Ligand-Modified Human Serum Albumin Nanoparticles for Enhanced Gene Delivery.

The development of nonviral gene delivery systems is a great challenge to enable safe gene therapy. In this study, ligand-modified nanoparticles based on human serum albumin (HSA) were developed and optimized for an efficient gene therapy. Different glutaraldehyde cross-linking degrees were investigated to optimize the HSA nanoparticles for gene delivery. The peptide sequence arginine-glycine-aspartate (RGD) and the HIV-1 transactivator of transduction sequence (Tat) are well-known as promising targeting ligands. Plasmid DNA loaded HSA nanoparticles were covalently modified on their surface with these different ligands. The transfection potential of the obtained plasmid DNA loaded RGD- and Tat-modified nanoparticles was investigated in vitro, and optimal incubation conditions for these preparations were studied. It turned out that Tat-modified HSA nanoparticles with the lowest cross-linking degree of 20% showed the highest transfection potential. Taken together, ligand-functionalized HSA nanoparticles represent promising tools for efficient and safe gene therapy.

New insights into stability of recombinant adenovirus vector genomes in mammalian cells.

Recombinant adenoviruses are widely used in basic virology research, therapeutic applications, vaccination studies or simply as a tool for genetic manipulation of eukaryotic cells. Dependent on the application, transient or stable maintenance of the adenoviral genome and transgene expression are required. The newest generation of recombinant adenoviral vectors is represented by high-capacity adenoviral vectors (HC-AdVs) which lack all viral coding sequences. HC-AdVs were shown to result in long-term persistence of transgene expression and phenotypic correction in small and large animal models with negligible toxicity. Although there is evidence that adenoviral vectors predominantly persist as episomal DNA molecules with a low integration frequency into the host genome, detailed information about the nuclear fate and the molecular status of the HC-AdV genome once inside the nucleus is lacking. In recent years we have focused on analyzing and modifying the nuclear fate of HC-AdVs after infection of mammalian cells. We have focused on investigating the molecular DNA forms of HC-AdV genomes and we have designed strategies to excise and stably integrate a transgene from an episomal adenovirus vector genome into the host chromosomes by recombinases. This review article provides a state-of-the art overview of the current knowledge of episomal HC-AdV persistence and it discusses strategies for changing the nuclear fate of a transgene inserted into the HC-AdV genome by somatic integration into host chromosomes.

Critical amino acid residues within the φC31 integrase DNA-binding domain affect recombination activities in mammalian cells.

The bacteriophage-derived ϕC31 integrase system represents an attractive tool for site-directed recombination in mammalian cells. Its integration reaction is based on recombination between the attachment site attB within an episomal substrate plasmid and either the bacteriophage-derived wild-type attachment site attP or pseudo-attP attachment sites (attP') present in the mammalian genome. In the present study we aimed at increasing the safety and efficiency of ϕC31 integrase-mediated recombination by mutating the DNA-binding domain located at the C terminus. Using an alanine mutagenesis approach, we generated 22 ϕC31 point mutants that were screened for activities in mammalian cells. Intramolecular excision assays based on recombination between attB and attP revealed five mutants with 2-fold enhanced excision activity. Importantly, we also identified mutants showing enhanced recombination activities between attB and three previously described attP' sites detected in the mammalian genome, indicating that there may be enhanced specificity for these hot spots. Several mutants showed, in mammalian cells, integration activities that increased in a cell line-dependent manner. The combination of beneficial mutations in addition to optimization of the integrase plasmid dose enhanced integration efficiencies up to 5.5-fold. We also identified three ϕC31 integrase mutants that were recombination defective in all applied assays, suggesting that these amino acid residues are essential for the functionality of ϕC31 integrase in mammalian cells. In summary, we identified critical amino acid residues within the ϕC31 DNA-binding domain. With respect to site-directed recombination and genome engineering these findings have important implications for improved ϕC31 protein design.

Pneumococcal interaction with human dendritic cells: phagocytosis, survival, and induced adaptive immune response are manipulated by PavA.

Dendritic cells (DCs) ingest and process bacteria for presenting their Ags to T cells. PavA (pneumococcal adherence and virulence factor A) is a key virulence determinant of pneumococci under in vivo conditions and was shown to modulate adherence of pneumococci to a variety of nonprofessional phagocytic host cells. Here, we demonstrated the role of PavA for the interaction of human DCs with live pneumococci and analyzed the induced host cell responses upon ingestion of viable pneumococci. Expression of PavA protected pneumococci against recognition and actin cytoskeleton-dependent phagocytosis by DCs compared with isogenic pavA mutants. A major proportion of internalized pneumococci were found in membrane-bound phagosomes. Pneumococcal phagocytosis promotes maturation of DCs, and both wild-type pneumococci and PavA-deficient pneumococci triggered production of proinflammatory cytokines such as IL-1beta, IL-6, IL-8, IL-12, and TNF-alpha and antiinflammatory IL-10. However, cytokine production was delayed and reduced when DCs encounter pneumococci lacking PavA, which also results in a less efficient activation of the adaptive immune response. Strikingly, purified PavA reassociates to pneumococci but not DCs and reduced phagocytosis of the pavA mutant to levels similar to those of wild-type pneumococci. Additionally, pavA mutants covered with exogenously provided PavA protein induced a DC cytokine profile similar to wild-type pneumococci. In conclusion, these results suggest that PavA is key factor for live pneumococci to escape phagocytosis and to induce optimal cytokine productions by DCs and adaptive immune responses as well.

Viral hybrid vectors for somatic integration - are they the better solution?

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.