Cre Reporter Mouse Research Articles

Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

ISuRe-HadCre is an essential tool for effective conditional genetics.

Methods for modifying gene function at high spatiotemporal resolution in mice have revolutionized biomedical research, with Cre-loxP being the most widely used technology. However, the Cre-loxP technology has several drawbacks, including weak activity, leakiness, toxicity, and low reliability of existing Cre-reporters. This is mainly because different genes flanked by loxP sites (floxed) vary widely in their sensitivity to Cre-mediated recombination. Here, we report the generation, validation, and utility of iSuRe-HadCre, a new dual Cre-reporter and deleter mouse line that avoids these drawbacks. iSuRe-HadCre achieves this through a novel inducible dual-recombinase genetic cascade that ensures that cells expressing a fluorescent reporter had only transient Cre activity, that is nonetheless sufficient to effectively delete floxed genes. iSuRe-HadCre worked reliably in all cell types and for the 13 floxed genes tested. This new tool will enable the precise, efficient, and trustworthy analysis of gene function in entire mouse tissues or in single cells.

Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Characterizing enteric neurons in dopamine transporter (DAT)-Cre reporter mice reveals dopaminergic subtypes with dual-transmitter content.

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease.

Efficacy and specificity of melanopsin reporters for retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are specialized retinal output neurons that mediate behavioral, neuroendocrine, and developmental responses to environmental light. There are diverse molecular strategies for marking ipRGCs, especially in mice, making them among the best characterized retinal ganglion cells (RGCs). With the development of more sensitive reporters, new subtypes of ipRGCs have emerged. We therefore tested high-sensitivity reporter systems to see whether we could reveal yet more. Substantial confusion remains about which of the available methods, if any, label all and only ipRGCs. Here, we compared many different methods for labeling of ipRGCs, including anti-melanopsin immunofluorescence, Opn4-GFP BAC transgenic mice, and Opn4cre mice crossed with three different Cre-specific reporters (Z/EG, Ai9, and Ai14) or injected with Cre-dependent (DIO) AAV2. We show that Opn4cre mice, when crossed with sensitive Cre-reporter mice, label numerous ganglion cell types that lack intrinsic photosensitivity. Though other methods label ipRGCs specifically, they do not label the entire population of ipRGCs. We conclude that no existing method labels all and only ipRGCs. We assess the appropriateness of each reporter for particular applications and integrate findings across reporters to estimate that the overall abundance of ipRGCs among mouse RGCs may approach 11%.

Region-specific targeting of microglia in vivo using direct delivery of tamoxifen metabolites via microfluidic polymer fibers

Region-specific genetic manipulation of glial cells remains challenging due to the lack of anatomically selective transgenic models. Although local transduction is achievable with viral vectors, uniform recombination can be challenging in larger brain regions. We investigated the efficacy of intraparenchymal delivery of the tamoxifen metabolite endoxifen using inducible cre reporter mice. After observing localized reporter induction following stereotaxic injections of endoxifen in CX3CR1creERT2 mice, we carried out chronic delivery via osmotic pumps attached to bilateral cannulas made of stainless steel or microfluidic polymer fibers. Analysis of reporter expression in sections or iDISCO-cleared brains from TMEM119creERT2 mice revealed widespread induction following chronic infusion. Neuronal damage and gliosis were more prevalent around steel cannulas than polymer fibers, and glial reactivity was further attenuated when devices were implanted two months before drug delivery. In summary, region-specific recombination is achievable in glia with minimal tissue damage after endoxifen delivery via microfluidic polymer implants.

Characterization of fetal microchimeric immune cells in mouse maternal hearts during physiologic and pathologic pregnancies.

Introduction: During pregnancy, fetal cells can be incorporated into maternal tissues (fetal microchimerism), where they can persist postpartum. Whether these fetal cells are beneficial or detrimental to maternal health is unknown. This study aimed to characterize fetal microchimeric immune cells in the maternal heart during pregnancy and postpartum, and to identify differences in these fetal microchimeric subpopulations between normal and pregnancies complicated by spontaneous preterm induced by ascending infection. Methods: A Cre reporter mouse model, which when mated with wild-type C57BL/6J females resulted in cells and tissues of progeny expressing red fluorescent protein tandem dimer Tomato (mT+), was used to detect fetal microchimeric cells. On embryonic day (E)15, 104 colony-forming units (CFU) E. coli was administered intravaginally to mimic ascending infection, with delivery on or before E18.5 considered as preterm delivery. A subset of pregnant mice was sacrificed at E16 and postpartum day 28 to harvest maternal hearts. Heart tissues were processed for immunofluorescence microscopy and high-dimensional mass cytometry by time-of-flight (CyTOF) using an antibody panel of immune cell markers. Changes in cardiac physiologic parameters were measured up to 60days postpartum via two-dimensional echocardiography. Results: Intravaginal E. coli administration resulted in preterm delivery of live pups in 70% of the cases. mT + expressing cells were detected in maternal uterus and heart, implying that fetal cells can migrate to different maternal compartments. During ascending infection, more fetal antigen-presenting cells (APCs) and less fetal hematopoietic stem cells (HSCs) and fetal double-positive (DP) thymocytes were observed in maternal hearts at E16 compared to normal pregnancy. These HSCs were cleared while DP thymocytes persisted 28days postpartum following an ascending infection. No significant changes in cardiac physiologic parameters were observed postpartum except a trend in lowering the ejection fraction rate in preterm delivered mothers. Conclusion: Both normal pregnancy and ascending infection revealed distinct compositions of fetal microchimeric immune cells within the maternal heart, which could potentially influence the maternal cardiac microenvironment via (1) modulation of cardiac reverse modeling processes by fetal stem cells, and (2) differential responses to recognition of fetal APCs by maternal T cells.

In utero delivery of mRNA to the heart, diaphragm and muscle with lipid nanoparticles.

Nanoparticle-based drug delivery systems have the potential to revolutionize medicine, but their low vascular permeability and rapid clearance by phagocytic cells have limited their medical impact. Nanoparticles delivered at the in utero stage can overcome these key limitations due to the high rate of angiogenesis and cell division in fetal tissue and the under-developed immune system. However, very little is known about nanoparticle drug delivery at the fetal stage of development. In this report, using Ai9 CRE reporter mice, we demonstrate that lipid nanoparticle (LNP) mRNA complexes can deliver mRNA in utero, and can access and transfect major organs, such as the heart, the liver, kidneys, lungs and the gastrointestinal tract with remarkable efficiency and low toxicity. In addition, at 4 weeks after birth, we demonstrate that 50.99±5.05%, 36.62±3.42% and 23.7±3.21% of myofiber in the diaphragm, heart and skeletal muscle, respectively, were transfected. Finally, we show here that Cas9 mRNA and sgRNA complexed to LNPs were able to edit the fetal organs in utero. These experiments demonstrate the possibility of non-viral delivery of mRNA to organs outside of the liver in utero, which provides a promising strategy for treating a wide variety of devastating diseases before birth.

Targeted postnatal knockout of Sclerostin using a bone-targeted adeno-associated viral vector increases bone anabolism and decreases canalicular density.

The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice, this study used a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector. 8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5×1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens. The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22% increase in bone volume and 21% in trabecular bone fraction compared to controls; this translated to a 17% increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25% increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation. This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.

Generation of Pmel-dependent conditional and inducible Cre-driver mouse line for melanocytic-targeted gene manipulation.

Conditional and inducible gene targeting using Cre/loxP-mediated recombination is a powerful reverse genetics approach used to study spatiotemporal gene functions in specified cell types. To enable temporal gene manipulation in the melanocyte lineage, we established a novel inducible Cre-driver mouse line by targeting an all-in-one tetracycline/doxycycline (Dox)-inducible Cre expression cassette into the Pmel locus (PmelP2A-TetON3G-TRE3G-iCre ), a gene locus preferentially expressed in pigment cells. By crossing these Cre-driver mice with a strong Cre-reporter mouse line, Gt(ROSA)26Sortm9(CAG-tdTomato)Hze , we show the effectiveness of the PmelP2A-TetON3G-TRE3G-iCre mouse line in facilitating Dox-inducible Cre/loxP recombination in a wide variety of pigment cell lineages including hair follicle melanocytes and their stem cells. Furthermore, to demonstrate proof of concept, we ablated Notch signaling postnatally in the PmelP2A-TetON3G-TRE3G-iCre mice. In agreement with the previously reported phenotype, induced ablation of Notch signaling in the melanocyte lineage resulted in premature hair graying, demonstrating the utility of the PmelP2A-TetON3G-TRE3G-iCre allele. Therefore, the PmelP2A-TetON3G-TRE3G-iCre mouse line is suitable for assessing gene functions in melanocytes using an in vivo inducible reverse genetics approach. Furthermore, we unexpectedly identified previously unrecognized PMEL-expressing cells in non-pigmentary organs in the mice, suggesting unanticipated functions of PMEL other than melanosome formation.

Adeno-associated virus (AAV)-mediated Cre recombinase expression in melanopsin ganglion cells without leaky expression in rod/cone photoreceptors

BackgroundConstituting about 5 % of mouse retinal ganglion cells (RGCs), intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin (gene symbol Opn4) and drive such photoresponses as pupil constriction, melatonin suppression, and circadian photoentrainment. Opn4Cre mice with Cre recombinase-expressing ipRGCs have enabled genetic manipulation of ipRGCs; unfortunately, while Cre expression within the inner retina is ipRGC-specific, leaky expression also occurs in some outer retinal photoreceptors, so Cre-induced alterations in the latter cells may confound certain studies of ipRGC function. Methods that express Cre in ipRGCs but not rods or cones are needed.New Method: We have constructed a recombinant serotype-2 adeno-associated virus, rAAV2-Opn4-Cre, with the improved Cre recombinase (iCre) gene under the control of a ∼3kbp Opn4 promoter sequence, and injected it intravitreally into mice to transduce inner retinal neurons while sparing the outer retina. ResultsWe introduced rAAV2-Opn4-Cre into Cre reporter mice in which enhanced green fluorescent protein (EGFP) expression indicates Cre expression. Single-cell electrophysiological recordings and intracellular dye fills showed that 84 % of the EGFP+ cells were ipRGCs including M1–M6 types, while 16 % were conventional RGCs.Comparison with Existing Methods: Whereas Opn4Cre mice express Cre in some rod/cone photoreceptors, intravitreally applied rAAV2-Opn4-Cre induces Cre only in the inner retina, albeit with leaky expression in some conventional RGCs. ConclusionsrAAV2-Opn4-Cre has overcome a significant limitation of Opn4Cre mice. We recommend usage scenarios where the Cre-expressing conventional RGCs should not pose a problem.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice

ObjectiveInsulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. MethodsWe generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. ResultsRxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. ConclusionThese findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

The role of the aging microenvironment on the fate of PDGFRβ lineage cells in skeletal muscle repair

BackgroundDuring aging, perturbation of muscle progenitor cell (MPC) constituents leads to progressive loss of muscle mass and accumulation of adipose and fibrotic tissue. Mesenchymal stem cells (MSCs) give rise to adipocytes and fibroblasts that accumulate in injured and pathological skeletal muscle through constitutive activation of platelet-derived growth factor receptors (PDGFRs). Although the role of the PDGFRα has been widely explored, there is a paucity of evidence demonstrating the role of PDGFRβ in aged skeletal muscle.MethodsIn this study, we investigated the role of PDGFRβ lineage cells in skeletal muscle during aging by using Cre/loxP lineage tracing technology. The PDGFR-Cre mice were crossed with global double-fluorescent Cre reporter mice (mTmG) that indelibly marks PDGFRβ lineage cells. Those cells were analyzed and compared at different ages in the skeletal muscle of the mice.ResultsOur results demonstrated that PDGFRβ lineage cells isolated from the muscles of young mice are MPC-like cells that exhibited satellite cell morphology, expressed Pax7, and undergo myogenic differentiation producing myosin heavy chain expressing myotubes. Conversely, the PDGFRβ lineage cells isolated from muscles of old mice displayed MSC morphology with a reduced myogenic differentiation potential while expressing adipogenic and fibrotic differentiation markers. PDGFRβ lineage cells also gave rise to newly regenerated muscle fibers in young mice after muscle injury, but their muscle regenerative process is reduced in old mice.ConclusionsOur data suggest that PDGFRβ lineage cells function as MPCs in young mice, while the same PDGFRβ lineage cells from old mice undergo a fate switch participating in adipose and fibrotic tissue infiltration in aged muscle. The inhibition of fate-switching in PDGFRβ lineage cells may represent a potential approach to prevent fibrosis and fatty infiltration in skeletal muscle during the aging process.

Abstract P1019: CycleTrack , A Genetic Method To Trace Cardiomyocyte Renewal In Small And Large Animal Models

To what extent cardiac renewal occurs in adulthood and can be stimulated by therapeutic interventions is still a matter of investigation. In particular, over the last few years, significant effort has been made into awakening the endogenous regenerative potential of the adult mammalian heart after injury using different strategies. However, these studies are significantly hindered by the lack of tools to properly assess cardiomyocyte renewal over time. We have developed a genetic strategy, which we named CycleTrack, allowing a cumulative and accurate estimate of cardiomyocyte divisions in vivo. This method is based on the expression, in cardiomyocytes, of the Cre recombinase under the control of a 312-bp fragment of the Cyclin B2 promoter, which is specifically sensitive to cell proliferation. Replication-activated Cre acts on floxed GFP that is either present in the genome of transgenic mice or is exogenously delivered using Adeno-Associated Viral (AAV) vectors. As a result, cardiomyocytes traversing G2/M become irreversibly labeled. Using CycleTrack, we could monitor cardiomyocyte turnover in several physiological and pathological conditions. These included the measurement of the rate of proliferation of cardiomyocytes after apical resection in neonatal mice, the pro-regenerative effect of AAV9-mediated delivery of miRNA-199a and miRNA-590 after myocardial infarction in adult mice and the effect of pregnancy on myocardial hyperplasia. We also delivered the CycleTrack vectors into pig hearts to visualize cardiomyocyte turnover after myocardial infarction. Considering the large availability of Cre reporter mouse lines and the efficacy of AAVs to transfer genes into cardiomyocytes, we propose CycleTrack as a robust and straightforward tool to trace mitotic events for cardiac regeneration studies in small and large animal models.

Smooth muscle protein 22α-Cre recombination in resting cardiac fibroblasts and hematopoietic precursors

The Cre-loxP system has been widely used for cell- or organ-specific gene manipulation, but it is important to precisely understand what kind of cells the recombination takes place in. Smooth muscle 22α (SM22α)-Cre mice have been utilized to alter genes in vascular smooth muscle cells (VSMCs), activated fibroblasts or cardiomyocytes (CMs). Moreover, previous reports indicated that SM22α-Cre is expressed in adipocytes, platelets or myeloid cells. However, there have been no report of whether SM22α-Cre recombination takes place in nonCMs in hearts. Thus, we used the double-fluorescent Cre reporter mouse in which GFP is expressed when recombination occurs. Immunofluorescence analysis demonstrated that recombination occurred in resting cardiac fibroblasts (CFs) or macrophages, as well as VSMCs and CMs. Flow cytometry showed that some CFs, resident macrophages, neutrophils, T cells, and B cells were positive for GFP. These results prompted us to analyze bone marrow cells, and we observed GFP-positive hematopoietic precursor cells (HPCs). Taken together, these results indicated that SM22α-Cre-mediated recombination occurs in resting CFs and hematopoietic cell lineages, including HPCs, which is a cautionary point when using SM22α-Cre mice.

Mapping transgene insertion sites reveals the \u03b1-Cre transgene expression in both developing retina and olfactory neurons

The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα0+ olfactory neurons. Most α-Cre+ olfactory neurons do not express Pax6, implicating the influence of neighboring regulatory elements. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

Tetracycline response element driven Cre causes ectopic recombinase activity independent of transactivator element

ObjectiveTamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity. MethodsAdipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity. ResultsTamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass. ConclusionsThese results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles

Lipid Nanoparticles (LNPs) are a promising drug delivery vehicle for clinical siRNA delivery. Modified mRNA (modRNA) has recently gained great attention as a therapeutic molecule in cardiac regeneration. However, for mRNA to be functional, it must first reach the diseased myocardium, enter the target cell, escape from the endosomal compartment into the cytosol and be translated into a functional protein. However, it is unknown if LNPs can effectively deliver mRNA, which is much larger than siRNA, to the ischemic myocardium. Here, we evaluated the ability of LNPs to deliver mRNA to the myocardium upon ischemia-reperfusion injury functionally. By exploring the bio-distribution of fluorescently labeled LNPs, we observed that, upon reperfusion, LNPs accumulated in the infarct area of the heart. Subsequently, the functional delivery of modRNA was evaluated by the administration of firefly luciferase encoding modRNA. Concomitantly, a significant increase in firefly luciferase expression was observed in the heart upon myocardial reperfusion when compared to sham-operated animals. To characterize the targeted cells within the myocardium, we injected LNPs loaded with Cre modRNA into Cre-reporter mice. Upon LNP infusion, Tdtomato+ cells, derived from Cre mediated recombination, were observed in the infarct region as well as the epicardial layer upon LNP infusion. Within the infarct area, most targeted cells were cardiac fibroblasts but also some cardiomyocytes and macrophages were found. Although the expression levels were low compared to LNP-modRNA delivery into the liver, our data show the ability of LNPs to functionally deliver modRNA therapeutics to the damaged myocardium, which holds great promise for modRNA-based cardiac therapies.

Screening of Chemically Distinct Lipid Nanoparticles In Vivo Using DNA Barcoding Technology Towards Effectively Delivering Messenger RNA to Hematopoietic Stem and Progenitor Cells

Using adenine base editors, we aim to treat sickle cell disease by generating single nucleotide polymorphisms in human CD34+ hematopoietic stem and progenitor cells (HSPCs) at specific target sites by mediating A-T to G-C base conversions. While ex vivo gene editing approaches show great therapeutic promise, access is limited due to the requirement of an autologous hematopoietic stem cell (HSC) transplant to deliver the ex vivo edited cells. To further increase the number of patients eligible for base editing therapy, we are developing an alternative approach to directly deliver base editors to HSCs in vivo through non-viral delivery methods. Lipid Nanoparticles (LNPs) are a clinically validated, non-viral approach that enables the delivery of nucleic acid payloads, which may avoid the challenges associated with ex vivo approaches including the transplantation of edited CD34+ HSPCs.Here we describe the development and characterization of LNPs for the delivery of messenger RNA (mRNA) to HSPCs in vivo in both mice and cynomolgus macaques. By screening >1,000 chemically distinct LNPs in vivo utilizing a DNA barcoding technology, we identified several hit LNPs capable of biodistribution to HSPCs. Upon individual validation of these hit LNPs by delivery of Cre recombinase mRNA in a Cre-reporter mouse model (Ai14), which expresses the fluorescent protein tdTomato under a constitutive CAG promoter following Cre-meditate gene editing, we confirmed that several LNPs efficiently delivered Cre recombinase mRNA to mouse Lin-Sca-1+c-Kit+ (LSK) HSPCs. We next confirmed the most potent hit LNP (LNP-HSC1) identified from the in vivo screen to transfect LSK HSPCs in a dose-dependent manner between 0.1 and 1.0 mg/kg Cre recombinase mRNA, transfecting over 40% of LSK HSPCs in Ai14 mice at 1.0mg/kg. In a transfection durability study using Ai14 mice, we observed maintenance of tdTomato+ LSK HSPCs levels in the bone marrow at 10 weeks post-LNP delivery.As LNP-HSC1 had been identified and validated in mice of a C57BL6/j background, we next confirmed its ability to transfect a reporter mRNA into HSPCs in Balb/c mice and in 5 cynomolgus macaques. LNP-HSC1 efficiently transfected LSK HSPCs in Balb/c mice at doses ranging from 0.3 to 1.0 mg/kg. In 5 cynomolgus macaques (n=5 across two experiments), we observed a dose-dependent increase in reporter mRNA delivery with an average of 19% of bone marrow-derived CD34+ HSPCs (n=3) expressing the reporter protein at the highest dose tested.Taken together, these data demonstrate the value of our in vivo high-throughput LNP screening approach to identify novel LNPs capable of delivering to HSPCs, providing a promising delivery platform for an in vivo HSC gene editing approach for the treatment of hemoglobinopathies. DisclosuresSago: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Campbell: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lutz: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patwardhan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamilton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keating: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Murray: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Singh: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.