Cilia are small microtubule-based structures found on the surface of most mammalian cells, which have key sensory and sometimes motile functions. Primary ciliary dyskinesia (PCD) is a type of ciliopathy caused by defects in motile cilia. The genetic basis of PCD is only partially understood. Studying a cohort of 11 human patients with PCD, we find that de novo mutations in TUBB4B , a beta tubulin isotype, cause three distinct classes of ciliopathic disease. In vivo studies in mice show that Tubb4b plays a specific role in cilia, building centrioles and axonemes in multiciliated cells. Examining the effects of specific TUBB4B variants in cells and in mice, we further demonstrate that distinct TUBB4B mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces. Importantly, these molecular differences correlate with disease features. We show that tubulin heterodimer-impairing TUBB4B variants underlie nonsyndromic PCD, whilst additional renal and sensorineural ciliopathic features in a syndromic PCD subtype arise from microtubule lumenal interface-impaired TUBB4B variants. These findings suggest that specific tubulin isotypes have distinct and non-redundant subcellular functions, and demonstrate that human tubulinopathies can be drivers of ciliopathic syndromes.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type– and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Introduction: CD38 is an ectoenzyme that was shown to mediate the age-dependent decrease in NAD + levels. We have shown that CD38 expression and enzymatic activity are increased in the brain of spontaneously hypertensive stroke-prone rats, a model of human cerebral small vessel disease. Therefore, we hypothesized that hypertension (HTN) induced by angiotensin II (ANG-II) infusion leads to an increase in CD38 expression and enzymatic activity. Methods: Male C57BL/6J mice (10-13) weeks of age underwent subcutaneous infusion of ANG-II (600 ng/kg/min) or saline for a 2-week duration through implanted osmotic pumps. CD38 expression was measured by immunofluorescence and Western Blotting. CD38 enzymatic activity was assessed in the brain homogenate by measuring the NAD + analogue conversion to the fluorescent product (ε-ADPR). Dihydroethidium, diaminofluorescein, JZL1707 sensor, and immunohistochemistry were used to measure the levels of superoxide, nitric oxide (NO), NAD(P)H, and endothelial nitric oxide synthase (eNOS) in the brain, respectively. Results: Ang-II infusion caused a slow rise of blood pressure leading to HTN. The brain of ANG-II infused mice showed 48% increase in the expression (P=0.013) and 73% increase in enzymatic activity (P=0.044) of CD38 compared to control mice. The expression of CD38 localized mainly to the endothelial cells in the ANG-II infused mice brains. The brains of ANG-II infused mice also had 26% lower levels of NAD(P)H (P<0.0001), 226% higher superoxide (P<0.0001), 32% lower NO (P<0.0001) and 30% lower eNOS levels (P=0.003) compared to control mice. Conclusions: ANG-II induced HTN increases CD38 expression and enzymatic activity with oxidative stress and lower levels of NAD(P)H. These results suggest a potential role of elevated CD38 enzymatic activity in the pathogenesis of cerebrovascular dysfunction induced by ANG-II as well as that seen in other models of HTN.

Acute respiratory distress syndrome (ARDS) represents a significant burden to the healthcare system, with ≈200 000 cases diagnosed annually in the USA. ARDS patients suffer from severe refractory hypoxemia, alveolar-capillary barrier dysfunction, impaired surfactant function, and abnormal upregulation of inflammatory pathways that lead to intensive care unit admission, prolonged hospitalization, and increased disability-adjusted life years. Currently, there is no cure or FDA-approved therapy for ARDS. This work describes the implementation of engineered extracellular vesicle (eEV)-based nanocarriers for targeted nonviral delivery of anti-inflammatory payloads to the inflamed/injured lung. The results show the ability of surfactant protein A (SPA)-functionalized IL-4- and IL-10-loaded eEVs to promote intrapulmonary retention and reduce inflammation, both in vitro and in vivo. Significant attenuation is observed in tissue damage, proinflammatory cytokine secretion, macrophage activation, influx of protein-rich fluid, and neutrophil infiltration into the alveolar space as early as 6 h post-eEVs treatment. Additionally, metabolomics analyses show that eEV treatment causes significant changes in the metabolic profile of inflamed lungs, driving the secretion of key anti-inflammatory metabolites. Altogether, these results establish the potential of eEVs derived from dermal fibroblasts to reduce inflammation, tissue damage, and the prevalence/progression of injury during ARDS via nonviral delivery of anti-inflammatory genes/transcripts.

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by PCM1. To study the requirement for centriolar satellites, we generated mice lacking PCM1. Pcm1 −/− mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, as well as variable expressivity of other ciliopathy features including cystic kidneys. Pcm1 −/− multiciliated ependymal cells and PCM1 −/− retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1 −/− RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromized early ciliogenesis. We show these molecular cascades are maintained in vivo , and we suggest that the cellular threshold to trigger ciliogenesis varies between cell types. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, inducing the departure of CP110 and CEP97 to initiate ciliogenesis.

Tumor-associated immune cells play a crucial role in cancer progression. Myeloid-derived suppressor cells (MDSCs), for example, are immature innate immune cells that infiltrate the tumor to exert immunosuppressive activity and protect cancer cells from the host's immune system and/or cancer-specific immunotherapies. While tumor-associated immune cells have emerged as a promising therapeutic target, efforts to counter immunosuppression within the tumor niche have been hampered by the lack of approaches that selectively target the immune cell compartment of the tumor, to effectively eliminate "tumor-protecting" immune cells and/or drive an "anti-tumor" phenotype. Here we report on a novel nanotechnology-based approach to target tumor-associated immune cells and promote "anti-tumor" responses in a murine model of breast cancer. Engineered extracellular vesicles (EVs) decorated with ICAM-1 ligands and loaded with miR-146a and Glut1, were biosynthesized (in vitro or in vivo) and administered to tumor-bearing mice once a week for up to 5 weeks. The impact of this treatment modality on the immune cell compartment and tumor progression was evaluated via RT-qPCR, flow cytometry, and histology. Our results indicate that weekly administration of the engineered EVs (i.e., ICAM-1-decorated and loaded with miR-146a and Glut1) hampered tumor progression compared to ICAM-1-decorated EVs with no cargo. Flow cytometry analyses of the tumors indicated a shift in the phenotype of the immune cell population toward a more pro-inflammatory state, which appeared to have facilitated the infiltration of tumor-targeting T cells, and was associated with a reduction in tumor size and decreased metastatic burden. Altogether, our results indicate that ICAM-1-decorated EVs could be a powerful platform nanotechnology for the deployment of immune cell-targeting therapies to solid tumors.

Gene/oligonucleotide therapies have emerged as a promising strategy for the treatment of different neurological conditions. However, current methodologies for the delivery of neurogenic/neurotrophic cargo to brain and nerve tissue are fraught with caveats, including reliance on viral vectors, potential toxicity, and immune/inflammatory responses. Moreover, delivery to the central nervous system is further compounded by the low permeability of the blood brain barrier. Extracellular vesicles (EVs) have emerged as promising delivery vehicles for neurogenic/neurotrophic therapies, overcoming many of the limitations mentioned above. However, the manufacturing processes used for therapeutic EVs remain poorly understood. Here, we conducted a detailed study of the manufacturing process of neurogenic EVs by characterizing the nature of cargo and surface decoration, as well as the transfer dynamics across donor cells, EVs, and recipient cells. Neurogenic EVs loaded with Ascl1, Brn2, and Myt1l (ABM) are found to show enhanced neuron-specific tropism, modulate electrophysiological activity in neuronal cultures, and drive pro-neurogenic conversions/reprogramming. Moreover, murine studies demonstrate that surface decoration with glutamate receptors appears to mediate enhanced EV delivery to the brain. Altogether, the results indicate that ABM-loaded designer EVs can be a promising platform nanotechnology to drive pro-neuronal responses, and that surface functionalization with glutamate receptors can facilitate the deployment of EVs to the brain.

Extracellular vesicles (EVs) have emerged as a promising carrier system for the delivery of therapeutic payloads in multiple disease models, including cancer. However, effective targeting of EVs to cancerous tissue remains a challenge. Here, it is shown that nonviral transfection of myeloid-derived suppressor cells (MDSCs) can be leveraged to drive targeted release of engineered EVs that can modulate transfer and overexpression of therapeutic anticancer genes in tumor cells and tissue. MDSCs are immature immune cells that exhibit enhanced tropism toward tumor tissue and play a role in modulating tumor progression. Current MDSC research has been mostly focused on mitigating immunosuppression in the tumor niche; however, the tumor homing abilities of these cells present untapped potential to deliver EV therapeutics directly to cancerous tissue. In vivo and ex vivo studies with murine models of breast cancer show that nonviral transfection of MDSCs does not hinder their ability to home to cancerous tissue. Moreover, transfected MDSCs can release engineered EVs and mediate antitumoral responses via paracrine signaling, including decreased invasion/metastatic activity and increased apoptosis/necrosis. Altogether, these findings indicate that MDSCs can be a powerful tool for the deployment of EV-based therapeutics to tumor tissue.

Ischemic stroke causes vascular and neuronal tissue deficiencies that could lead to substantial functional impairment and/or death. Although progenitor-based vasculogenic cell therapies have shown promise as a potential rescue strategy following ischemic stroke, current approaches face major hurdles. Here, we used fibroblasts nanotransfected with Etv2, Foxc2, and Fli1 (EFF) to drive reprogramming-based vasculogenesis, intracranially, as a potential therapy for ischemic stroke. Perfusion analyses suggest that intracranial delivery of EFF-nanotransfected fibroblasts led to a dose-dependent increase in perfusion 14 days after injection. MRI and behavioral tests revealed ~70% infarct resolution and up to ~90% motor recovery for mice treated with EFF-nanotransfected fibroblasts. Immunohistological analysis confirmed increases in vascularity and neuronal cellularity, as well as reduced glial scar formation in response to treatment with EFF-nanotransfected fibroblasts. Together, our results suggest that vasculogenic cell therapies based on nanotransfection-driven (i.e., nonviral) cellular reprogramming represent a promising strategy for the treatment of ischemic stroke.

While gene and cell therapies have emerged as promising treatment strategies for various neurological conditions, heavy reliance on viral vectors can hamper widespread clinical implementation. Here, the use of tissue nanotransfection as a platform nanotechnology to drive nonviral gene delivery to nerve tissue via nanochannels, in an effective, controlled, and benign manner is explored. TNT facilitates plasmid DNA delivery to the sciatic nerve of mice in a voltage-dependent manner. Compared to standard bulk electroporation (BEP), impairment in toe-spread and pinprick response is not caused by TNT, and has limited to no impact on electrophysiological parameters. BEP, however, induces significant nerve damage and increases macrophage immunoreactivity. TNT is subsequently used to deliver vasculogenic cell therapies to crushed nerves via delivery of reprogramming factor genes Etv2, Foxc2, and Fli1 (EFF). The results indicate the TNT-based delivery of EFF in a sciatic nerve crush model leads to increased vascularity, reduced macrophage infiltration, and improved recovery in electrophysiological parameters compared to crushed nerves that are TNT-treated with sham/empty plasmids. Altogether, the results indicate that TNT can be a powerful platform nanotechnology for localized nonviral gene delivery to nerve tissue, in vivo, and the deployment of reprogramming-based cell therapies for nerve repair/regeneration.

Advances in genome editing technologies have created opportunities to treat rare genetic diseases, which are often overlooked in terms of therapeutic development. Nonetheless, substantial challenges remain: namely, achieving therapeutically beneficial levels and kinds of editing in the right cell type(s). Here we describe the development of FIVER (fluorescent in vivo editing reporter) — a modular toolkit for in vivo detection of genome editing with distinct fluorescent read-outs for non-homologous end-joining (NHEJ), homology-directed repair (HDR) and homology-independent targeted integration (HITI). We demonstrate that fluorescent outcomes reliably report genetic changes following editing with diverse genome editors in primary cells, organoids and in vivo. We show the potential of FIVER for high-throughput unbiased screens, from small molecule modulators of genome editing outcomes in primary cells through to genome-wide in vivo CRISPR cancer screens. Importantly, we demonstrate its in vivo application in postnatal organ systems of interest for genetic therapies — retina and liver. FIVER will broadly help expedite the development of therapeutic genome surgery for many genetic disorders.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of ‘client’ proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.