Gene transfer into human cord blood-derived CD34(+) cells by adeno-associated viral vectors.

Objective: 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.

Optimization of stealth adeno-associated virus vectors by randomization of immunogenic epitopes.

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.

Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations.

Background: 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.

Influence of lipopolysaccharide and interleukin-6 on RANKL and OPG expression and release in human periodontal ligament cells.

Recent research into periodontal disease pathology focuses on the role of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in periodontal bone destruction processes. RANKL regulates the differentiation of osteoclast by binding to its specific receptor RANK, while OPG inhibits the differentiation of osteoclasts by binding RANKL and therefore preventing RANKL to bind RANK. The aim of the present study was to investigate the influence of Porphyromonas gingivalis lipopolysaccharide (LPS) and interleukin-6 (IL-6) on RANKL and OPG expression and release in periodontal ligament (PDL) cells.

Efficient gene transfer to periodontal ligament cells and human gingival fibroblasts by adeno-associated virus vectors.

Objectives: 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.

Methods: 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.

Recent developments in adeno-associated virus vector technology.

Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery.

Artificial evolution with adeno-associated viral libraries.

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.

Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism.

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype.

Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus.

Background: Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles.

Methods: Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype.

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency.

Background: Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism.

AAV-based gene transfer.

Gene therapy remains an attractive form of treatment for a variety of diseases, both inherited and acquired. Recent experience in clinical gene therapy has highlighted important safety issues pertaining to gene delivery in humans. As such, the choice of gene delivery system for individual applications is fundamentally important and must afford efficiency and safety.

In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.

Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers.

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors.

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 x 10(11)/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection.