Reduction of Filamin C Results in Altered Proteostasis, Cardiomyopathy, and Arrhythmias.

BACKGROUND: Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. METHODS AND RESULTS: We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. CONCLUSIONS: FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

New semi-automated tool for the quantitation of MR imaging to estimate in vivo muscle disease severity in mice.

The pathology in Duchenne muscular dystrophy (DMD) is characterized by degenerating muscle fibers, inflammation, fibro-fatty infiltrate, and edema, and these pathological processes replace normal healthy muscle tissue. The mdx mouse model is one of the most commonly used preclinical models to study DMD. Mounting evidence has emerged illustrating that muscle disease progression varies considerably in mdx mice, with inter-animal differences as well as intra-muscular differences in pathology in individual mdx mice. This variation is important to consider when conducting assessments of drug efficacy and in longitudinal studies. Magnetic resonance imaging (MRI) is a non-invasive method that can be used qualitatively or quantitatively to measure muscle disease progression in the clinic and in preclinical models. Although MR imaging is highly sensitive, image acquisition and analysis can be time intensive. The purpose of this study was to develop a semi-automated muscle segmentation and quantitation pipeline that can quickly and accurately estimate muscle disease severity in mice. Herein, we show that the newly developed segmentation tool accurately divides muscle. We show that measures of skew and interdecile range based on segmentation sufficiently estimate muscle disease severity in healthy wildtype and diseased mdx mice. Moreover, the semi-automated pipeline reduced analysis time by nearly 10-fold. Use of this rapid, non-invasive, semi-automated MR imaging and analysis pipeline has the potential to transform preclinical studies, allowing for pre-screening of dystrophic mice prior to study enrollment to ensure more uniform muscle disease pathology across treatment groups, improving study outcomes.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

COVID-19 symptom severity predicts neutralizing antibody activity in a community-based serological study.

Serological testing for SARS-CoV-2 IgG antibodies is used to assess their presence in blood samples from exposed individuals and provides a measure of the magnitude of immune response to infection. The measurement of neutralizing antibodies (NAbs) in particular provides information about the severity of prior infection and level of protective immunity against re-infection. Much of the work investigating the association between prior infection severity and NAb levels has been conducted among clinical populations, and less is known about this relationship in the general population. Accordingly, we utilize data from a large (n = 790) community-based cohort of unvaccinated, seropositive participants. We analyzed the association between NAb response, measured via surrogate virus neutralization assay, with patterns of symptoms and household exposure. Our results indicate no detectable NAb activity in 63.8% of the seropositive participants (n = 504). Those with detectable NAb levels demonstrated a positive relationship between NAb activity and both self-reported previous symptom severity and household exposure. These findings are significant in light of recent concerns about degree of protective immunity conferred by prior infection or vaccination, and we highlight the value of community-based research for investigating variation in immune response.

Low Levels of Neutralizing Antibodies After Natural Infection With Severe Acute Respiratory Syndrome Coronavirus 2 in a Community-Based Serological Study.

BACKGROUND: Confidence in natural immunity after infection with severe acute respiratory syndrome coronavirus 2 is one reason for vaccine hesitancy. METHODS: We measured antibody-mediated neutralization of spike protein-ACE2 receptor binding in a large community-based sample of seropositive individuals who differed in severity of infection (N = 790). RESULTS: A total of 39.8% of infections were asymptomatic, 46.5% were symptomatic with no clinical care, 13.8% were symptomatic with clinical care, and 3.7% required hospitalization. Moderate/high neutralizing activity was present after 41.3% of clinically managed infections, in comparison with 7.9% of symptomatic and 1.9% of asymptomatic infections. CONCLUSIONS: Prior coronavirus disease 2019 infection does not guarantee a high level of antibody-mediated protection against reinfection in the general population.

Geographic disparities in COVID-19 case rates are not reflected in seropositivity rates using a neighborhood survey in Chicago.

To date, COVID-19 case rates are disproportionately higher in Black and Latinx communities across the US, leading to more hospitalizations, and deaths in those communities. These differences in case rates are evident in comparisons of Chicago neighborhoods with differing race and/or ethnicities of their residents. Disparities could be due to neighborhoods with more adverse health outcomes associated with poverty and other social determinants of health experiencing higher prevalence of SARS-CoV-2 infection or due to greater morbidity and mortality resulting from equivalent SARS-CoV-2 infection prevalence. We surveyed five pairs of adjacent ZIP codes in Chicago with disparate COVID-19 case rates for highly specific and quantitative serologic evidence of any prior infection by SARS-CoV-2 to compare with their disparate COVID-19 case rates. Dried blood spot samples were self-collected at home by internet-recruited participants in summer 2020, shortly after Chicago's first wave of the COVID-19 pandemic. Pairs of neighboring ZIP codes with very different COVID-19 case rates had similar seropositivity rates for anti-SARS-CoV-2 receptor binding domain IgG antibodies. Overall, these findings of comparable exposure to SARS-CoV-2 across neighborhoods with very disparate COVID-19 case rates are consistent with social determinants of health, and the co-morbidities related to them, driving differences in COVID-19 rates across neighborhoods.

Anti-latent TGFß binding protein 4 antibody improves muscle function and reduces muscle fibrosis in muscular dystrophy.

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor ß (TGFß) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFß. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein's hinge region, was linked to increased sequestration of latent TGFß, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-ß, and together, these data underscored the functional role of LTBP4's hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4's hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mdx mice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy.

Comparison of IgG and neutralizing antibody responses after one or two doses of COVID-19 mRNA vaccine in previously infected and uninfected individuals.

BACKGROUND: Recent reports have suggested that among individuals previously infected with SARS-CoV-2, a single mRNA vaccine dose is sufficient to elicit high levels of immunity. METHODS: We compared anti-SARS-CoV-2 spike receptor binding domain (RBD) IgG antibody concentrations and antibody-mediated neutralization of spike-angiotensin-converting enzyme (ACE2) receptor binding in vitro following vaccination of non-hospitalized participants by sero-status and acute virus diagnosis history. Participants were analysed before and after mRNA vaccination (BNT162b2/Pfizer or mRNA-1273/Moderna) in a community-based, home-collected, longitudinal serosurvey in the Chicago area (USA); none reported hospitalization for COVID-19. Samples were collected in January and February 2021. Before vaccination, some reported prior positive acute viral diagnostic testing and were seropositive (COVID-19+); the others who did not report acute viral diagnostic testing were categorized as seropositive or seronegative based on anti-spike RBD IgG test results. FINDINGS: Of 307 unique vaccine recipients, 46 reported a prior COVID-19 diagnosis and were seropositive (COVID-19 +). Of the 261 with no history of acute viral diagnostic testing, 117 were seropositive and 144 seronegative before vaccination. The median age was 38 years (range 21-83) with 67 female and 33% male; 40% were non-White. Responses were evaluated after one (n = 142) or two (n = 191) doses of BNT162b2 or mRNA-1273 vaccine. After one dose, median post-vaccine IgG concentration and percent surrogate neutralization were each significantly higher among the COVID-19+ (median 48·2 µg/ml, IgG; > 99.9% neutralization) compared to the seropositives (3·6 µg /ml IgG; 56.5% neutralization) and seronegatives (2·6 µg /ml IgG; 38·3% neutralization). The latter two groups reached > 95% neutralization after the second vaccine dose. INTERPRETATION: After one dose of mRNA vaccine, individuals previously diagnosed with COVID-19 responded with high levels of anti-RBD IgG and surrogate neutralization of spike-ACE2 interaction. One dose of mRNA vaccine was not sufficient to generate comparably high responses among most persons previously infected with SARS-CoV-2 without a clinical COVID-19 diagnosis, nor among seronegative persons. FUNDING: National Science Foundation 2035114, NIH 3UL1TR001422-06S4, and Northwestern University Office of Research.

Cohabitation With a Known Coronavirus Disease 2019 Case Is Associated With Greater Antibody Concentration and Symptom Severity in a Community-Based Sample of Seropositive Adults.

In a community-based sample of seropositive adults (n = 1101), we found that seropositive individuals who lived with a known coronavirus disease 2019 (COVID-19) case exhibited higher blood anti-severe acute respiratory syndrome coronavirus 2 spike receptor-binding domain immunoglobulin G concentrations and greater symptom severity compared to seropositive individuals who did not live with a known COVID-19 case.

COVID-19 mRNA Vaccination Generates Greater Immunoglobulin G Levels in Women Compared to Men.

We investigated whether the antibody response to coronavirus disease 2019 (COVID-19) mRNA vaccination is similar in women and men. In a community cohort without prior COVID-19, first vaccine dose produced higher immunoglobulin G (IgG) levels and percent inhibition of spike-ACE2 receptor binding, a surrogate measure of virus neutralization, in women compared to men (7.0 µg/mL, 51.6% vs 3.3 µg/mL, 36.4%). After 2 doses, IgG levels remained significantly higher for women (30.4 µg/mL) compared to men (20.6 µg/mL), while percent inhibition was similar (98.4% vs 97.7%). Sex-specific antibody response to mRNA vaccination informs future efforts to understand vaccine protection and side effects.

Patterns and persistence of SARS-CoV-2 IgG antibodies in Chicago to monitor COVID-19 exposure.

BACKGROUNDEstimates of seroprevalence to SARS-CoV-2 vary widely and may influence vaccination response. We ascertained IgG levels across a single US metropolitan site, Chicago, from June 2020 through December 2020.METHODSParticipants (n = 7935) were recruited through electronic advertising and received materials for a self-sampled dried-blood spot assay through the mail or a minimal contact in-person method. IgG against the receptor-binding domain of SARS-CoV-2 was measured using an established highly sensitive and highly specific assay.RESULTSOverall seroprevalence was 17.9%, with no significant difference between method of contact. Only 2.5% of participants reported having had a diagnosis of COVID-19 based on virus detection, consistent with a 7-fold greater exposure to SARS-CoV-2 measured by serology than that detected by viral testing. The range of IgG level observed in seropositive participants from this community survey overlapped with the range of IgG levels associated with COVID-19 cases having a documented positive PCR test. From a subset of those who participated in repeat testing, half of seropositive individuals retained detectable antibodies for 3 to 4 months.CONCLUSIONQuantitative IgG measurements with a highly specific and sensitive assay indicated more widespread exposure to SARS-CoV-2 than observed by viral testing. The range of IgG concentrations produced from these asymptomatic exposures was similar to IgG levels occurring after documented nonhospitalized COVID-19, which were considerably lower than those produced from hospitalized COVID-19 cases. The differing ranges of IgG response, coupled with the rate of decay of antibodies, may influence response to subsequent viral exposure and vaccine.FundingNational Science Foundation grant 2035114, NIH grant 3UL1TR001422-06S4, NIH National Center for Advancing Translational Sciences grants UL1 TR001422 and UL1 TR002389, Dixon Family Foundation, Northwestern University Cancer Center (NIH grant P30 CA060553), and Walder Foundation's Chicago Coronavirus Assessment Network.

Exposure to SARS-CoV-2 within the household is associated with greater symptom severity and stronger antibody responses in a community-based sample of seropositive adults.

Magnitude of SARS-CoV-2 virus exposure may contribute to symptom severity. In a sample of seropositive adults (n=1101), we found that individuals who lived with a known COVID-19 case exhibited greater symptom severity and IgG concentrations compared to individuals who were seropositive but did not live with a known case (P<0.0001).

Patterns and persistence of SARS-CoV-2 IgG antibodies in Chicago to monitor COVID-19 exposure.

Background: Estimates of seroprevalence to SARS-CoV-2 vary widely and may influence vaccination response. We ascertained IgG levels across a single US metropolitan site, Chicago, from June 2020 through December 2020. Methods: Participants (n=7935) were recruited through electronic advertising and received materials for a self-sampled dried blood spot assay through the mail or a minimal contact in person method. IgG to the receptor binding domain of SARS-CoV-2 was measured using an established highly sensitive and highly specific assay. Results: Overall seroprevalence was 17.9%, with no significant difference between method of contact. Only 2.5% of participants reported having had a diagnosis of COVID-19 based on virus detection, consistent with a 7-fold greater exposure to SARS-CoV-2 measured by serology than detected by viral testing. The range of IgG level observed in seropositive participants from this community survey overlapped with the range of IgG levels associated with COVID-19 cases having a documented positive PCR positive test. From a subset of those who participated in repeat testing, half of seropositive individuals retained detectable antibodies for 3-4 months. Conclusions: Quantitative IgG measurements with a highly specific and sensitive assay indicate more widespread exposure to SARS-CoV-2 than observed by viral testing. The range of IgG concentration produced from these asymptomatic exposures is similar to IgG levels occurring after documented non-hospitalized COVID-19, which is considerably lower than that produced from hospitalized COVID-19 cases. The differing ranges of IgG response, coupled with the rate of decay of antibodies, may influence response to subsequent viral exposure and vaccine.

High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay.

OBJECTIVE: Serological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS). We describe assay performance and demonstrate its utility for remote sampling with results from a community-based study. METHODS: An ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members. RESULTS: Among 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain. CONCLUSION: DBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.

An Amino Acid-Based Oral Rehydration Solution Regulates Radiation-Induced Intestinal Barrier Disruption in Mice.

BACKGROUND: Radiotherapy inadvertently affects gastrointestinal (GI) epithelial cells, causing intestinal barrier disruption and increased permeability. OBJECTIVE: We examined the effect of amino acid-based oral rehydration solution (AA-ORS) on radiation-induced changes of intestinal barrier function and epithelial tight junctions (TJs) in a randomized experimental study using a total-body irradiation (TBI) mouse model. METHODS: Eight-week-old male Swiss mice received a single-dose TBI (0, 1, 3, or 5 Gy), and subsequent gastric gavage with AA-ORS (threonine, valine, serine, tyrosine, and aspartic acid) or saline for 2 or 6 d. Intestinal barrier function of mouse ileum was characterized by electrophysiological analysis of conductance, anion selectivity, and paracellular permeability [fluorescein isothiocyanate (FITC)-dextran]. Ultrastructural changes of TJs were evaluated by transmission electron microscopy. Membrane protein and mRNA expression of claudin-1, -2, -3, -5, and -7, occludin, and E-cadherin were analyzed with western blot, qPCR, and immunohistochemistry. Nonparametric tests were used to compare treatment-dose differences for each time point. RESULTS: Saline-treated mice had a higher conductance at doses as low as 3 Gy, and as early as 2 d post-TBI compared with 0 Gy (P < 0.001). Paracellular permeability and dilution potential were increased 6 d after 5 Gy TBI (P < 0.001). Conductance decreased with AA-ORS after 2 d in 3-Gy and 5-Gy mice (P < 0.05 and P < 0.001), and on day 6 after 5 Gy TBI (P < 0.001). Anion selectivity and FITC permeability decreased from 0.73 ± 0.02 to 0.61 ± 0.03 pCl/pNa (P < 0.01) and from 2.7 ± 0.1 × 105 to 2.1 ± 0.1 × 105 RFU (P < 0.001) in 5-Gy mice treated with AA-ORS for 6 d compared with saline. Irradiation-induced ultrastructural changes of TJs characterized by decreased electron density and gap formation improved with AA-ORS. Reduced claudin-1, -3, and -7 membrane expression after TBI recovered with AA-ORS within 6 d, whereas claudin-2 decreased indicating restitution of TJ proteins. CONCLUSIONS: Radiation-induced functional and structural disruption of the intestinal barrier in mice is reversed by AA-ORS rendering AA-ORS a potential treatment option in prospective clinical trials in patients with gastrointestinal barrier dysfunction.

AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice.

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa -/- ) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.

Pompe disease gene therapy: neural manifestations require consideration of CNS directed therapy.

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span.

Impact of obesity on breast cancer recurrence and minimal residual disease.

BACKGROUND: Obesity is associated with an increased risk of breast cancer recurrence and cancer death. Recurrent cancers arise from the pool of residual tumor cells, or minimal residual disease (MRD), that survives primary treatment and persists in the host. Whether the association of obesity with recurrence risk is causal is unknown, and the impact of obesity on MRD and breast cancer recurrence has not been reported in humans or in animal models. METHODS: Doxycycline-inducible primary mammary tumors were generated in intact MMTV-rtTA;TetO-HER2/neu (MTB/TAN) mice or orthotopic recipients fed a high-fat diet (HFD; 60% kcal from fat) or a control low-fat diet (LFD; 10% kcal from fat). Following oncogene downregulation and tumor regression, mice were followed for clinical recurrence. Body weight was measured twice weekly and used to segregate HFD mice into obese (i.e., responders) and lean (i.e., nonresponders) study arms, and obesity was correlated with body fat percentage, glucose tolerance (measured using intraperitoneal glucose tolerance tests), serum biomarkers (measured by enzyme-linked immunosorbent assay), and tissue transcriptomics (assessed by RNA sequencing). MRD was quantified by droplet digital PCR. RESULTS: HFD-Obese mice weighed significantly more than HFD-Lean and LFD control mice (p < 0.001) and had increased body fat percentage (p < 0.001). Obese mice exhibited fasting hyperglycemia, hyperinsulinemia, and impaired glucose tolerance, as well as decreased serum levels of adiponectin and increased levels of leptin, resistin, and insulin-like growth factor 1. Tumor recurrence was accelerated in HFD-Obese mice compared with HFD-Lean and LFD control mice (median relapse-free survival 53.0 days vs. 87.0 days vs. 80.0 days, log-rank p < 0.001; HFD-Obese compared with HFD-Lean HR 2.52, 95% CI 1.52-4.16; HFD-Obese compared with LFD HR 2.27, 95% CI 1.42-3.63). HFD-Obese mice harbored a significantly greater number of residual tumor cells than HFD-Lean and LFD mice (12,550 ± 991 vs. 7339 ± 2182 vs. 4793 ± 1618 cells, p < 0.001). CONCLUSION: These studies provide a genetically engineered mouse model for study of the association of diet-induced obesity with breast cancer recurrence. They demonstrate that this model recapitulates physiological changes characteristic of obese patients, establish that the association between obesity and recurrence risk is causal in nature, and suggest that obesity is associated with the increased survival and persistence of residual tumor cells.

Amino Acid Hydration Decreases Radiation-Induced Nausea in Mice: A Pica Model.

Nausea and diarrhea are common yet inconsistent side effects of abdominal and pelvic irradiation. Their frequency, chronicity, and severity vary greatly, and the reasons for inter-subject variability are unknown. We studied the potential for radiation-induced changes in amino acid absorption and mucosal barrier function to lead to gastrointestinal toxicity. We found profound and prolonged changes in the absorption and secretion of several electrolytes and nutrients, caused by changes in transporter function, after radiation doses as low as 1 to 3 Gy. After identifying absorbed and non-absorbed amino acids, we demonstrated the role of a beneficial amino acid drink to alleviate radiation-related gastrointestinal symptoms in a mouse model.