Periodic Reporting for period 3 - IBAF (Inhibiting BAF to Improve Gene Delivery)
Berichtszeitraum: 2023-11-01 bis 2025-04-30
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 a successful gene therapy, DNA has to reach in sufficient amounts the nucleus of target cells, 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 mediated by the protein barrier-to-autointegration factor (BAF) preventing their 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, which would boost the transfection efficiency of current and novel transfection systems. Our project aims to develop a potent gene delivery system that can overcome the critical hurdles in the transfection process, including BAF sequestration. We are addressing the BAF problem in two ways. First, by identifying small molecule inhibitors of BAF. The application of these together with any gene therapy could increase the number of DNA molecules transported from the cytoplasm to the nucleus. We developed a high-throughput assay to find inhibitors of BAF through the screening diversified compound libraries. Any identified BAF inhibitor would then be validated its ability to increase transfection. The second approach is the codelivering BAF-inhibiting enzymes with the DNA, using a protein-based transfection system. The enzymes are intended to protect the DNA and promote its nuclear uptake.
We have screened 30’000 compounds and detected one potential hit that inhibits the function of BAF in the assay. It is currently being assessed in cell culture experiments for its potential transfection enhancing effect. To increase our chances to find a suitable BAF inhibitor, we collaborate with the group of Dr. Scheuermann (ETH Zurich) who develops large DNA-encoded libraries of small molecules and peptide-like structures and obtained 6 binders. In the next step, we will test the successful candidates for their impact on cytoplasmic DNA clustering by BAF. We have also engineered a highly promising protein-based DNA transfection agent that is based on the human mitochondrial transcription factor A (TFAM). We showed that a mutated version of TFAM forms stable nanocomplexes (TFAMoplexes) with DNA and transfect cells under challenging conditions with negligible cytotoxicity and at low DNA concentrations (Burger et al., Adv. Sci. 2022, 9, 2104987). To improve the performance of the TFAMoplexes, we have been working on the bottlenecks of the transfection process. These are the stability of the complexes in body fluids, uptake into the target cell, endosomal escape and the mobility of the transfected DNA in the cytoplasm. Regarding the stability of the complexes, we developed a marker system based on split luciferase and split GFP, which allows us to track particle formation and stability. We further implemented tools to understand the DNA complexation processes and directly characterize the complex in relevant environments.
We performed mutational screenings of the TFAM protein to increase the transfection efficiency of the TFAMoplex. To this end, we have inserted single, double, or triple point mutations at specific positions in the TFAM sequence. The initial mutational screening did, however, not result in TFAM variants with significantly improved activities. Therefore, we added to TFAM a secondary DNA binding domain from the human transcription factor cAMP response element-binding protein (CREB). This resulted in an improvement of TFAMoplex transfection efficiency by 60%. Currently, we are performing confocal microscopy for DNA tracking experiments to better understand how the modified TFAM variants in complex with DNA act in the cell. We are further trying to increase the uptake of the TFAMoplexes into the target cells and tested various TFAM fusion proteins with human uptake factors in combination with specific uptake pathway inhibitors. We found that the TFAMoplex is most productively internalized by the cells via lipid raft mediated uptake pathways.
The original version of the TFAMoplex is relying on a potent bacterial phospholipase from Listeria monocytogenes that is used to facilitate the endosomal escape of the complex. Our goal is to replace this phospholipase with a human variant in order to reduce the potential immunogenicity and increase the safety of the system. We produced several distinct human phospholipases fused to TFAM and tested their potency in transfection assays. We have identified two human enzymes, which are able to trigger endosomal escape and result in TFAMoplex transfection. However, since the transfection efficiency of these enzymes is lower than the original bacterial phospholipase, we are currently evolving the human enzymes towards higher activity under the conditions encountered in the endosomal compartment. Further, we have strong indication, that the phospholipase must be released from the TFAMoplex in the endosome to reach the endosomal membrane and achieve efficient endosomal escape. We have developed a calcium sensitive release mechanism, which permits the detachment of phospholipase from the TFAMoplex in the low-calcium conditions of the early endosome. This system allowed to decrease the required phospholipase concentration by one order of magnitude.
We performed mutational screenings of the TFAM protein to increase the transfection efficiency of the TFAMoplex. To this end, we have inserted single, double, or triple point mutations at specific positions in the TFAM sequence. The initial mutational screening did, however, not result in TFAM variants with significantly improved activities. Therefore, we added to TFAM a secondary DNA binding domain from the human transcription factor cAMP response element-binding protein (CREB). This resulted in an improvement of TFAMoplex transfection efficiency by 60%. Currently, we are performing confocal microscopy for DNA tracking experiments to better understand how the modified TFAM variants in complex with DNA act in the cell. We are further trying to increase the uptake of the TFAMoplexes into the target cells and tested various TFAM fusion proteins with human uptake factors in combination with specific uptake pathway inhibitors. We found that the TFAMoplex is most productively internalized by the cells via lipid raft mediated uptake pathways.
The original version of the TFAMoplex is relying on a potent bacterial phospholipase from Listeria monocytogenes that is used to facilitate the endosomal escape of the complex. Our goal is to replace this phospholipase with a human variant in order to reduce the potential immunogenicity and increase the safety of the system. We produced several distinct human phospholipases fused to TFAM and tested their potency in transfection assays. We have identified two human enzymes, which are able to trigger endosomal escape and result in TFAMoplex transfection. However, since the transfection efficiency of these enzymes is lower than the original bacterial phospholipase, we are currently evolving the human enzymes towards higher activity under the conditions encountered in the endosomal compartment. Further, we have strong indication, that the phospholipase must be released from the TFAMoplex in the endosome to reach the endosomal membrane and achieve efficient endosomal escape. We have developed a calcium sensitive release mechanism, which permits the detachment of phospholipase from the TFAMoplex in the low-calcium conditions of the early endosome. This system allowed to decrease the required phospholipase concentration by one order of magnitude.
One of the main obstacles of DNA delivery is the BAF-dependent DNA retention in the cytoplasm. We tackle this problem with both, small molecule inhibitors, and with BAF inactivating kinases of human origin (VRK1), which we fused to TFAM. However, the incorporation of VRK1 into the TFAMoplex is not sufficient to prevent BAF/DNA clustering in the cytoplasm, indicating that most DNA that enters the cytoplasm cannot reach the nucleus. Until the end of the project, we want to answer the question why VRK1 is not sufficiently protecting the DNA on its journey through the cytoplasm. We are currently evolving the BAF kinase to become resistant against known cellular inhibitors of VRK1, such as the protein LEM4, and test the impact of the constitutively active VRK1 protein on cytoplasmic DNA clustering. Another reason for the lack of efficacy of VRK1 could be that the TFAMoplex quickly dissociates in the cytoplasm and the TFAM proteins (together with VRK1) are removed from the DNA. We are performing confocal imaging studies to understand the behaviour of the TFAMoplex inside the cell and to determine whether some of the TFAM proteins remain attached to the DNA in the cytoplasm. By modifying TFAM with additional high affinity DNA binding domains, we try to ensure that TFAM-VRK1 remains associated with the DNA to protect it from BAF. Once the BAF-dependent DNA retention in the cytoplasm can be mitigated, we expect to be able to drastically reduce the DNA required to achieve transfection in cell culture.