mRNA Vaccine for Alzheimer’s Disease: Pilot Study

The escalating global healthcare challenge posed by Alzheimer's Disease (AD) and compounded by the lack of effective treatments emphasizes the urgent need for innovative approaches to combat this devastating disease. Currently, passive and active immunotherapies remain the most promising strategy for AD. FDA-approved lecanemab significantly reduces Aβ aggregates from the brains of early AD patients administered biweekly with this humanized monoclonal antibody. Although the clinical benefits noted in these trials have been modest, researchers have emphasized the importance of preventive immunotherapy. Importantly, data from immunotherapy studies have shown that antibody concentrations in the periphery of vaccinated people should be sufficient for targeting Aβ in the CNS. To generate relatively high concentrations of antibodies in vaccinated people at risk of AD, we generated a universal vaccine platform, MultiTEP, and, based on it, developed a DNA vaccine, AV-1959D, targeting pathological Aβ, completed IND enabling studies, and initiated a Phase I clinical trial with early AD volunteers. Our current pilot study combined our advanced MultiTEP technology with a novel mRNA approach to develop an mRNA vaccine encapsulated in lipid-based nanoparticles (LNPs), AV-1959LR. Here, we report our initial findings on the immunogenicity of 1959LR in mice and non-human primates, comparing it with the immunogenicity of its DNA counterpart, AV-1959D

Controlled Release of Stem Cell Secretome Attenuates Inflammatory Response against Implanted Biomaterials

Inflammatory response against implanted biomaterials impairs their functional integration and induces medical complications in the host's body. To suppress such immune responses, one approach is the administration of multiple drugs to halt inflammatory pathways. This challenges patient's adherence and can cause additional complications such as infection. Alternatively, biologics that regulate multiple inflammatory pathways are attractive agents in addressing the implants immune complications. Secretome of mesenchymal stromal cells (MSCs) is a multipotent biologic, regulating the homeostasis of lymphocytes and leukocytes. Here, it is reported that alginate microcapsules loaded with processed conditioned media (pCM-Alg) reduces the infiltration and/or expression of CD68+ macrophages likely through the controlled release of pCM. In vitro cultures revealed that alginate can dose dependently induce macrophages to secrete TNFα, IL-6, IL-1β, and GM-CSF. Addition of pCM to the cultures attenuates the secretion of TNFα (p = 0.023) and IL-6 (p < 0.0001) by alginate or lipopolysaccharide (LPS) stimulations. Mechanistically, pCM suppressed the NfκB pathway activation of macrophages in response to LPS (p < 0.0001) in vitro and cathepsin activity (p = 0.005) in response to alginate in vivo. These observations suggest the efficacy of using MSC-derived secretome to prevent or delay the host rejection of implants

Absence of microglia promotes diverse pathologies and early lethality in Alzheimer’s disease mice

Microglia are strongly implicated in the development and progression of Alzheimer's disease (AD), yet their impact on pathology and lifespan remains unclear. Here we utilize a CSF1R hypomorphic mouse to generate a model of AD that genetically lacks microglia. The resulting microglial-deficient mice exhibit a profound shift from parenchymal amyloid plaques to cerebral amyloid angiopathy (CAA), which is accompanied by numerous transcriptional changes, greatly increased brain calcification and hemorrhages, and premature lethality. Remarkably, a single injection of wild-type microglia into adult mice repopulates the microglial niche and prevents each of these pathological changes. Taken together, these results indicate the protective functions of microglia in reducing CAA, blood-brain barrier dysfunction, and brain calcification. To further understand the clinical implications of these findings, human AD tissue and iPSC-microglia were examined, providing evidence that microglia phagocytose calcium crystals, and this process is impaired by loss of the AD risk gene, TREM2

Engineering human induced pluripotent stem cells to enable microglial replacement therapy in the central nervous system

Ongoing studies have demonstrated that a high proportion of neurodegenerative risk genes are highly expressed by microglia, the resident immune cell of the central nervous system (CNS). Polymorphisms within many of these risk genes have further been shown to alter microglial response to neuropathology, impacting disease onset and progression. These findings highlight the importance of microglia in maintaining neural health and the pressing need to develop approaches to therapeutically target dysfunctional microglia in neurodegenerative disease. However, achieving brain-specific uptake of large therapeutic molecules remains largely elusive, limiting effective concentrations within the brain and often promoting peripheral side-effects. More invasive techniques including direct injection of peptides or viral gene therapy have also been examined. However, these methods often require multiple treatments for long-term therapeutic efficacy, provide limited diffusion beyond the injection site, and inefficiently target resident microglia. Progress in the development of immune cell therapies (ICTs) has begun to offer a promising approach to treating a variety of peripheral blood diseases including lymphoma, leukemia, and sickle cell disease. When coupled with advancements in gene editing techniques, they offer long-term therapeutic efficacy and potentially address many of the limitations of previously developed gene therapy approaches. Most recently, researchers have begun to propose the use of bone marrow-derived hematopoietic stem cell (HSC) transplantation as a source of peripherally derived macrophage populations to supplement or potentially replace diseased microglia. These populations have been shown to infiltrate and reside within the CNS following injury and degeneration and are often described as having adopted a ‘microglial’ or microglia-like’ identity. However, recent studies have shown that even after long-term CNS engraftment, these myeloid populations remain transcriptionally and functionally distinct from endogenous microglia. Furthermore, large scale infiltration and engraftment of peripherally derived macrophage populations requires toxic bone marrow preconditioning treatments, which have been shown to cause considerable patient mortality. As the resident immune cell of the brain, microglia stand out as the ideal candidate for ICT in the brain. They are long-lived, self-renew without contribution from the periphery, and can be readily differentiated from patient-derived induced pluripotent stem cells (iPSC). Studies have shown that iPSC-derived microglia (iMG) transcriptionally resemble ex vivo human microglia and respond to both injury and neuropathology when transplanted into xenotolerant mice. For this reason, iMG have proven to be an effective preclinical tool for the investigation of human microglia function and their role in disease progression. However, studies have also shown that human iMG transplanted into an adult murine brain engraft minimally and remain relatively near the injection site. This is due to the well-regulated “microglial niche” that manages microglia numbers and hampers donor microglial engraftment.The focus of this dissertation is to develop a microglia replacement strategy that allows for robust engraftment into an occupied “microglial niche” and to explore the therapeutic potential of iMG to prevent and/or reverse neurodegenerative disease. To this end, we engineered an inhibitor-resistant CSF1R that enables CNS-wide microglial replacement. CSF1R is necessary for microglia viability and is implicated in regulating microglia homeostasis and function. Therefore, inhibition of CSF1R has been shown to deplete microglia in vivo. We identified a single glycine to alanine substitution at position 795 of human CSF1R (G795A) that confers resistance to multiple CSF1R inhibitors (CSF1Ri), including PLX3397 and PLX5622, with no discernable gain or loss of function. Xenotransplantation studies show G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand under constant CSF1Ri treatment, replacing endogenous microglia to fully occupy the brain. Importantly, G795A-iMG remain in newly engrafted regions of the brain and return to a homeostatic state one month after cessation of CSF1Ri treatment. These findings, discussed in detail within Chapter 1, demonstrate a novel platform and robust approach to enable the investigation and future development of microglia replacement therapies with iMG. Transplantation of microglia could have therapeutic implications for a variety of neurological diseases. However, primary microgliopathies, a group of rare diseases that specifically result from microglial dysfunction, represent the most logical indication in which to first develop and test this novel approach. One example of a primary microgliopathies is Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). ALSP is a rare autosomal dominant neurodegenerative disease caused by mutations in CSF1R, a receptor that is critical for the development and survival of microglia. Thus far, the therapeutic potential of microglia replacement to treat ALSP has been limited by the unavailability of mouse models that recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. We therefore generated and examined a humanized xenotolerant mouse model lacking a conserved enhancer (fms-intronic regulatory element, FIRE) within the mouse Csf1r locus (hFIRE) that develops nearly all the hallmark pathologies including axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Remarkably, transplantation of human iMG progenitors restores a homeostatic microglial gene signature in hFIRE mice and prevents the development of each of these ALSP-related neuropathologies. To further examine a potential autologous approach, we generated and CRISPR-corrected ALSP-patient-derived iPSCs. We found genetic correction of CSF1R rescues mutation-induced deficits in microglial proliferation and enables brain-wide microglial engraftment. Surprisingly, transplantation of CSF1R-corrected iMG reduces pre-existing spheroid, astrogliosis, and calcification pathologies within just 6 weeks. These results, which are discussed in detail within Chapter 2, provide compelling evidence that transplantation of iMG could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders. For the treatment of neurodegenerative diseases other than primary microgliopathies, replacing dysfunctional microglia with CRISPR-corrected microglia may not suffice to achieve therapeutically favorable outcomes. However, our ability to deploy genetically modified iMG in patients could permit the delivery of therapeutic peptides previously prevented by the functional qualities of the blood brain barrier. Therefore, as proof of principle, we CRISPR-engineered iMG to produce neprilysin, a well-characterized beta-amyloid degrading protease, under the control of the endogenous CD9 promoter for transplantation into a xenotolerant, amyloid-accumulating transgenic mouse model of Alzheimer’s Disease. We demonstrate CD9, a plaque-associated microglia marker, is capable of driving membrane-bound neprilysin (NEP) and secreted neprilysin (sNEP) specifically in response to plaque deposition without off-target degradation of homeostatic neuropeptides bradykinin and somatostatin. Biochemical analysis of the transplanted hippocampus and overlying cortex reveals both NEP and sNEP iMG reduce levels of soluble and insoluble amyloid proteins while only sNEP iMG prevents synaptic degradation and significantly reduces levels of astrogliosis in vivo. Importantly, no significant differences in these levels were observed by transplantation of human WT iMG.To better understand if CNS-wide microglia replacement is necessary for a secreted payload like sNEP to achieve brain-wide therapeutic efficacy, sNEP-G795A iMG precursors were transplanted into a second cohort of mice treated with or without CSF1Ri. Remarkably, biochemical analysis of whole brain lysates reveals sNEP iMG without treatment not only significantly reduces amyloid protein levels, including Aβ oligomers, as effectively as CSF1Ri-treated mice, but also significantly prevents synaptic degradation, reduces levels of astrogliosis, lowers peripheral plasma NfL, and decreases levels of inflammatory markers in comparison to untreated mice. Taken together, these results, which are further discussed in Chapter 3, indicate iMG can be engineered ex vivo to produce and deliver therapeutics such as amyloid-targeting peptides in response to neuropathology and even achieve brain-wide therapeutically favorable outcomes with only partial microglial engraftment. In conclusion, these studies provide compelling evidence that iPSC-derived microglia provide an optimal platform to deliver immune cell therapies to the central nervous system. We show that iMG can be deployed to replace dysfunctional microglia in vivo, prevent the development of ALSP-associated neuropathologies, reverse already formed neuropathologies, and be genetically modified to enable brain-wide delivery of therapeutic peptides. These advancements address many of the limitations currently faced by peripheral delivery of therapeutic peptides to the brain and offer a novel approach for the treatment of rare neurodegenerative diseases, like ALSP, which currently lack any FDA-approved treatment

Cytoplasmic and Nuclear TAZ Exert Distinct Functions in Regulating Primed Pluripotency

Mouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of β-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic β-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of β-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization

CRISPR generation of CSF1R-G795A human microglia for robust microglia replacement in a chimeric mouse model

Summary: Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia.For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Plaque-associated human microglia accumulate lipid droplets in a chimeric model of Alzheimer's disease.

BackgroundDisease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer's disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown.MethodsTo determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed.ResultsHere we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE.ConclusionsAltogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity

TREM2 regulates purinergic receptor-mediated calcium signaling and motility in human iPSC-derived microglia.

The membrane protein TREM2 (Triggering Receptor Expressed on Myeloid cells 2) regulates key microglial functions including phagocytosis and chemotaxis. Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD). Because abnormalities in Ca2+ signaling have been observed in several AD models, we investigated TREM2 regulation of Ca2+ signaling in human induced pluripotent stem cell-derived microglia (iPSC-microglia) with genetic deletion of TREM2. We found that iPSC-microglia lacking TREM2 (TREM2 KO) show exaggerated Ca2+ signals in response to purinergic agonists, such as ADP, that shape microglial injury responses. This ADP hypersensitivity, driven by increased expression of P2Y12 and P2Y13 receptors, results in greater release of Ca2+ from the endoplasmic reticulum stores, which triggers sustained Ca2+ influx through Orai channels and alters cell motility in TREM2 KO microglia. Using iPSC-microglia expressing the genetically encoded Ca2+ probe, Salsa6f, we found that cytosolic Ca2+ tunes motility to a greater extent in TREM2 KO microglia. Despite showing greater overall displacement, TREM2 KO microglia exhibit reduced directional chemotaxis along ADP gradients. Accordingly, the chemotactic defect in TREM2 KO microglia was rescued by reducing cytosolic Ca2+ using a P2Y12 receptor antagonist. Our results show that loss of TREM2 confers a defect in microglial Ca2+ response to purinergic signals, suggesting a window of Ca2+ signaling for optimal microglial motility