Spliceosome malfunction causes neurodevelopmental disorders with overlapping features.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Genomic profiling informs diagnoses and treatment in vascular anomalies.

Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies.

Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2­dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Treatment of severe Kaposiform lymphangiomatosis positive for NRAS mutation by MEK inhibition.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly involving most commonly the mediastinum, lung, skin and bones with few effective treatments. In recent years, RAS-MAPK pathway mutations were shown to underlie the pathogenesis of several complex lymphatic anomalies. Specifically, an activating NRAS mutation (p.Q61R) was found in the majority of KLA patients. Recent reports demonstrated promising results of treatment with the MEK inhibitor, Trametinib, in patients with complex lymphatic anomalies harboring gain of function mutations in ARAF and SOS1, as well as loss of function mutation in the CBL gene, a negative regulator of the RAS-MAPK pathway. We present a 9-year-old child with a severe case of KLA harboring the typical NRAS (p.Q61R) mutation detected by plasma-derived cell free DNA, responsive to trametinib therapy. METHODS: The NRAS somatic mutation was detected from plasma cfDNA using droplet digital PCR. Concurrent in-vitro studies of trametinib activity on mutant NRAS affected lymphatic endothelial cells were performed using a three-dimensional spheroid sprouting assay. RESULTS: Trametinib treatment lead to resolution of lifelong thrombocytopenia, improvement of pulmonary function tests and wellbeing, as well as weaning from prolonged systemic steroid treatment. Concurrent studies of mutant NRAS-expressing cells showed enhanced lymphangiogenic capacity along with over activation of the RAS-MAPK and PI3K-AKT-mTOR pathways, both reversed by trametinib. CONCLUSIONS: Trametinib treatment can substantially change the prognosis of patients with RAS pathway associated lymphatic anomalies. IMPACT: This is the first description of successful trametinib treatment of a patient with KLA harboring the most characteristic NRAS p.Q61R mutation. Treatment can significantly change the prognosis of patients with RAS pathway-associated lymphatic anomalies. We devised an in vitro model of KLA enabling a reproducible method for the continued study of disease pathogenesis. Mutated NRAS p.Q61R cells demonstrated increased lymphangiogenic capacity.

Experimental Single-Platform Approach to Enhance the Functionalization of Magnetically Targetable Cells.

Magnetic guidance shows promise as a strategy for improving the delivery and performance of cell therapeutics. However, clinical translation of magnetically guided cell therapy requires cell functionalization protocols that provide adequate magnetic properties in balance with unaltered cell viability and biological function. Existing methodologies for characterizing cells functionalized with magnetic nanoparticles (MNP) produce aggregate results, both distorted and unable to reflect variability in either magnetic or biological properties within a preparation. In the present study, we developed an inverted-plate assay allowing determination of these characteristics using a single-platform approach, and applied this method for a comparative analysis of two loading protocols providing highly uniform vs. uneven MNP distribution across cells. MNP uptake patterns remarkably different between the two protocols were first shown by fluorimetry carried out in a well-scan mode on endothelial cells (EC) loaded with BODIPY558/568-labeled MNP. Using the inverted-plate assay we next demonstrated that, in stark contrast to unevenly loaded cells, more than 50% of uniformly functionalized EC were captured within 5 min over a broad range of MNP doses. Furthermore, magnetically captured cells exhibited unaltered viability, substrate attachment, and proliferation rates. Conducted in parallel, magnetophoretic mobility studies corroborated the markedly superior guidance capacity of uniformly functionalized cells, confirming substantially faster cell capture kinetics on a clinically relevant time scale. Taken together, these results emphasize the importance of optimizing cell preparation protocols with regard to loading uniformity as key to efficient site-specific delivery, engraftment, and expansion of the functionalized cells, essential for both improving performance and facilitating translation of targeted cell therapeutics.

ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

Optimizing endothelial cell functionalization for cell therapy of vascular proliferative disease using a direct contact co-culture system.

Increased susceptibility to thrombosis, neoatherosclerosis, and restenosis due to incomplete regrowth of the protective endothelial layer remains a critical limitation of the interventional strategies currently used clinically to relieve atherosclerotic obstruction. Rapid recovery of endothelium holds promise for both preventing the thrombotic events and reducing post-angioplasty restenosis, providing the rationale for developing cell delivery strategies for accelerating arterial reendothelialization. The successful translation of experimental cell therapies into clinically viable treatment modalities for restoring vascular endothelium critically depends on identifying strategies for enhancing the functionality of endothelial cells (EC) derived from high cardiovascular risk patients, the target group for the majority of angioplasty procedures. Enhancing EC-associated nitric oxide (NO) synthesis by inducing overexpression of NO synthase (NOS) has shown promise as a way of increasing paracrine activity and restoring function of EC. In the present study, we developed a direct contact co-culture approach compatible with highly labile effectors, such as NO, and applied it for determining the effect of EC functionalization via NOS gene transfer on the growth of co-cultured arterial smooth muscle cells (A10 cell line) exhibiting the defining characteristics of neointimal cells. Bovine aortic endothelial cells magnetically transduced with inducible NOS-encoding adenovirus (Ad) formulated in zinc oleate-based magnetic nanoparticles (MNP[iNOSAd]) strongly suppressed growth of proliferating A10 and attenuated the stimulatory effect of a potent mitogen, platelet-derived growth factor (PDGF-BB), whereas EC functionalization with free iNOSAd or MNP formulated with a different isoform of the enzyme, endothelial NOS, was associated with lower levels of NO synthesis and less pronounced antiproliferative activity toward co-cultured A10 cells. These results show feasibility of applying magnetically facilitated gene transfer to potentiate therapeutically relevant effects of EC for targeted cell therapy of restenosis. The direct contact co-culture methodology provides a sensitive and reliable tool with potential utility for a variety of biomedical applications.

Development of a Dual-Functional Hydrogel Using RGD and Anti-VEGF Aptamer.

Synthetic molecular libraries hold great potential to advance the biomaterial development. However, little effort is made to integrate molecules with molecular recognition abilities selected from different libraries into a single biomolecular material. The purpose of this work is to incorporate peptides and nucleic acid aptamers into a porous hydrogel to develop a dual-functional biomaterial. The data show that an anti-integrin peptide can promote the attachment and growth of endothelial cells in a 3D porous poly(ethylene glycol) hydrogel and an antivascular endothelial growth factor aptamer can sequester and release VEGF of high bioactivity. Importantly, the dual-functional porous hydrogel enhances the growth and survival of endothelial cells. This work demonstrates that molecules selected from different synthetic libraries can be integrated into one system for the development of novel biomaterials.

Chimeric Aptamer-Gelatin Hydrogels as an Extracellular Matrix Mimic for Loading Cells and Growth Factors.

It is important to synthesize materials to recapitulate critical functions of biological systems for a variety of applications such as tissue engineering and regenerative medicine. The purpose of this study was to synthesize a chimeric hydrogel as a promising extracellular matrix (ECM) mimic using gelatin, a nucleic acid aptamer, and polyethylene glycol. This hydrogel had a macroporous structure that was highly permeable for fast molecular transport. Despite its high permeability, it could strongly sequester and sustainably release growth factors with high bioactivity. Notably, growth factors retained in the hydrogel could maintain ∼ 50% bioactivity during a 14-day release test. It also provided cells with effective binding sites, which led to high efficiency of cell loading into the macroporous hydrogel matrix. When cells and growth factors were coloaded into the chimeric hydrogel, living cells could still be observed by day 14 in a static serum-reduced culture condition. Thus, this chimeric aptamer-gelatin hydrogel constitutes a promising biomolecular ECM mimic for loading cells and growth factors.

Polymerization of affinity ligands on a surface for enhanced ligand display and cell binding.

Surfaces functionalized with affinity ligands have been widely studied for applications such as biological separations and cell regulation. While individual ligands can be directly conjugated onto a surface, it is often important to conjugate polyvalent ligands onto the surface to enhance ligand display. This study was aimed at exploring a method for surface functionalization via polymerization of affinity ligands, which was achieved through ligand hybridization with DNA polymers protruding from the surface. The surface with polyvalent ligands was evaluated via aptamer-mediated cell binding. The results show that this surface bound target cells more effectively than a surface directly functionalized with individual ligands in situations with either equal amounts of ligand display or equal amounts of surface reaction sites. Therefore, this study has demonstrated a new strategy for surface functionalization to enhance ligand display and cell binding. This strategy may find broad applications in settings where surface area is limited or the surface of a material does not possess sufficient reaction sites.

Aptamer-functionalized superporous hydrogels for sequestration and release of growth factors regulated via molecular recognition.

While the discovery of highly potent biologics has led to the development of promising therapies for various human diseases, biologics can cause severe toxicity if delivered inappropriately. Thus, great efforts have been made to synthesize polymeric systems for safe and efficient delivery of biologics. However, the application of polymeric delivery systems is often limited by problems such as harsh reaction conditions, low drug sequestration efficiency, and difficult drug release regulation. This study was aimed at developing a superporous material system with a hydrogel and an aptamer to overcome these challenges. The results have shown that the superporous hydrogel is capable of instantaneously and fully sequestering a large amount of growth factors, owing to the presence of superporous architectures and aptamers. Moreover, the sequestering and loading procedure does not involve any harsh conditions. The release kinetics of growth factors can be molecularly modulated by either changing the binding affinity of the aptamer or by using a triggering effector. Therefore, this study presents a promising superporous material for the delivery of highly potent biologics such as growth factors for clinical applications.

Programmable hydrogels for the controlled release of therapeutic nucleic acid aptamers via reversible DNA hybridization.

Reversible DNA hybridization can be used as a new mechanism to control the sustained and triggered release of therapeutic oligonucleotides from hydrogels.

Programmable hydrogels for controlled cell catch and release using hybridized aptamers and complementary sequences.

The ability to regulate cell-material interactions is important in various applications such as regenerative medicine and cell separation. This study successfully demonstrates that the binding states of cells on a hydrogel surface can be programmed by using hybridized aptamers and triggering complementary sequences (CSs). In the absence of the triggering CSs, the aptamers exhibit a stable, hybridized state in the hydrogel for cell-type-specific catch. In the presence of the triggering CSs, the aptamers are transformed into a new hybridized state that leads to the rapid dissociation of the aptamers from the hydrogel. As a result, the cells are released from the hydrogel. The entire procedure of cell catch and release during the transformation of the aptamers is biocompatible and does not involve any factor destructive to either the cells or the hydrogel. Thus, the programmable hydrogel is regenerable and can be applied to a new round of cell catch and release when needed.

Programmable release of multiple protein drugs from aptamer-functionalized hydrogels via nucleic acid hybridization.

Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer-protein interactions and aptamer-CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

Aptamer-based molecular recognition for biosensor development.

Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.