Adding functional entities to plasmids.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

In vivo nuclear delivery of oligonucleotides via hybridizing bifunctional peptides.

Linking proteins directly to nucleic acids has been a complex task. By hybridizing a bifunctional peptide nucleic acid (PNA) consisting of a nucleic acid binding moiety and a nuclear localization signal (NLS) we have previously demonstrated that it is possible to link protein functions directly to nucleic acids containing a PNA target site. By hybridizing fluorescently labeled oligonucleotides to PNA-NLS molecules and subsequently transfecting different organs in vivo we demonstrate an active nuclear translocation of the PNA-NLS/oligonucleotide complex in different mouse organs.

BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

BACKGROUND: It has been shown previously that mutations in the cytoplasmic protein kinase, Bruton's tyrosine kinase (BTK) lead to X-linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy. METHODS: Producer cell lines using retroviral BTK constructs were generated and retroviral titers determined. Southern blot analysis was performed to check for pro-viral integrity in the respective clones. Furthermore, cotransfection of green fluorescent protein (GFP) with BTK expression plasmids was used in HeLa cells to establish and characterize the role of BTK in apoptosis. RESULTS: Following the attempt to generate retroviral producer clones by conventional methods, we observed that the BTK gene is deleted from neomycin-resistant high titer clones. We show that BTK mediated apoptosis in GP#E86 and HeLa cells. Furthermore, membrane targeting and kinase activity are required for this effect. In addition, BTK induced apoptosis could be inhibited by using a specific inhibitor for p38 mitogen-activated protein kinase (MAPK), SB203580. CONCLUSION: Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA.

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html