Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

Human Pluripotent Stem Cells as In Vitro Models of Neurodegenerative Diseases.

My main research focused in the last years has been the reprogramming of differentiated cell types, such as human fibroblasts, into pluripotent stem cells called induced pluripotent stem cells (iPSCs) and the application of this technology to studies of the nervous system and the diseases that affect it. We have been working on the generation of iPSC lines from Alzheimer's disease (AD) patients using recent developments in reprogramming strategies such as non-integrating episomal vectors to produce virus-free, clinical safe hiPSC. Our study shows that neurons differentiated from these cells display important disease properties and, thus, have the potential to serve as cellular models to explore various aspects of Alzheimer's pathogenesis. One of the lab's scientific goal is to use lines of familial Alzheimer's disease (FAD)-derived induced pluripotent stem cells (iPSCs) to generate brain-like structures ("organoids") mimicking native brains. Three-dimensional (3D) systems, called cerebral organoids, can recapitulate distinct architectures of the human brain, such as fluid-filled cavities resembling brain ventricles and tissues organized in layers including progenitor ventricular and subventricular zones present in the native brain. Recently, we have extended our research interests in the rapidly emerging field of exosomes and micro-vesicles (called as EMVs). Extracellular vesicles of either 50-200 nm in size (called exosomes) or 200 nm-1 µm in size (called micro-vesicles) are membrane-bounded vesicles that can carry RNAs, proteins, and other metabolites and are secreted from all cell types and are present in biological fluids such as serum and plasma. We have examined properties and functions of EMVs from human iPSCs that can be cultured infinitely under a chemically defined medium and compared them with the ones secreted by human mesenchymal stem cells (MSCs). Purified EVs produced by both stem cell types have similar sizes, but human iPSCs produced 16-fold more EVs than MSCs. When iPSC-EMVs were applied in culture to senescent MSCs, they reduced their elevated cellular ROS levels and alleviated aging phenotypes. We are currently exploring the potential application of EMVs in diagnostics, pathology, and therapeutics of AD. Extracellular vesicles secreted from AD patient derived neurons contain a relatively low amount of Aß but have an increased Aß42/ Aß40 ratio; the majority of Aß is located on the surface of the EVs. The results of our research can contribute substantially to the successful translation of stem cell biology into clinical therapy by improving our understanding of the pathogenesis and treatment of Alzheimer's disease.

Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.

Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.

Extracellular Vesicle-Associated Aß Mediates Trans-Neuronal Bioenergetic and Ca2+-Handling Deficits in Alzheimer's Disease Models.

Alzheimer's Disease (AD) is an age-related neurodegenerative disorder in which aggregation-prone neurotoxic amyloid ß-peptide (Aß) accumulates in the brain. Extracellular vesicles (EVs) are small 50-150 nanometer membrane vesicles that have recently been implicated in the prion-like spread of self-aggregating proteins. Here we report that EVs isolated from AD patient CSF and plasma, from the plasma of two AD mouse models, and from the medium of neural cells expressing familial AD presenilin 1 mutations, destabilize neuronal Ca2+ homeostasis, impair mitochondrial function, and sensitize neurons to excitotoxicity. EVs contain a relatively low amount of Aß but have an increased Aß42/ Aß40 ratio; the majority of Aß is located on the surface of the EVs. Impairment of lysosome function results in increased generation EVs with elevated Aß42 levels. EVs may mediate transcellular spread of pathogenic Aß species and that impair neuronal Ca2+ handling and mitochondrial function, and may thereby render neurons vulnerable to excitotoxicity.