For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes’ synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or “proteoDNAsome”, which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

Loss of pancreatic beta cells is a central feature of type 1 (T1D) and type 2 (T2D) diabetes, but a therapeutic strategy to preserve beta cell mass remains to be established. Here we show that the death receptor TMEM219 is expressed on pancreatic beta cells and that signaling through its ligand insulin-like growth factor binding protein 3 (IGFBP3) leads to beta cell loss and dysfunction. Increased peripheral IGFBP3 was observed in established and at-risk T1D/T2D patients and was confirmed in T1D/T2D preclinical models, suggesting that dysfunctional IGFBP3/TMEM219 signaling is associated with abnormalities in beta cells homeostasis. In vitro and in vivo short-term IGFBP3/TMEM219 inhibition and TMEM219 genetic ablation preserved beta cells and prevented/delayed diabetes onset, while long-term IGFBP3/TMEM219 blockade allowed for beta cell expansion. Interestingly, in several patients’ cohorts restoration of appropriate IGFBP3 levels was associated with improved beta cell function. The IGFBP3/TMEM219 pathway is thus shown to be a physiological regulator of beta cell homeostasis and is also demonstrated to be disrupted in T1D/T2D. IGFBP3/TMEM219 targeting may therefore serve as a therapeutic option in diabetes.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure–activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor (HIF). The activity of this transcriptional regulator is mainly linked to the oxygen-dependent hydroxylation of conserved proline residues in its α subunit, carried out by Prolyl-Hydroxylases (PHDs), and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor (pVHL), which targets HIF-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis thaliana mesophyll protoplasts indicated that a combination of the yeast Gal4/UAS system and the mammalian oxygen sensor machinery can be effectively used to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device was also shown to be selectively controlled by oxygen in whole plants, when its components were stably expressed in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof-of-concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.