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

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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

Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor

Designing lentiviral gene vectors

Lentiviral gene vectors are an important tool in gene therapy and basic biomedical research. They are transducing viral particles, normally replication defective, which are generated using the packaging machinery of lentiviruses. These vectors are used to deliver the encapsidated payload genes to the nuclei of the target cells, offering stable transgene expression in many settings in vitro and in vivo. Successful generation of high-titre lentiviral vectors capable of efficiently expressing transgenes over long period of time is governed by a number of vector design rules, some of which are common to all gene vectors while others are specific to lentiviral vectors. Construction of lentiviral vectors with the cargo genes driven by tissue-specific promoters is a particular challenge. This review focuses both on the guiding principles and the technical know-how of the lentiviral gene vector design.Published versio

Linear structure on a finite Hecke category in type A

For the group GL(n), we construct an action of the derived category of coherent sheaves on the Grothendieck-Springer resolution on a certain subcategory of a finite monodromic Hecke category. We use this to construct a partial categorification of the projection from the extened affine to the finite Hecke algebra of GL(n). As a crucial intermediate step, we compute the exterior powers, with respect to the perversely truncated multiplicative convolution, of a parabolic Springer sheaf corresponding to a maximal parabolic subgroup fixing a line in the defining $n$-dimensional representation of GL(n).Comment: Comments welcome! arXiv admin note: substantial text overlap with arXiv:2209.0160

Exterior powers of a parabolic Springer sheaf on a Lie algebra

We compute the exterior powers, with respect to the additive convolution on the general linear Lie algebra, of a parabolic Springer sheaf corresponding to a maximal parabolic subgroup of type (1, n -- 1). They turn out to be isomorphic to the semisimple perverse sheaves attached by the Springer correspondence to the exterior powers of the permutation representation of the symmetric group.Comment: Content reorganised. Proofs for the Lie algebra statement significantly simplified. Material related to the convolution on the group was moved to the preprint "Linear structure on a finite Hecke category in type A" by the second autho

Methods of Transfection with Messenger RNA Gene Vectors

Non-viral gene delivery vectors with messenger RNA (mRNA) as a carrier of genetic information are among the staple gene transfer vectors for research in gene therapy, gene vaccination and cell fate reprogramming. As no passage of genetic cargo in and out of the nucleus is required, mRNA-based vectors typically offer the following five advantages: 1) fast start of transgene expression; 2) ability to express genes in non-dividing cells with an intact nuclear envelope; 3) insensitivity to the major gene silencing mechanisms, which operate in the nucleus; 4) absence of potentially mutagenic genomic insertions; 5) high cell survival rate after transfection procedures, which do not need to disturb nuclear envelope. In addition, mRNA-based vectors offer a simple combination of various transgenes through mixing of several mRNAs in a single multi-gene cocktail or expression of a number of proteins from a single mRNA molecule using internal ribosome entry sites (IRESes), ribosome skipping sequences and proteolytic signals. However, on the downside, uncontrolled extracellular and intracellular decay of mRNA can be a substantial hurdle for mRNA-mediated gene transfer. Procedures for mRNA delivery are analogous to DNA transfer methods, which are well-established. In general, there are three actors in the gene delivery play, namely, the vector, the cell and the transfer environment. The desired outcome, that is, the efficient delivery of a gene to a target cell population, depends on the efficient interaction of all three parties. Thus, the vector should be customised for the target cell population and presented in a form that is resistant to the aggressive factors in the delivery milieu. At the same time, the delivery environment should be adjusted to be more vector-friendly and more cell-friendly. The recipient cells should be subjected to a specific regimen or artificially modified to become receptive to gene transfer with a particular vector and resistant to the environment. As a rule, barriers outside tissues (e.g. mucus) and an aggressive intercellular environment complicate gene delivery in vivo, which, therefore, requires more complex gene transfer procedures than transfection of tissue culture cells. This review is focused on transfection methods for mRNA vectors, which rely either on the forceful propulsion of mRNA inside the target cells (e.g. by electroporation or gene gun) or on the complexing of mRNA with other substances (e.g. polycationic transfection reagents) for delivery via endocytic pathways

Silencing of Transgene Expression: A Gene Therapy Perspective

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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