Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaa c.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaa c.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics. Generation of transgenic murine knock-in models of human disease once relied solely upon targeted insertion of the desired sequence via Cre-Lox recombination and embryonic stem cell implantation. Though this strategy has resulted in many successful murine model systems, it is labor-intensive, time-consuming, and expensive. The advent of genome editing via engineered nucleases, especially clustered regularly interspaced short palindromic repeats (CRISPR)-based systems has allowed for a potentially accurate, efficient, and relatively inexpensive alternative to the traditional method of transgenic knock-in model generation 1,2. As an intriguing example of disorders that may uniquely benefit from genome editing, inherited metabolic disorders (IMDs) are a diverse group of genetic diseases affecting the proper breakdown or synthesis of essential compounds such as carbohydrates, amino acids, or organic acids. Many of these disorders are caused by single gene defects that alter the expression and/or activity of critical metabolic enzymes. Given the monogenic nature of IMD pathogenesis, this class of genetic disorders is quickly becoming an area of high interest for CRISPR-mediated genome editing therapeutics 3-5. Pompe disease, caused by the deficiency of acid α-glucosidase (GAA; EC 3.2.1.20), is characterized by lysosomal accumulation of glycogen in body tissues, primarily cardiac and skeletal muscle. Muscle lysosomal glycogen storage results in muscle weakness varying in age of onset and severity according to residual GAA enzymatic

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.

The goal of this study is to generate and characterize a knock-in model of Pompe disease (PD) a rare, progressive, fatal disorder primarily affecting the cardiac and musculoskeletal systems. While a murine model of PD exists, it bears a Cre/loxP induced exonic insertion of a neomycin cassette and does not completely recapitulate severe human PD -displaying nonfatal hypertrophic cardiomyopathy only late in its natural history. We therefore designed a CRISPR-Cas9 knock-in system targeting the Gaa gene to introduce the known pathogenic CRIM negative Gaa mutation c.1826insA (p.Y609*).Following optimization of our knock-in strategy in cultured murine myoblasts, we successfully generated a Gaa c1826insA mouse model using a dual sgRNA with ssODN donor template approach. Whole genome sequencing and analysis of the Gaa c1826insA murine model establishes that our system is highly specific for the Gaa c1826 target locus and does not induce any off-target mutations or genomic rearrangements. Next, we examined GAA mRNA transcript, protein expression and enzymatic activity levels in our PD knock-in mice. Gaa c1826insA mice display significantly reduced levels of GAA expression and enzymatic activity relative to wild-type mice.We performed echocardiography on Gaa c1826insA mice to assess cardiac structure and function.Gaa c1826insA mice exhibit early-onset, progressive cardiac hypertrophy as measured by significant increases in left ventricular wall thickness and mass index by 3 months of age. We also conducted functional testsgrip strength, inverted screen, gait analysison Gaa c1826insA mice every 3 months to assess overall motor performance. Gaa c1826insA mice display impaired motor strength and coordination relative to wild-type mice. Altogether, our results demonstrate that the Gaa c1826insA murine model recapitulates human infantile-onset Pompe disease and is better suited for evaluation of therapeutic strategies such as genome correction.

Splenectomy in an animal model requires a standardized technique utilizing best practice to avoid variability which can result in adverse impact to the animal resulting in flawed physiologic responses simply due to technique rather than to the studied variables. In the case of the spleen, often investigators are analyzing the animal immune or inflammatory responses. Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. This operation, in turn, impacts blood loss and insensible fluid losses, sterile technique, unintended trauma to the spleen and surrounding organs, the length of the incision and the duration of the operation with more prolonged exposure to anesthetic agents. All these variables ultimately play a significant role in the experiment since they directly affect the response of the model in terms of inflammation, immune activation, or even suppression. Undesired variables such as these go unnoticed and lead to inaccurate and misleading data.

Neurologic Diseases (iNcluDiNg ophthalmic aND auDitory Diseases) iii transcription. Clinically this presents as progressive degeneration of the central nervous system, retina, cardiovascular system, and cochlea, causing mental retardation, post-natal growth defects, pigmented retinopathy, cataracts, dermal UV sensitivity, organ dysfunction and shortened life expectancy.CSA and CSB single mutant mice display retinal degeneration with age and ocular sensitivity to ionizing radiation. Because limited publications on these mouse models exist we began by characterizing the retinal dysfunction through histology and electroretinography (ERG). We found that the retinal degeneration in CSA and CSB mutant mice (C57Bl6 background) was occurring over a slower time course (over 1 year) than previously reported. Therefore we aimed to increase the rate of retinal degeneration in these animals using UV light exposure, whole body radiation, or high intensity light exposure. Our goal was to increase the speed of retinal degeneration to a timeframe that is more readily studied for the purposes of gene therapy correction. Preliminary data indicates that we can cause a decrease in the scotopic ERG (b-wave) 2 weeks after high-dose ultraviolet light exposure (100000J/m^2), a change not seen in C57Bl6 mice at the same UV dose. There was also a decrease in b-wave amplitude following radiation exposure (2-3 gray) in the CSB mouse model that was not seen in C57Bl6 mice. Histology on these mice is currently pending. Improving the rate of photoreceptor loss will allow us to perform rAAV based gene therapy to deliver the CSB gene back to the retina and ascertain whether UV and radiation resistance can be restored.While single mutant CSA and CSB mice do not have significant neurological deficits, when crossed with XPA deficient mice they develop clinical signs similar to severely affected human patients. Signs in these mice include neurologic deficits, poor weight gain and death around postnatal day 21. These mice could be ideal candidates to show that correction of both the central nervous system disease as well as the retinal degeneration is possible in a single model. To determine if both the brain and retina could be targeted in the same animal (and therefore model what may be necessary in the human patients) we have looked at targeting both organs simultaneously using either a single IV injection in neonatal mice or a combined intra-cerebral ventricular (ICV) injection and IV injection in the same animal. We found that delivery of the rAAV vector (regardless of serotype tested) into the retro-orbital sinus at postnatal day 1-3 leads to widespread transduction of the retina. Temporal vein delivery postnatal day 1-3 only resulted in transduction of the inner layer of the retina, not the photoreceptors. When ICV and IV injections were combined in neonates widespread brain and retinal transduction was achieved.These preliminary data indicate that a murine model for retinal gene therapy of CS is feasible and that potentially both the retina an...

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