Rescue of auditory function by a single administration of AAV-TMPRSS3 gene therapy in aged mice of human recessive deafness DFNB8.

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knockin mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knockin mouse inner ear results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. A single AAV2-hTMPRSS3 injection in Tmprss3A306T/A306T mice of an average age of 18.5 months leads to sustained rescue of the auditory function to a level similar to wild-type mice. AAV2-hTMPRSS3 delivery rescues the hair cells and the spiral ganglions neurons. This study demonstrates successful gene therapy in an aged mouse model of human genetic deafness. It lays the foundation to develop AAV2-hTMPRSS3 gene therapy to treat DFNB8 patients, as a standalone therapy or in combination with cochlear implantation.

Muc4 loss mitigates epidermal growth factor receptor activity essential for PDAC tumorigenesis.

Mucin4 (MUC4) appears early during pancreatic intraepithelial neoplasia-1 (PanIN1), coinciding with the expression of epidermal growth factor receptor-1 (EGFR). The EGFR signaling is required for the onset of Kras-driven pancreatic ductal adenocarcinoma (PDAC); however, the players and mechanisms involved in sustained EGFR signaling in early PanIN lesions remain elusive. We generated a unique Esai-CRISPR-based Muc4 conditional knockout murine model to evaluate its effect on PDAC pathology. The Muc4 depletion in the autochthonous murine model carrying K-ras and p53 mutations (K-rasG12D; TP53R172H; Pdx-1cre, KPC) to generate the KPCM4-/- murine model showed a significant delay in the PanIN lesion formation with a significant reduction (p < 0.01) in EGFR (Y1068) and ERK1/2 (T202/Y204) phosphorylation. Further, a significant decrease (p < 0.01) in Sox9 expression in PanIN lesions of KPCM4-/- mice suggested the impairment of acinar-to-ductal metaplasia in Muc4-depleted cells. The biochemical analyses demonstrated that MUC4, through its juxtamembrane EGF-like domains, interacts with the EGFR ectodomain, and its cytoplasmic tail prevents EGFR ubiquitination and subsequent proteasomal degradation upon ligand stimulation, leading to sustained downstream oncogenic signaling. Targeting the MUC4 and EGFR interacting interface provides a promising strategy to improve the efficacy of EGFR-targeted therapies in PDAC and other MUC4-expressing malignancies.

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism.

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

CD4+ effector T cells accelerate Alzheimer's disease in mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aß) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. METHODS: In this report, we developed and characterized cloned lines of amyloid beta (Aß) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aß-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aß T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb-KLVFFAEDVGSNKGA) tetramer binding. Aß-Th1 and Aß-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. RESULTS: The propagated Aß-Th1 and Aß-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aß reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aß-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. CONCLUSIONS: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aß reactive Tregs.

Generation and characterization of a P2rx2 V60L mouse model for DFNA41.

P2RX2 encodes the P2X2 receptor, which is an adenosine triphosphate (ATP) gated (purinoreceptor) ion channel. P2RX2 c. 178G > T (p.V60L) mutation was previously identified in two unrelated Chinese families, as the cause of human DFNA41, a form of dominant, early-onset and progressive sensorineural hearing loss. We generated and characterized a knock-in mouse model based on human p.V60L mutation that recapitulates the human phenotype. Heterozygous KI mice started to exhibit hearing loss at 21-day-old and progressed to deafness by 6-month-old. Vestibular dysfunction was also observed in mutant mice. Abnormal morphology of the inner hair cells and ribbon synapses was progressively observed in KI animals suggesting that P2rx2 plays a role in the membrane spatial location of the ribbon synapses. These results suggest that P2rx2 is essential for acoustic information transfer, which can be the molecular mechanism related to hearing loss.

Distinct subtypes of proprioceptive dorsal root ganglion neurons regulate adaptive proprioception in mice.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Human genome-edited hematopoietic stem cells phenotypically correct Mucopolysaccharidosis type I.

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD.

Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.

Human-like NSG mouse glycoproteins sialylation pattern changes the phenotype of human lymphocytes and sensitivity to HIV-1 infection.

BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins' chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγc-/- (NSG) mouse strain, which is widely used for the transplantation of human cells, using the CRISPR/Cas9 system. The new strain provides a better environment for human immune cells. Transplantation of human hematopoietic stem cells leads to broad B cells repertoire, higher sensitivity to HIV-1 infection, and enhanced proliferation of transplanted peripheral blood lymphocytes. The mice showed no effect on the clearance of human immunoglobulins and enhanced transduction efficiency of recombinant adeno-associated viral vector rAAV2/DJ8. CONCLUSION: NSG-cmah-/- mice expand the mouse models suitable for human cells transplantation, and this new model has advantages in generating a human B cell repertoire. This strain is suitable to study different aspects of the human immune system development, provide advantages in patient-derived tissue and cell transplantation, and could allow studies of viral vectors and infectious agents that are sensitive to human-like sialylation of mouse glycoproteins.

Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

Cleavage by Caspase 8 and Mitochondrial Membrane Association Activate the BH3-only Protein Bid during TRAIL-induced Apoptosis.

The BH3-only protein Bid is known as a critical mediator of the mitochondrial pathway of apoptosis following death receptor activation. However, since full-length Bid possesses potent apoptotic activity, the role of a caspase-mediated Bid cleavage is not established in vivo In addition, due to the fact that multiple caspases cleave Bid at the same site in vitro, the identity of the Bid-cleaving caspase during death receptor signaling remains uncertain. Moreover, as Bid maintains its overall structure following its cleavage by caspase 8, it remains unclear how Bid is activated upon cleavage. Here, Bid-deficient (Bid KO) colon cancer cells were generated by gene editing, and were reconstituted with wild-type or mutants of Bid. While the loss of Bid blocked apoptosis following treatment by TNF-related apoptosis inducing ligand (TRAIL), this blockade was relieved by re-introduction of the wild-type Bid. In contrast, the caspase-resistant mutant Bid(D60E) and a BH3 defective mutant Bid(G94E) failed to restore TRAIL-induced apoptosis. By generating Bid/Bax/Bak-deficient (TKO) cells, we demonstrated that Bid is primarily cleaved by caspase 8, not by effector caspases, to give rise to truncated Bid (tBid) upon TRAIL treatment. Importantly, despite the presence of an intact BH3 domain, a tBid mutant lacking the mitochondrial targeting helices (α6 and α7) showed diminished apoptotic activity. Together, these results for the first time establish that cleavage by caspase 8 and the subsequent association with the outer mitochondrial membrane are two critical events that activate Bid during death receptor-mediated apoptosis.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function.

Generation of a Retinoblastoma (Rb)1-inducible dominant-negative (DN) mouse model.

Retinoblastoma 1 (Rb1) is an essential gene regulating cellular proliferation, differentiation, and homeostasis. To exert these functions, Rb1 is recruited and physically interacts with a growing variety of signaling pathways. While Rb1 does not appear to be ubiquitously expressed, its expression has been confirmed in a variety of hematopoietic and neuronal-derived cells, including the inner ear hair cells (HCs). Studies in transgenic mice demonstrate that complete germline or conditional Rb1 deletion leads to abnormal cell proliferation, followed by massive apoptosis; making it difficult to fully address Rb1's biochemical activities. To overcome these limitations, we developed a tetracycline-inducible TetO-CB-myc6-Rb1 (CBRb) mouse model to achieve transient and inducible dominant-negative (DN) inhibition of the endogenous RB1 protein. Our strategy involved fusing the Rb1 gene to the lysosomal protease pre-procathepsin B (CB), thus allowing for further routing of the DN-CBRb fusion protein and its interacting complexes for proteolytic degradation. Moreover, reversibility of the system is achieved upon suppression of doxycycline (Dox) administration. Preliminary characterization of DN-CBRb mice bred to a ubiquitous rtTA mouse line demonstrated a significant inhibition of the endogenous RB1 protein in the inner ear and in a number of other organs where RB1 is expressed. Examination of the postnatal (P) DN-CBRb mice inner ear at P10 and P28 showed the presence of supernumerary inner HCs (IHCs) in the lower turns of the cochleae, which corresponds to the described expression domain of the endogenous Rb1 gene. Selective and reversible suppression of gene expression is both an experimental tool for defining function and a potential means to medical therapy. Given the limitations associated with Rb1-null mice lethality, this model provides a valuable resource for understanding RB1 activity, relative contribution to HC regeneration and its potential therapeutic application.

The notch regulator MAML1 interacts with p53 and functions as a coactivator.

Members of the evolutionarily conserved Mastermind (MAM) protein family, including the three related mammalian Mastermind-like (MAML) proteins MAML1-3, function as crucial coactivators of Notch-mediated transcriptional activation. Given the recent evidence of cross-talk between the p53 and Notch signal transduction pathways, we have investigated whether MAML1 may also be a transcriptional coactivator of p53. Indeed, we show here that MAML1 is able to interact with p53. We show that MAML1-p53 interaction involves the N-terminal region of MAML1 and the DNA-binding domain of p53, and we use a chromatin immunoprecipitation assay to show that MAML1 is part of the activator complex that binds to native p53-response elements within the promoter of the p53 target genes. Overexpression of wild-type MAML1 as well as a mutant, defective in Notch signaling, enhanced the p53-dependent gene induction in mammalian cells, whereas MAML1 knockdown reduced the p53-dependent gene expression. MAML1 increases the half-life of p53 protein and enhances its phosphorylation/acetylation upon DNA damage of cells. Finally, RNA interference-mediated knockdown of the single Caenorhabditis elegans MAML homolog, Lag-3, led to substantial abrogation of p53-mediated germ-cell apoptotic response to DNA damage and markedly reduced the expression of Ced-13 and Egl-1, downstream pro-apoptotic targets of the C. elegans p53 homolog Cep-1. Thus, we present evidence for a novel coactivator function of MAML1 for p53, independent of its function as a coactivator of Notch signaling pathway.

The human orthologue of Drosophila ecdysoneless protein interacts with p53 and regulates its function.

Biochemical mechanisms that control the levels and function of key tumor suppressor proteins are of great interest as their alterations can lead to oncogenic transformation. Here, we identify the human orthologue of Drosophila melanogaster ecdysoneless (hEcd) as a novel p53-interacting protein. Overexpression of hEcd increases the levels of p53 and enhances p53 target gene transcription whereas hEcd knockdown has the opposite effects on p53 levels and target gene expression. Furthermore, hEcd interacts with murine double minute-2 and stabilizes p53 by inhibiting murine double minute-2-mediated degradation of p53. Thus, hEcd protein represents a novel regulator of p53 stability and function. Our studies also represent the first demonstration of a biochemical function for hEcd protein and raise the possibility that altered hEcd levels and/or function may contribute to oncogenesis.