Arginase 1 (ARG1) deficiency (ARG1-D) is a rare genetic disorder due to loss of ARG1, the final enzyme in the urea cycle. ARG1-D hepatocytes are impaired in converting arginine into urea, resulting in elevated peripheral arginine and ammonia, which leads to progressive neurological symptoms. Current therapeutic strategies mainly focus on managing plasma arginine and ammonia level, but long-term outcomes remain poor. While no approved treatment specific for ARG1-D is available in the United States, a recombinant protein-based enzyme replacement therapy is available in Europe. Recently, extracellular vesicles (EVs) are emerging as a powerful therapeutic vehicle. By using Capricor's StealthX (TM) platform, EVs were engineered to express human ARG1 on their surface or encapsulated within. Regardless of their localization on the EV membrane, nanograms of ARG1 carried by EVs were biologically active and able to convert arginine into urea as potent as micrograms of human recombinant ARG1 (rHuArg1). Furthermore, ARG1-encapsulating EVs (STX-Arg1-in) were able to deliver ARG1 intracellularly but not EVs carrying ARG1 on their surface or rHuArg1. STX-Arg1-in EVs were further evaluated in a series of in vivo studies, and the results showed that STX-Arg1-in EVs were non-toxic and able to restore arginase activities in the liver of Arg1(-/-) mice, which led to a lowered plasma arginine concentration similar to that in wild-type mice. Most importantly, Arg1-in EVs expanded the lifespan of the lethal neonatal Arg1 deficiency mouse model. Taken together, our data suggested StealthX (TM)-engineered STX-Arg1-in EVs have a better safety profile due to the extremely low dosage and have great potential as a novel enzyme replacement strategy for patients suffering from ARG1-D. Significance statement: Intracellular delivery of recombinant protein and improved llifespanare endpoints of successful enzyme replacement therapy for the treatment of ARG1-D. Using the StealthX platform, a fully functional ARG1 enzyme was engineered to be carried inside of the extracellular vesicles, which allowed for the intracellular delivery of ARG1 protein in vitro and in vivo, with an improvement of lifespan in a lethal neonatal mouse model of Arg1 deficiency. More importantly, no toxicity was observed, and efficacy was achieved with a low dose, setting the base for an improved therapeutic approach.

Arginase 1 (ARG1) deficiency (ARG1-D) is a rare genetic disorder due to loss of ARG1, the final enzyme in the urea cycle. ARG1-D hepatocytes are impaired in converting arginine into urea, resulting in elevated peripheral arginine and ammonia, which leads to progressive neurological symptoms. Current therapeutic strategies mainly focus on managing plasma arginine and ammonia level, but long-term outcomes remain poor. While no approved treatment specific for ARG1-D is available in the United States, a recombinant protein based enzyme replacement therapy is available in Europe. Recently, extracellular vesicles (EVs) are emerging as a powerful therapeutic vehicle. By using Capricor’s StealthX™ platform, EVs were engineered to express human ARG1 on their surface or encapsulated within. Regardless of its localization on the EV membrane, nanograms of ARG1 carried by EVs were biologically active and able to convert arginine into urea as potent as micrograms of human recombinant ARG1 (rHuArg1). Furthermore, ARG1- encapsulating EVs (STX-Arg1-in) were able to deliver ARG1 intracellularly but not EVs carrying ARG1 on their surface or rHuArg1. STX-Arg1-in EVs were further evaluated in a series of in vivo studies and results showed that STX-Arg1-in EVs were non-toxic and able to restore the arginase activities in the liver of the Arg1-/- mice, which leads to a lowered plasma arginine concentration similar to wildtype mice. Most importantly, Arg1-in expanded the life span of the lethal neonatal Arg1 deficiency mouse model. Taken together, our data suggested StealthX™-engineered STX-Arg1-in EVs have a better safety profile due to extreme low dosage and had great potential as a novel enzyme replacement strategy for patients suffering from ARG1-D. Significance statement Intracellular delivery of recombinant protein and improved life span are endpoints of a successful enzyme replacement therapy for the treatment of ARG1-D. Using StealthX platform, a fully functional ARG1 enzyme was engineered to be carried inside of the extracellular vesicles, which allowed intracellular delivery of ARG1 protein in vitro and in vivo, with improvement of life span in a lethal neonatal mouse model of Arg1 deficiency. More importantly, no toxicity was observed, and efficacy was achieved with low dose, setting the base for an improved therapeutic approach. ### Competing Interest Statement All authors are employee of Capricor Therapeutics, Inc

Background: Endemic viruses are becoming increasingly the norm, and the development of a rapid and effective vaccine is necessary. Methods: Here, we used our StealthXTM exosome platform to express either Influenza H3 (StealthXTM-Hemagglutinin, STX-H3), SARS-CoV-2 Delta spike (StealthXTM-Spike, STX-S) or respiratory syncytial virus proteins (StealthXTM-RSV fusion protein, STX-RSV) protein on the membrane surface and facilitate their trafficking to the exosomes. Results: The administration of exosomes carrying one of the antigens by themselves resulted in a strong immune response with the production of a potent humoral and cellular immune response in mice. Interestingly, these effects were obtained with the administration of nanograms of protein and without adjuvant. We tested the possibility of manufacturing a multivalent vaccine by combining exosomes expressing either STX-H3, STX-RSV or STX-S exosomes in the same formulation, in a “mix and match” approach. Mice immunized with the cocktail vaccine showed an increased immune response against all three antigens received. Conclusions: The results further demonstrated that our STX trivalent cocktail vaccine elicited a strong immune response, and the magnitude of the responses was comparable to the single antigen administered individually. Our data show that our exosome platform has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, to tackle the fast-evolving viral infections.

Background Among the anthropoids, humans uniquely have apolipoprotein E (ApoE) isoforms that modulate Alzheimer's disease (AD) risk and accelerate aspects of brain aging. While chimpanzee and human ApoE4 share R112 and R158, the oldest chimps do not show symptoms of advanced AD. Another key structural difference is T61 in chimps instead of R61 found in humans predicted to be structurally similar to ApoE3.Objective Besides their impact on later life brain health, ApoE isoforms influence the development of brain regions relevant to AD. We explored the functional impact of ApoE isoforms produced by astrocytes on neuronal morphology and considered structural predictions for their differences.Methods Astrocyte conditioned media (ACM) was collected from primary astrocytes cultured from mice with targeted replacement of mouse ApoE with human ApoE3, ApoE4, or chimp. Neuron morphology was then examined in neonatal rat hippocampal neurons cultured in ACM. In vitro data was complemented by structural analysis of ApoE isoforms.Results ApoE-chimp ACM stimulated 30% more neurites per neuron than human ApoE ACM. In contrast, ACM from ApoE-chimp more closely resembled human ApoE4 than ApoE3, yielding 40% shorter neurites and spines. Structural modeling confirmed that chimpanzee ApoE differs from both ApoE4 than ApoE3, consistent with the predicted evolutionary trajectory.Conclusions Chimpanzee ApoE is structurally and functionally closer to ApoE4 than ApoE3 but still differs for neuronal development and protein folding. These findings provide insight into species-specific ApoE evolution, with implications for AD susceptibility and neuronal development.

Background:Among anthropoids, humans uniquely possess ApoE isoforms that modulate Alzheimer's disease (AD) risk and brain aging. While chimpanzee and human ApoE4 share R112 and R158, chimps do not exhibit advanced AD. A key difference is T61 in chimps versus R61 in humans, structurally resembling ApoE3. Objective:We examined how astrocyte-derived ApoE isoforms impact neuronal morphology and used structural modeling to explore functional divergence. Methods:Neonatal rat hippocampal neurons were cultured with astrocyte-conditioned media (ACM) from mice expressing human ApoE3, ApoE4, or chimpanzee ApoE. Neuronal outgrowth was quantified after 72 hours. Results:Chimpanzee ACM increased neurite number by 30% over human ApoE isoforms. However, chimpanzee ACM resembled ApoE4 functionally, producing 40% shorter neurites and spines. Structural modeling supported greater similarity to ApoE4. Conclusions:Chimpanzee ApoE is structurally and functionally more similar to ApoE4 than ApoE3, revealing evolutionary distinctions relevant to AD risk and neurodevelopment.

INTRODUCTION:Chronic air pollution (AirPoll) is associated with accelerated cognitive decline and risk of Alzheimer's disease (AD). Correspondingly, wild-type and AD-transgenic rodents exposed to AirPoll have increased amyloid peptides and behavioral impairments. METHODS:We examined the γ-secretase modulator GSM-15606 for potential AirPoll protection by its attenuating of amyloid beta (Aβ)42 peptide production. Male and female wild-type mice were fed GSM-15606 during an 8-week inhalation exposure to AirPoll subfractions, ambient nanoparticulate matter (nPM), and diesel exhaust particles (DEP). RESULTS:GSM-15606 decreased Aβ42 during nPM and DEP exposure without changing beta- or gamma-secretase activity or BACE1 and PS1 protein levels. DEP increased lateral ventricle volume by 25%. DISCUSSION:These enzyme responses are relevant to AD drug treatments, as well as to the physiological functions of the Aβ42 peptide. GSM-15606 attenuation of Aβ42 may benefit human exposure to AirPoll. HIGHLIGHTS:Gamma-secretase modulator (GSM-15606) attenuates the amyloidogenic amyloid beta (Aβ)42 peptide during exposure to air pollution, which may be a mechanism by which air pollution increases Alzheimer's disease (AD) risk. AD drug treatments may also consider Aβ homeostasis among the chronic effects of GSM-15606 and other amyloid reduction treatments on secretase enzymes.

Exosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor's StealthXTM platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+ and CD8+ T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthXTM exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements necessary for mRNA vaccines. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.

Background: Epidemiological studies have variably linked air pollution to increased risk of Parkinson’s disease (PD). However, there is little experimental evidence linking air pollution particles to PD-like neuropathology. Methods: Mice were exposed to three different batches of nano-particulate matter (nPM) (300 μg/m3, 5 h/d, 3 d/week), collected at different dates, 2017-2019, in the same urban site. After these experiments, these nPM batches were found to vary in activity. C57BL/6 female mice (3 mo) were injected with pre-formed murine α-synuclein fibrils (PFFs) (0.4 μg), which act as seeds for α-synuclein aggregation. Two exposure paradigms were used: Exposure 1, PFFs were injected into olfactory bulb (OB) prior to nPM exposure for 4 weeks, followed by 10 weeks normal housing condition before euthanasia; Exposure 2 and 3, mice were exposed to nPM for 3 weeks, then PFFs were injected in right OB, then mice were continuously exposed to nPM for another 7 weeks before euthanasia. α-Synuclein (α-syn) pSer129, microglia Iba1, and Gria1 (glutamate receptor A1) expression were measured by immunohistochemistry or qPCR.Results: As expected, α-syn pSer129 was detected in ipsilateral OB, anterior olfactory nucleus, amygdala and piriform cortex, but not in contralateral counterparts in both paradigms. None of the three batches of nPM changed α-syn pSer129 signal intensity or its distribution between brain regions. However, the combination of nPM and PFF significantly decreased Gria1 mRNA in both the ipsi- and contralateral OB and cortex for the most active two nPM batches. Neither nPM nor PFFs alone induced responses of microglia Iba1 and expression of Gria1 in the OB and cortex.Conclusion: Exposure to ambient nPM did not affect spread of α-syn pSer129 pathology in the brain, but when mice were exposed to both nPM and PFF injections, the expression of Gria1 was synergistically reduced in both OB and cortex.

ABSTRACT Endemic viruses are becoming increasingly the norm, and the development of a rapid and effective vaccine is emergent. Here, we used our StealthX ™ exosome platform to express either Influenza H3 (Stealth™ X-Hemagglutinin, STX-H3) or SARS-CoV-2 Delta spike (Stealth™ X-Spike, STX-S) protein on the surface and facilitate their trafficking to the exosomes. When administered as single product, both STX-H3 and STX-S induced a strong immunization with the production of a potent humoral and cellular immune response in mice. Interestingly, these effects were obtained with administration of nanograms of protein and without adjuvant. Therefore, we tested the possibility of a multivalent vaccine: STX-H3 and STX-S exosomes were formulated together in a “mix and match” approach and the immune response was further evaluated. We showed that our STX-H3+S cocktail vaccine is as effective as the single components administered separately, resulting in a strong antibody and T-cell response. Our data show that our exosome platform has an enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation, and for therapeutics by enabling cell and tissue specific targeting.

The pandemic emergency has brought to light the need for a new generation of rapidly developed vaccines that induce longer-lasting, potent, and broader immune responses. While the mRNA vaccines played a critical role during the emergency in reducing SARS-CoV-2 hospitalization rates and deaths, more efficient approaches are needed.

AD is associated with increases in lipid peroxidation and decreased antioxidant defense. Little is known about antioxidant defense of brain lipid rafts which are the site of APP processing. Oxidative damage to lipid rafts increases amyloid production. Lipid hydroperoxidases, glutathione peroxidase 4 (GPx4) and peroxiredoxin 6 (Prdx6) are potential detoxifying enzymes capable of reducing oxidized phospholipids and oxysterols within cellular membranes. We hypothesized that lipid hydroperoxidases would be reduced in ApoE4 carriers, which also have deficient cholesterol transport. To determine this, we investigated the status of the AD lipid raft by ApoE isoform in human frontal cortex. Lipid rafts were isolated (Invent Biotechnologies; LR-039) from postmortem AD (ApoE3,3, ApoE3,4, and ApoE4,4) and age-matched controls (ApoE3,3) and were analyzed by western blot and enzymatic assay. Lipid raft ApoE protein level of AD brains showed large decreases: 43% (Ctl vs AD3,3; p<0.01), 53% (Ctl vs AD3,4; p<0.001) and 70% (Ctl vs AD4,4; p<0.0001). Total cholesterol content of lipid rafts decreased 25% (Ctl vs AD; p<0.01). GPx4 protein of lipid rafts decreased 30% (Ctl vs AD3,3; p<0.01), 45% (Ctl vs AD3,4; p<0.0001) and 57% (Ctl vs AD4,4; p<0.001). AD brains from ApoE4,4 carriers had 36%(p<0.01) lower GPx4 localization than ApoE3,3. Prdx6 localization was not detected in the lipid rafts. Reductions in ApoE, total cholesterol, and GPx4 in the AD lipid raft suggests its vulnerability during AD. Furthermore, many of these decreases were ApoE isoform dependent, suggesting that antioxidant defense may differ by ApoE isoform.

Cerebral microbleeds (MBs) increase at later ages in association with increased cognitive decline and Alzheimer Disease (AD). MB prevalence is also increased by APOE4 and hypertension. In EFAD mice (5XFAD(+)(/-)/human APOE(+/+)), cerebral cortex MBs are most prevalent in E4 females at 6 months, paralleling plaque amyloid. We evaluated MBs at 2, 4, and 6 months in relation to amyloid in plaques and cerebral amyloid angiopathy (CAA) by age, sex, APOE allele, and blood pressure. At 2 mo, MBs were 50% more numerous than plaques, followed by decreased ratio of MBs:A beta plaques with female excess to 6 mo. The stable size of MBs suggests MBs arise as single events of extravasation, which may "seed" plaque formation. Blood pressure was normal from 2 to 6 months, minimizing a role of hypertension. Memory, assessed by fear conditioning, decreased with age in correlation with MBs and amyloid. Cortical layer analysis showed prevalent MBs and plaque in layers 4 and 5. Contrarily, CAA was prevalent in layers 1 and 2, discounting its contribution to MBs. (C) 2021 Elsevier Inc. All rights reserved.

BACKGROUND:Air pollution is widely associated with accelerated cognitive decline at later ages and risk of Alzheimer's disease (AD). Correspondingly, rodent models demonstrate the neurotoxicity of ambient air pollution and its components. Our studies with nano-sized particulate matter (nPM) from urban Los Angeles collected since 2009 have shown pro-amyloidogenic and pro-inflammatory responses. However, recent batches of nPM have diminished induction of the glutamate receptor GluA1 subunit, Iba1, TNFα, Aβ42 peptide, and white matter damage. The same methods, materials, and mouse genotypes were used throughout. OBJECTIVE:Expand the nPM batch comparisons and evaluate archived brain samples to identify the earliest change in nPM potency. METHODS:Batches of nPM were analyzed by in vitro cell assays for NF-κB and Nrf2 induction for comparison with in vivo responses of mouse brain regions from mice exposed to these batches, analyzed by PCR and western blot. RESULTS:Five older nPM batches (2009-2017) and four recent nPM batches (2018, 2019) for NF-κB and Nrf2 induction showed declines in nPM potency after 2017 that paralleled declines of in vivo activity from independent exposures in different years. CONCLUSION:Transcription-based in vitro assays of nPM corresponded to the loss of in vivo potency for inflammatory and oxidative responses. These recent decreases of nPM neurotoxicity give a rationale for evaluating possible benefits to the risk of dementia and stroke in Los Angeles populations.

Functional delivery of mRNA has high clinical potential. Previous studies established that mRNAs can be delivered to cells in vitro and in vivo via RNA-loaded lipid nanoparticles (LNPs). Here we describe an alternative approach using exosomes, the only biologically normal nanovesicle. In contrast to LNPs, which elicited pronounced cellular toxicity, exosomes had no adverse effects in vitro or in vivo at any dose tested. Moreover, mRNA-loaded exosomes were characterized by efficient mRNA encapsulation (∼90%), high mRNA content, consistent size, and a polydispersity index under 0.2. Using an mRNA encoding the red light-emitting luciferase Antares2, we observed that mRNA-loaded exosomes were superior to mRNA-loaded LNPs at delivering functional mRNA into human cells in vitro. Injection of Antares2 mRNA-loaded exosomes also led to strong light emission following injection into the vitreous fluid of the eye or into the tissue of skeletal muscle in mice. Furthermore, we show that repeated injection of Antares2 mRNA-loaded exosomes drove sustained luciferase expression across six injections spanning at least 10 weeks, without evidence of signal attenuation or adverse injection site responses. Consistent with these findings, we observed that exosomes loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins induced long-lasting cellular and humoral responses to both. Taken together, these results demonstrate that exosomes can be used to deliver functional mRNA to and into cells in vivo.

The neurotoxicity of air pollution is undefined for sex and APOE alleles. These major risk factors of Alzheimer's disease (AD) were examined in mice given chronic exposure to nPM, a nano-sized subfraction of urban air pollution. In the cerebral cortex, female mice had two-fold more genes responding to nPM than males. Transcriptomic responses to nPM had sex-APOE interactions in AD-relevant pathways. Only APOE3 mice responded to nPM in genes related to Abeta deposition and clearance (Vav2, Vav3, S1009a). Other responding genes included axonal guidance, inflammation (AMPK, NFKB, APK/JNK signaling), and antioxidant signaling (NRF2, HIF1A). Genes downstream of NFKB and NRF2 responded in opposite directions to nPM. Nrf2 knockdown in microglia augmented NFKB responses to nPM, suggesting a critical role of NRF2 in air pollution neurotoxicity. These findings give a rationale for epidemiologic studies of air pollution to consider sex interactions with APOE alleles and other AD-risk genes.