Anke Huber

University of Cologne, Köln, North Rhine-Westphalia, Germany

Are you Anke Huber?

Claim your profile

Publications (7)23.33 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid-engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvß8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.Molecular Therapy (2014); doi:10.1038/mt.2014.14.
    Molecular Therapy 01/2014; · 7.04 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clinical application of viral vectors is often hampered by the lack of selectivity of viral particles for the targeted tissue. This drawback decreases the efficiency of gene delivery and raises safety concerns. We successfully established a novel in vitro evolution protocol to engineer adeno-associated virus vectors with increased selectivity for designated target cells. Subjecting a peptide-display library of AAV capsids to negative selection cycles on human primary fibroblasts and to positive selection cycles on a human melanoma cell line, we isolated several variants with up to 3.7-fold increased specificity for malignant cells in comparison to fibroblasts and other cell types. These mutants can be used to achieve high levels of gene transfer to target cells reducing undesired transduction of neighbouring tissues.
    Combinatorial chemistry & high throughput screening 11/2010; 13(9):807-12. · 2.46 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bone marrow-derived CD34(+) cells are currently used in clinical trials in patients with ischemic heart disease. An option to enhance activity of injected progenitors may be offered by genetic engineering of progenitor cells with angiogenic growth factors. Recombinant adeno-associated viral vectors (rAAV) have emerged as a leading gene transfer systems. In contrast to other vector systems in use for genetic engineering of CD34(+) cells, rAAV-mediated gene expression does not depend on vector integration. This is relevant for application in regenerative medicine of ischemic tissues, where transient transgene expression is likely sufficient to achieve therapeutic benefits. We compared three different human AAV serotypes, packaged as pseudotypes by a helper virus-free production method, for their transduction efficiency in human cord blood-derived CD34(+) cells. We further assessed the impact of vector genome conformation, of alpha(v)beta(5) and alpha(5)beta(1) integrin availability and of the transcription-modulating drugs retinoic acid and Trichostatin A on rAAV-mediated human CD34(+) cell transduction. We provide, for the first time, evidence that hCD34(+) cells can be reproducibly transduced with high efficiency by self-complementary rAAV2 without inducing cytotoxicity or interfering with their differentiation potential. We further show the involvement of alpha(5)beta(1) integrin as a crucial AAV2 internalization receptor and a function for transcription-modulating drugs in enhancing rAAV-mediated transgene expression. This study represents a first step toward translation of a combined cellular/rAAV-based therapy of ischemic disease.
    Experimental hematology 05/2010; 38(9):707-17. · 3.11 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity. Molecular engineering was recently used to identify key immunogenic amino acid residues of the viral capsid and generate mutants with decreased antibody recognition. Here we explored the importance of finely tuning amino acid identity at immunogenic sites to optimize vector phenotype. A capsid library was generated by codon randomization at five positions where substitutions were shown to yield antibody evading phenotypes. Screening this library to isolate immune-escaping mutants allowed an exhaustive scan of combinations of the 20 natural amino acids at each position and yielded variants that remained infectious when incubated with serum or IVIG concentrations that completely neutralize AAV-2. Clones obtained replacing different residues at the same positions displayed strikingly different phenotypes, demonstrating that a precise choice of amino acid substitutions is fundamental to optimize immune-escaping, packaging ability, infectivity and tropism.
    Virology 02/2010; · 3.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site. Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent assay, whereas the transduction efficiency and specificity of receptor binding were assayed by gene transfer and competition experiments, respectively. Vector biodistribution was determined in mice by quantitative polymerase chain reaction analysis. Initially, RGD-4C, inserted at position 453, failed to efficiently bind its target receptor. R585 and R588, located at the neighboring peak and known to mediate primary receptor binding, were identified as interfering residues. R585A and R588A substitutions rendered position 453 mutants superior to those with the ligand in position 587 in target receptor binding and cell transduction efficiency. The in vivo biodistribution was independent of the insertion site, but directed by the inserted ligand when primary receptor binding was avoided. Position 453 emerged as a prominent site for the development of targeting mutants. Furthermore, we show for the first time that linearly distant residues can be critical for the efficiency of inserted peptide ligands.
    The Journal of Gene Medicine 09/2009; 11(12):1103-13. · 2.16 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. AAV serotypes 1-5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.
    Journal of dentistry 05/2009; 37(7):502-8. · 3.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: After attracting the attention of the scientific community due to a number of favourable characteristics that make it an attractive vector for human gene therapy [1,2], AAV has been thoroughly investigated in the past two decades. Standard technologies for the manipulation of the viral genome and for efficient packaging and purification protocols have paved the road for trial and error manipulation by educated guesses to study viral infectious biology by reverse genetics and to generate improved vectors for human gene transfer. However, despite remarkable progress, our limited knowledge of molecular mechanisms implicated in virus-cell interactions has been a limiting factor. Combinatorial engineering and high-throughput selection techniques hold the potential to boost technological improvement by offering the possibility to screen large numbers of randomly generated clones by appropriate selection protocols. These approaches not only require lesser knowledge of viral biology, but can also be employed as valuable tools to investigate molecular mechanisms that drive the infection process. In this review we recapitulate the rationale for employment of combinatorial methods in AAV vector development and the accomplishments achieved so far, discussing current limitations and interesting developments that are in sight.
    Combinatorial Chemistry & High Throughput Screening 03/2008; 11(2):118-26. · 2.00 Impact Factor