothesise that a successful assembly of the retroviral transducing particles can be accomplished intracellularly inside yeast cells with a normal cell wall. Firstly, it is possible that some of the intracellular yeast compartments have all the necessary host factors that are required for successful RNA packaging, budding and maturation of infectious retroviral vector particles. Secondly, it might be possible to improve intracellular viral vector production by artificially targeting viral cores to bud at specific intracellular vesicular structures using appropriate targeting or retention signals. A suitable envelope protein, conferring infectivity and specific cellular tropism to the vector particles, can be expressed in yeast or, alternatively, 'bald' viral particles without envelope protein can be produced in yeast and later complexed with a desired envelope protein in vitro. Retroviral budding on yeast intracellular membranes can rely on the same host factors that are used by yeast retrotransposons driving intracellular formation of non-infectious virus-like particles. It is likely that optimal vector packaging system can be found as a result of the dedicated screening of the various retroviral vectors against an array of yeast species. The central implication of the hypothesis is that budding of retroviral vector particles inside yeast cells, with no need for obtaining and maintaining yeast spheroplasts or protoplasts, can be an efficient and economical method of mass production of these valuable gene therapy vectors.

Biotechnology↗
PubMed2006

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.

Animals↗
PubMed2006

RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA.

BACKGROUND: Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. RESULTS: We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination. CONCLUSION: Using transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- minicircle DNA producer strain HB101Cre was used to obtain pure minicircle DNA, consisting of monomeric and multimeric minicircle forms. The obtained recA- minicircle DNA producer strain is expected to decrease the risk of undesired deletions within minicircle producer plasmids and, therefore, to improve production of the therapeutic minicircle vectors.

Chromosomes, Bacterial↗
PubMed2006

Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo.

BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.

Animals↗
PubMed2005

Coupled analysis of bacterial transformants and ligation mixture by duplex PCR enables detection of fatal instability of a nascent recombinant plasmid.

When a DNA cloning experiment fails, it is often difficult to distinguish between an inadequate cloning protocol and instability of the new recombinant plasmid. The identification of plasmid instability is particularly challenging when the instability is fatal and no DNA of the expected construct can be isolated. We have effectively addressed this problem by employment of duplex PCR (insert-insert, vector-insert) to analyse both the ligation mixture and the resultant bacterial transformants. Using this approach we found a fatal maintenance instability of one of the plasmids generated during subcloning of the cDNA for human LDLR in Escherichia coli STBL2. The described duplex PCR screening method allows monitoring of the fate of nascent recombinant plasmid from ligation, through the initial bacterial colony and the subsequent overnight culture.

Cloning, Molecular↗
PubMed2003

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein.

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.

Active Transport, Cell Nucleus↗