Genetic disorders are found in about 2% of all live human births, and they account for up to 30% of pediatric hospital admissions and around 50% of childhood deaths in industrialized countries. Gene therapy has the potential to treat and even cure some of these diseases. For successful gene therapy, DNA has to reach the nucleus of target cells in sufficient amounts, which is a challenging endeavor.
In this project, we aim to address this problem by developing novel non-viral approaches that overcome the main hurdles in the delivery process, potentially leading to the development of more effective therapies against genetic disorders. At the intracellular level, DNA delivery is, among others, hindered by endo/lysosomal sequestration and a cytoplasmic DNA retention mechanism that is thought to be mediated by the protein barrier-to-autointegration factor (BAF), which prevents DNA transport into the nucleus. Consequently, a significant fraction of DNA is inactivated in the cytoplasm and therefore never reaches the nucleus.
Inhibiting BAF-dependent DNA retention could improve the ability of DNA to be transported into the nucleus, thereby boosting the transfection efficiency of current and novel transfection systems. Our project aims to develop a potent gene delivery system that can overcome critical hurdles in the transfection process, including BAF sequestration. We address the BAF problem in two ways.
First, we aimed at identifying small-molecule inhibitors of BAF. The application of these inhibitors together with gene therapy could increase the number of DNA molecules transported from the cytoplasm to the nucleus. We developed a high-throughput assay to identify inhibitors of BAF through the screening of diversified compound libraries. However, this strategy did not lead to the identification of compounds with cellular transfection activity.
The second approach is the co-delivery of BAF-inhibiting enzymes with DNA using a protein-based transfection system. The enzymes are intended to protect the DNA and promote its nuclear uptake. The developed and tested protein-based nanoparticulate systems efficiently transfected cells at very low concentrations under highly challenging conditions. For the optimized system, the efficacy was of the same order of magnitude as viral transduction. However, in dividing cells, the data suggest that BAF inhibition reduced transfection activity, highlighting the complex role of BAF in gene delivery.
