Preclinical Therapeutic Testing Research Articles

P258 Development of a liquid polymicrobial biofilm model for preclinical testing of novel cystic fibrosis therapeutics

P258 Development of a liquid polymicrobial biofilm model for preclinical testing of novel cystic fibrosis therapeutics

Advances in Gluten Hypersensitivity: Novel Dietary-Based Therapeutics in Research and Development.

Gluten hypersensitivity is characterized by the production of IgE antibodies against specific wheat proteins (allergens) and a myriad of clinical allergic symptoms including life-threatening anaphylaxis. Currently, the only recommended treatment for gluten hypersensitivity is the complete avoidance of gluten. There have been extensive efforts to develop dietary-based novel therapeutics for combating this disorder. There were four objectives for this study: (i) to compile the current understanding of the mechanism of gluten hypersensitivity; (ii) to critically evaluate the outcome from preclinical testing of novel therapeutics in animal models; (iii) to determine the potential of novel dietary-based therapeutic approaches under development in humans; and (iv) to synthesize the outcomes from these studies and identify the gaps in research to inform future translational research. We used Google Scholar and PubMed databases with appropriate keywords to retrieve published papers. All material was thoroughly checked to obtain the relevant data to address the objectives. Our findings collectively demonstrate that there are at least five promising dietary-based therapeutic approaches for mitigating gluten hypersensitivity in development. Of these, two have advanced to a limited human clinical trial, and the others are at the preclinical testing level. Further translational research is expected to offer novel dietary-based therapeutic options for patients with gluten hypersensitivity in the future.

Development of Orthotopic Patient-Derived Xenograft Models of Pediatric Intracranial Tumors.

The development of clinically relevant and reliable models of central nervous system tumors has been instrumental in advancing the field of Neuro-Oncology. The orthotopic intracranial injection is widely used to study the growth, invasion, and spread of tumors in a controlled environment. Orthotopic models are performed to examine tumor cells isolated from a specific region in a patient in the same site or location in an animal model. Orthotopic brain tumor models are also utilized for preclinical testing of therapeutics as they closely recapitulate the behavior of such cancer and the brain environment of patients. Below, we describe our experiences in the development of murine models of pediatric brain tumors including diffuse midline glioma (DMG), glioblastoma (GBM), and medulloblastoma. The method provides an overview of intracranial stereotactic injections in mice.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

A novel panel of Drosophila TAFAZZIN mutants in distinct genetic backgrounds as a resource for therapeutic testing.

Barth Syndrome is a rare, X-linked disorder caused by mutation of the gene TAFAZZIN (TAZ). The corresponding Tafazzin protein is involved in the remodeling of cardiolipin, a phospholipid with critical roles in mitochondrial function. While recent clinical trials have been promising, there is still no cure for Barth Syndrome. Because TAZ is highly conserved, multiple animal and cell culture models exist for pre-clinical testing of therapeutics. However, since the same mutation in different patients can lead to different symptoms and responses to treatment, isogenized experimental models can't fully account for human disease conditions. On the other hand, isogenized animal models allow for sufficient numbers to thoroughly establish efficacy for a given genetic background. Therefore, a combined method for testing treatments in a panel of isogenized cohorts that are genetically distinct from each other would be transformative for testing emerging pre-clinical therapies. To aid in this effort, we've created a novel panel of 10 Drosophila lines, each with the same TAZ mutation in highly diverse genetic backgrounds, to serve as a helpful resource to represent natural variation in background genetics in pre-clinical studies. As a proof of principle, we test our panel here using nicotinamide riboside (NR), a treatment with established therapeutic value, to evaluate how robust this therapy is across the 10 genetic backgrounds in this novel reference panel. We find substantial variation in the response to NR across backgrounds. We expect this resource will be valuable in pre-clinical testing of emerging therapies for Barth Syndrome.

Aqueous Humor TGF-β2 and Its Association With Intraocular Pressure in a Naturally Occurring Large Animal Model of Glaucoma.

Transforming growth factor (TGF)-β2 has been widely implicated in human glaucoma pathology. The purpose of this study was to determine the source of TGF-β2 in aqueous humor (AH) and its relationship with intraocular pressure (IOP) in an inherited large animal model of glaucoma. Sixty-six glaucomatous cats homozygous for LTBP2 mutation, and 42 normal cats were studied. IOP was measured weekly by rebound tonometry. AH was collected by anterior chamber paracentesis from each eye under general anesthesia, and serum samples collected from venous blood concurrently. Concentrations of total, active and latent TGF-β2 in AH and serum samples were measured by quantitative sandwich immunoassay. For comparisons between groups, unpaired t-test or Mann Whitney test were used, with P < 0.05 considered significant. The relationships between TGF-β2 concentrations and IOP values were examined by Pearson's correlation coefficient and generalized estimating equation. IOP and AH TGF-β2 concentrations were significantly higher in glaucomatous than in normal cats. AH TGF-β2 showed a significant, robust positive correlation with IOP in glaucomatous cats (r = 0.83, R2 = 0.70, P < 0.0001). Serum TGF-β2 did not correlate with AH TGF-β2 and was not significantly different between groups. TGF-β2 mRNA and protein expression were significantly increased in local ocular tissues in glaucomatous cats. Enhanced, local ocular production of TGF-β2 with a robust positive association with IOP was identified in this spontaneous feline glaucoma model, providing a foundation for preclinical testing of novel therapeutics to limit disease-associated AH TGF-β2 elevation and signaling in glaucoma.

Feasibility of probe washing after stereotactic needle biopsy as a novel technique for developing cell lines and xenografts of H3 K27-altered diffuse midline gliomas.

H3 K27-altered diffuse midline gliomas (DMGs) are frequently biopsied to obtain tissue diagnosis, inform clinical decision-making, and determine clinical trial eligibility. Tissue yield from biopsies is typically low, leaving little material available for research. To advance understanding of disease biology and promote preclinical testing of novel therapeutics, collecting viable cellular material from treatment-naive tumors is of paramount importance. Here, the authors report the feasibility of a practicable technique for creating DMG cell lines and patient-derived xenografts (PDXs) without the need for additional biopsy specimens. Tumor cells are obtained by probe washing immediately after completion of biopsy. Wash fluid is collected, and viable cells are expanded in vitro. Cultured cells are used to establish PDX rodent models. A total of 5 patient samples were collected by this technique. Viable tumor cells were obtained from 3 of the 5 samples, and cell lines suitable for experiments were obtained within 6-8 months. Orthotopic implantation and flank engraftment was successful in 1 of the 3 established cell lines. Animals harboring intracranial tumors were euthanized due to disease burden 6-7 months after stereotactic injection. Flank tumors formed within 4-5 months and were serially passaged. Molecular and tissue analyses confirmed retention of H3 K27M expression and loss of H3 K27me3 in all cell lines and PDXs.

A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

BIOL-19. DIFFUSE MIDLINE GLIOMA CEREBRAL ORGANOID MODEL AND MULTIOMICS CHARACTERIZATION

Abstract Diffuse midline glioma (DMG) are highly aggressive malignancies of the central nervous system that primarily affect the pediatric population. These tumors are historically universally fatal with no curative treatment options available. There is a need to identify more targeted and optimal treatments for these patients. Current approaches to pre-clinical therapeutic testing have been limited by many obstacles to effectively translate theses to patients. It is known that the interactions between tumors and the other components of the tumor microenvironment (TME) can change the response to therapeutic interventions. This especially holds true for brain tumors and the complex neural network encompassed within their TME. Given this, it is crucial to develop more realistic DMG models that integrate this to conduct therapeutic testing rather than relying upon conventional cell culture models. The goal of our study was to develop a three-dimensional DMG cerebral organoid model derived from human induced pluripotent stem cells (iPSCs) co-cultured with three different DMG patient-derived xenograft (PDX) cell lines to better mimic the TME for therapeutic testing. We were able to successfully integrate our three cell lines into the cerebral organoids, capturing TME interactions along with performing multiomic profiling for better characterization. We next plan to perform therapeutic testing to further validate the model and improve preclinical drug screening for DMG.

DIPG-13. NEW IMMUNOCOMPETENT GENETICALLY-ENGINEERED MOUSE MODELS OF PEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMAS REVEAL MECHANISMS OF TUMOR INITIATION AND PROGRESSION

Abstract Pediatric-type diffuse high-grade gliomas (pHGGs) form a group of aggressive brain tumors with very poor prognosis. Recurrent driver mutations in TP53 and histone H3-encoding genes, most commonly H3F3A, are observed in a large proportion of these tumors. H3-K27M substitutions are particularly prevalent in midline gliomas, and H3-G34R mutations occur in supratentorial tumors. To better understand the molecular mechanisms driving pHGG emergence and progression, we set out to develop immunocompetent mouse models recapitulating the oncogenic lesions observed in human tumors. Given that oligodendrocyte progenitor cells (OPCs) may be the cells of origin of some pHGGs, we investigated the effect of mutation induction in the OLIG2-expressing cell lineage, using the Olig2Cre driver. Interestingly, deletion of the P53-encoding gene, Trp53, in Trp53flox/flox;Olig2Cre/+ mice led to the development of diffuse gliomas in nearly all animals. Latency to humane endpoint was relatively long (&amp;gt;1 year), and tumors developed throughout the brain with a mild preference for infratentorial (e.g. brainstem) localization. The gliomas diffusely infiltrated the brain parenchyma, and were heterogeneously immuno-reactive for OPC, astrocytic, and neural progenitor cell (NPC) markers. Based on RNA sequencing, the tumors segregated in two groups: one enriched in expression of OPC genes, and another with a prominent NPC-like signature. Whole exome sequencing revealed frequent Ccnd1 and Kras/Hras copy number gains in advanced tumors. To model additional oncogenic lesions, we generated knock-in mice in which the H3f3aK27M or the H3f3aG34R mutations can be induced in any cell type with Cre recombinase, and extinguished with Flp recombinase. These were combined with conditional Trp53 loss-of-function alleles in various cell lineages, and their effect on tumor initiation and progression was monitored. Collectively, our studies provide new reagents for accurate disease modeling of pHGG in mice, opening opportunities to better understand tumor initiation and progression at high resolution, and for pre-clinical therapeutic testing.

Astrocytes: Dissecting Their Diverse Roles in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders often co-occurring in the same patient, a feature that suggests a common origin of the two diseases. Consistently, pathological inclusions of the same proteins as well as mutations in the same genes can be identified in both ALS/FTD. Although many studies have described several disrupted pathways within neurons, glial cells are also regarded as crucial pathogenetic contributors in ALS/FTD. Here, we focus our attention on astrocytes, a heterogenous population of glial cells that perform several functions for optimal central nervous system homeostasis. Firstly, we discuss how post-mortem material from ALS/FTD patients supports astrocyte dysfunction around three pillars: neuroinflammation, abnormal protein aggregation, and atrophy/degeneration. Furthermore, we summarize current attempts at monitoring astrocyte functions in living patients using either novel imaging strategies or soluble biomarkers. We then address how astrocyte pathology is recapitulated in animal and cellular models of ALS/FTD and how we used these models both to understand the molecular mechanisms driving glial dysfunction and as platforms for pre-clinical testing of therapeutics. Finally, we present the current clinical trials for ALS/FTD, restricting our discussion to treatments that modulate astrocyte functions, directly or indirectly.

Advances in Immunocompetent Mouse and Rat Models.

Rodent models of breast cancer have played critical roles in our understanding of breast cancer development and progression as well as preclinical testing of cancer prevention and therapeutics. In this article, we first review the values and challenges of conventional genetically engineered mouse (GEM) models and newer iterations of these models, especially those with inducible or conditional regulation of oncogenes and tumor suppressors. Then, we discuss nongermline (somatic) GEM models of breast cancer with temporospatial control, made possible by intraductal injection of viral vectors to deliver oncogenes or to manipulate the genome of mammary epithelial cells. Next, we introduce the latest development in precision editing of endogenous genes using in vivo CRISPR-Cas9 technology. We conclude with the recent development in generating somatic rat models for modeling estrogen receptor-positive breast cancer, something that has been difficult to accomplish in mice.

Contemporary and emerging technologies for research in children's rare and interstitial lung disease.

Although recent decades have seen the identification, classification and discovery of the genetic basis of many children's interstitial and rare lung disease (chILD) disorders, detailed understanding of pathogenesis and specific therapies are still lacking for most of them. Fortunately, a revolution of technological advancements has created new opportunities to address these critical knowledge gaps. High-throughput sequencing has facilitated analysis of transcription of thousands of genes in thousands of single cells, creating tremendous breakthroughs in understanding normal and diseased cellular biology. Spatial techniques allow analysis of transcriptomes and proteomes at the subcellular level in the context of tissue architecture, in many cases even in formalin-fixed, paraffin-embedded specimens. Gene editing techniques allow creation of "humanized" animal models in a shorter time frame, for improved knowledge and preclinical therapeutic testing. Regenerative medicine approaches and bioengineering advancements facilitate the creation of patient-derived induced pluripotent stem cells and their differentiation into tissue-specific cell types which can be studied in multicellular "organoids" or "organ-on-a-chip" approaches. These technologies, singly and in combination, are already being applied to gain new biological insights into chILD disorders. The time is ripe to systematically apply these technologies to chILD, together with sophisticated data science approaches, to improve both biological understanding and disease-specific therapy.

Advancements in pre-clinical development of gene editing-based therapies to treat inherited retinal diseases

One of the major goals in the inherited retinal disease (IRD) field is to develop an effective therapy that can be applied to as many patients as possible. Significant progress has already been made toward this end, with gene editing at the forefront. The advancement of gene editing-based tools has been a recent focus of many research groups around the world. Here, we provide an update on the status of CRISPR/Cas-derived gene editors, promising options for delivery of these editing systems to the retina, and animal models that aid in pre-clinical testing of new IRD therapeutics.

475 Local Conditional Induction of Brain Arteriovenous Malformations in Alk1-Inducible Knock-Out Mice

INTRODUCTION: Current mouse models of familial brain arteriovenous malformations (AVMs) have limitations in long-term survival. These drawbacks are mainly attributed to systemic and ubiquitous genetic recombinations with development of AVMs in peripheral organs that result in hemorrhagic conditions. METHODS: Transgenic mice with ubiquitous CreER expression were used (ROSA26CreER;Alk1f/f; Alk1-iKO). 4-hydroxytamoxifen (4-OHTM) was stereotactically delivered into the striatum, parietal cortex, or cerebellum of neonatal mice at postnatal day 1. Mice were evaluated for brain AVMs with systemic latex dye perfusion and 3D time-of-flight magnetic resonance angiography. RESULTS: Alk1-iKO mice injected stereotactically with 4-OHTM developed brain AVMs with 68% (21/31) frequency in the striatum, 72% (13/18) in the parietal cortex, and 44% (4/9) in the cerebellum. Brain AVMs formed in or near the injection site with 95% accuracy (36/38). A subset of mice (8%, [3/38]) presented with a communicating hydrocephalus from CSF malabsorption. The formation of AVMs in these Alk1-iKO mice was restricted to the brain, without AVMs developing in peripheral organs (n = 38). The hemoglobin levels were not significantly different between mutant and control Alk1-iKO mice after stereotactic, intracerebral 4-OHTM injection (p = 0.27). The 4-week mortality was 5% (2/38). Longitudinally monitored mice demonstrated good survival at 3 months of age (86%, [6/7]). CONCLUSIONS: We present the first experimental mouse model with local, CreER-mediated induction of brain AVMs. The model is advantageous in generating brain AVMs with high efficiency, along with an improved long-term survival and reduced early lethality of mice. Collectively, these features may serve as a framework to accelerate the ongoing investigations of brain AVM pathogenesis, rupture, and preclinical testing of novel therapeutics.

Development and characterization of a chronic hepatitis B murine model with a mutation in the START codon of an HBV polymerase.

Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

A new FRDA mouse model [Fxn null:YG8s(GAA) > 800] with more than 800 GAA repeats.

Friedreich's ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing. We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxn null:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models. We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls. These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.

Brain Alterations in Aged OVT73 Sheep Model of Huntington's Disease: An MRI Based Approach.

Huntington's disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

LW-10 PRECLINICAL THERAPEUTIC TESTING FOR BRAIN TUMOR USING PATIENT-DERIVED XENOGRAFT (PDX) MODEL

Abstract I have been working on the brain tumor research about 20 years in the United State. My primary research interests in understanding of the mechanisms of genetic and epigenetic regulation that promote tumor development and progression, and identify novel therapeutic targets for the treatment of human brain cancer. For these purposes, I have developed many unique patient-derived xenograft (PDX) models for human brain tumors in the brains of immunocompromised mice. This animal model system has been shown to better preserve tumor characteristics, as manifested in patients, than tumor cell propagation in cell culture. In association with the development of these models, my laboratory utilizes numerous techniques to investigate brain tumor growth and response to novel agents that are administered singularly or in combinations, including with radiation. My laboratory employs the latest genomic/epigenomic technologies to identify novel genetic and epigenetics alteration that would reveal rational therapeutic targets, and test the anti-tumor activity of the therapeutics using PDX mouse model including diffuse intrinsic pontine glioma (DIPG) harboring histone H3 gene mutation (H3K27M). We have investigated the role of H3K27M mutations and histone modifications, and preclinical therapeutic testing in DIPG PDX models. In addition to the molecular targeted therapy, we recently implement AI machine learning approach and emerging patient genomic data to discover more effective and less toxic candidates from billions of compounds for brain tumor therapy. Moreover, we investigate new drug delivery systems to bypass the blood-brain barrier using convection-enhanced delivery and intranasal delivery. My long-term goal of my work further tests the efficacy of my approach preclinically and, if validated, move this therapeutic approach to clinical trial. In the meeting, I will present current advances in experimental therapeutics in my laboratory.

Practical Mouse Model to Investigate Therapeutics for Staphylococcusaureus Contaminated Surgical Mesh Implants

IntroductionThe use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus–infected surgical mesh and provide results from antibiotic and immunotherapeutic testing. Materials and MethodsImplantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 “don't eat me” molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points. ResultsA single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection. ConclusionsThis fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.