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

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Title: Hypothesis: Inserting bacterial natural transformation protein complexes into human cells for efficient gene therapy using naked DNA
Authors: Tolmachov, O
Item Type: Journal Article
Abstract: 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.
Content Version: Published version
Issue Date: 9-Sep-2010
Citation: WebmedCentral HUMAN GENETICS Vol.( 1(9):WMC00577 ) No.( ) pp 1 - 8
URI: http://hdl.handle.net/10044/1/5950
Publisher Link: http://www.webmedcentral.com/article_view/577
Start Page: 1
End Page: 8
Copyright Statement: © The author
Volume: 1(9):WMC00577
Appears in Collections:Heart Science

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