Interfacing gene delivery vehicles with biomaterials has the potential to play a key role in diversifying gene transfer capabilities, including localized, patterned, and controlled delivery. However, strategies for modifying biomaterials to interact with delivery vectors must be redesigned whenever new delivery vehicles and applications are explored. We have developed a vector-independent biomaterial platform capable of interacting with various adeno-associated viral (AAV) serotypes. A water-soluble, cysteine-tagged, recombinant protein version of the recently discovered multi-AAV serotype receptor (AAVR), referred to as cys-AAVR, was conjugated to maleimide-displaying polycaprolactone (PCL) materials using click chemistry. The resulting cys-AAVR-PCL system bound to a broad range of therapeutically relevant AAV serotypes, thereby providing a platform capable of modulating the delivery of all AAV serotypes. Intramuscular injection of cys-AAVR-PCL microspheres with bound AAV vectors resulted in localized and sustained gene delivery as well as reduced spread to off-target organs compared to a vector solution. This cys-AAVR-PCL system is thus an effective approach for biomaterial-based AAV gene delivery for a broad range of therapeutic applications.

BACKGROUND:Brain-computer interfaces (BCIs) have demonstrated the potential to provide paralyzed individuals with new means of communication, but an electroencephalography (EEG)-based endogenous BCI has never been successfully used for communication with a patient in a completely locked-in state (CLIS). METHODS:In this study, we investigated the possibility of using an EEG-based endogenous BCI paradigm for online binary communication by a patient in CLIS. A female patient in CLIS participated in this study. She had not communicated even with her family for more than one year with complete loss of motor function. Offline and online experiments were conducted to validate the feasibility of the proposed BCI system. In the offline experiment, we determined the best combination of mental tasks and the optimal classification strategy leading to the best performance. In the online experiment, we investigated whether our BCI system could be potentially used for real-time communication with the patient. RESULTS:An online classification accuracy of 87.5% was achieved when Riemannian geometry-based classification was applied to real-time EEG data recorded while the patient was performing one of two mental-imagery tasks for 5 s. CONCLUSIONS:Our results suggest that an EEG-based endogenous BCI has the potential to be used for online communication with a patient in CLIS.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by defective β-galactosylceramidase (GALC), a lysosomal enzyme responsible for cleavage of several key substrates including psychosine. Accumulation of psychosine to the cytotoxic levels in KD patients is thought to cause dysfunctions in myelinating glial cells based on a comprehensive study of demyelination in KD. However, recent evidence suggests myelin-independent neuronal death in the murine model of KD, thus indicating defective GALC in neurons as an autonomous mechanism for neuronal cell death in KD. These observations prompted us to generate induced neurons (iNeurons) from two adult-onset KD patients carrying compound heterozygous mutations (p.[K563*];[L634S]) and (p.[N228_S232delinsTP];[G286D]) to determine the direct contribution of autonomous neuronal toxicity to KD. Here we report that directly converted KD iNeurons showed not only diminished GALC activity and increased psychosine levels, as expected, but also neurite fragmentation and abnormal neuritic branching. The lysosomal-associated membrane proteins 1 (LAMP1) was expressed at higher levels than controls, LAMP1-positive vesicles were significantly enlarged and fragmented, and mitochondrial morphology and its function were altered in KD iNeurons. Strikingly, we demonstrated that psychosine was sufficient to induce neurite defects, mitochondrial fragmentation, and lysosomal alterations in iNeurons derived in healthy individuals, thus establishing the causal effect of the cytotoxic GALC substrate in KD and the autonomous neuronal toxicity in KD pathology.

Background

Mutations in the fused in sarcoma (FUS) gene have been linked to amyotrophic lateral sclerosis (ALS). ALS patients with FUS mutations exhibit neuronal cytoplasmic mislocalization of the mutant FUS protein. ALS patients' fibroblasts or induced pluripotent stem cell (iPSC)-derived neurons have been developed as models for understanding ALS-associated FUS (ALS-FUS) pathology; however, pathological neuronal signatures are not sufficiently present in the fibroblasts of patients, whereas the generation of iPSC-derived neurons from ALS patients requires relatively intricate procedures.

Results

Here, we report the generation of disease-specific induced neurons (iNeurons) from the fibroblasts of patients who carry three different FUS mutations that were recently identified by direct sequencing and multi-gene panel analysis. The mutations are located at the C-terminal nuclear localization signal (NLS) region of the protein (p.G504Wfs*12, p.R495*, p.Q519E): two de novo mutations in sporadic ALS and one in familial ALS case. Aberrant cytoplasmic mislocalization with nuclear clearance was detected in all patient-derived iNeurons, and oxidative stress further induced the accumulation of cytoplasmic FUS in cytoplasmic granules, thereby recapitulating neuronal pathological features identified in mutant FUS (p.G504Wfs*12)-autopsied ALS patient. Importantly, such FUS pathological hallmarks of the patient with the p.Q519E mutation were only detected in patient-derived iNeurons, which contrasts to predominant FUS (p.Q519E) in the nucleus of both the transfected cells and patient-derived fibroblasts.

Conclusions

Thus, iNeurons may provide a more reliable model for investigating FUS mutations with disrupted NLS for understanding FUS-associated proteinopathies in ALS.