Structural characterization of antibody-responses from Zolgensma treatment provides the blueprint for the engineering of an AAV capsid suitable for redosing.

Monoclonal antibodies (mAbs) are useful tools to dissect the neutralizing antibody response against the adeno-associated virus (AAV) capsids used as gene therapy delivery vectors. This study structurally characterizes the interactions of 21 human-derived antibodies from patients treated with the AAV9 vector, Zolgensma ® , utilizing high-resolution cryo-electron microscopy. The majority of the bound antibodies do not conform to the icosahedral symmetry of the capsid, thus requiring localized reconstructions. These complex structures provide unprecedented details of the mAbs binding interfaces, with some antibodies inducing structural perturbations of the capsid upon binding. Key surface capsid amino acid residues were identified facilitating the design of capsid variants with an antibody escape phenotype, with the potential to expand the patient cohort treatable with AAV9 vectors to include those that were previously excluded due to their pre-existing neutralizing antibodies, and possibly also to those requiring redosing.

Production and characterization of an AAV1-VP3-only capsid: An analytical benchmark standard.

Adeno-associated viruses (AAVs) are non-enveloped ssDNA icosahedral T = 1 viruses used as vectors for clinical gene delivery. Currently, there are over 200 AAV-related clinical trials and six approved biologics on the market. As such new analytical methods are continually being developed to characterize and monitor the quality and purity of manufactured AAV vectors, these include ion-exchange chromatography and Direct Mass Technology. However, these methods require homogeneous analytical standards with a high molecular weight standard comparable to the mass of an AAV capsid. Described here is the design, production, purification, characterization, and the cryo-electron microscopy structure of an AAV1-VP3-only capsid that fulfills this need as a calibrant to determine capsid mass, charge, homogeneity, and transgene packaging characteristics.

Structural and functional characterization of capsid binding by anti-AAV9 monoclonal antibodies from infants after SMA gene therapy.

Success in the treatment of infants with spinal muscular atrophy (SMA) underscores the potential of vectors based on adeno-associated virus (AAV). However, a major obstacle to the full realization of this potential is pre-existing natural and therapy-induced anti-capsid humoral immunity. Structure-guided capsid engineering is one possible approach to surmounting this challenge but necessitates an understanding of capsid-antibody interactions at high molecular resolution. Currently, only mouse-derived monoclonal antibodies (mAbs) are available to structurally map these interactions, which presupposes that mouse and human-derived antibodies are functionally equivalent. In this study, we have characterized the polyclonal antibody responses of infants following AAV9-mediated gene therapy for SMA and recovered 35 anti-capsid mAbs from the abundance of switched-memory B (smB) cells present in these infants. For 21 of these mAbs, seven from each of three infants, we have undertaken functional and structural analysis measuring neutralization, affinities, and binding patterns by cryoelectron microscopy (cryo-EM). Four distinct patterns were observed akin to those reported for mouse-derived mAbs, but with early evidence of differing binding pattern preference and underlying molecular interactions. This is the first human and largest series of anti-capsid mAbs to have been comprehensively characterized and will prove to be powerful tools for basic discovery and applied purposes.

EFFECTS OF SARS COVID-19 POSITIVITY STATUS ON VENOUS THROMBOSIS AND PULMONARY EMBOLISM RATES IN TRAUMA PATIENTS.

ABSTRACT: Introduction : COVID-19-induced coagulopathy (CIC) can increase the risk of thromboembolism without underlying clotting disorders, even when compared with other respiratory viruses. Trauma has a known association with hypercoagulability. Trauma patients with concurrent COVID-19 infection potentially have an even greater risk of thrombotic events. The purpose of this study was to evaluate venous thromboembolism (VTE) rates in trauma patients with COVID-19. Methods : This study reviewed all adult patients (≥18 years of age) admitted to the Trauma Service from April through November 2020 for a minimum of 48 hours. Patients were grouped based off COVID-19 status and compared for inpatient VTE chemoprophylaxis regimen, thrombotic complications defined as deep vein thrombosis, pulmonary embolism, myocardial infarction, and cerebrovascular accident, intensive care unit (ICU) length of stay, hospital length of stay, and mortality. Results : A total of 2,907 patients were reviewed and grouped into COVID-19-positive (n = 110) and COVID-19-negative (n = 2,797) groups. There was no difference in terms of receiving deep vein thrombosis chemoprophylaxis or type, but a longer time to initiation in the positive group ( P = 0.0012). VTE occurred in 5 (4.55%) positive and 60 (2.15%) negative patients without a significant difference between the groups, as well as no difference in type of VTE observed. Mortality was higher ( P = 0.009) in the positive group (10.91%). Positive patients had longer median ICU LOS ( P = 0.0012) and total LOS ( P < 0.001). Conclusion : There were no increased rates of VTE complications between COVID-19-positive and -negative trauma patients, despite a longer time to initiation of chemoprophylaxis in the COVID-19-positive group. COVID-19-positive patients had increased ICU LOS, total LOS, and mortality, which are likely due to multifactorial causes but primarily related to their underlying COVID-19 infection.

A pilot study using simvastatin in the treatment of nonalcoholic steatohepatitis: A randomized placebo-controlled trial.

GOALS: This study was designed to assess the utility of statin therapy in patients with biopsy proven nonalcoholic steatohepatitis (NASH) and hyperlipidemia. BACKGROUND: Nonalcoholic fatty liver disease, as the hepatic manifestation of the metabolic syndrome, has become a growing public health concern. Nonalcoholic steatohepatitis (NASH) represents a subset of nonalcoholic fatty liver disease manifested by hepatic fatty infiltration and inflammation which may progress to cirrhosis and its subsequent complications, to include hepatocellular carcinoma. As the metabolic syndrome is thought to be central in the pathogenesis of NASH, it has been speculated that medications that improve metabolic profiles may be beneficial in treatment. In fact, recent studies have demonstrated potential benefit of 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins), which are used in clinical practice to improve lipid panels. STUDY: This double-blinded randomized placebo-controlled trial compared the HMG-CoA reductase inhibitor, simvastatin, with placebo in the treatment of NASH over a 12-month period using serum aminotransferases and repeat liver biopsy to assess for improvement. RESULTS: Sixteen patients with biopsy proven NASH were enrolled: 14 completed the study and 10 underwent 1-year repeat liver biopsy. Mean age: 53 years (+/-10.1), mean body mass index: 32.4 (+/-6.1) with 11 male and 5 female patients. Although a 26% reduction in low-density lipoprotein was seen in the simvastatin group compared with placebo, there was no statistically significant improvement in serum aminotransferases, hepatic steatosis, necroinflammatory activity or stage of fibrosis within or between groups. CONCLUSIONS: In this pilot trial, monotherapy with simvastatin does not seem to be an effective treatment for NASH.

B vitamins alleviate indices of neuropathic pain in diabetic rats.

There are sporadic reports that assorted combinations of B vitamins can alleviate pain in diabetic patients, but there is neither agreement on the relative efficacy of individual B vitamins nor understanding of the mechanisms involved. We therefore investigated the efficacy of a cocktail of the vitamins B1, B6 and B12 in alleviating behavioral indices of sensory dysfunction such as allodynia and hyperalgesia in diabetic rats and also the relative contribution of individual components of the cocktail. Repeated daily treatment with the cocktail of B vitamins for 7-9 days ameliorated tactile allodynia and formalin-evoked hyperalgesia in a dose-dependent manner and also improved sensory nerve conduction velocity in diabetic rats. Investigation of the contribution of individual B vitamins suggested that all three participated with variable efficacy in the alleviation of allodynia after protracted, but not single dose treatment. Only vitamin B6 improved sensory nerve conduction velocity slowing in diabetic rats when given alone. To address potential mechanisms of action, we measured markers of oxidative stress (lipid and protein oxidation) and inflammation (cyclooxygenase-2 (COX-2) and TNFalpha protein) in the nerve but treatment with the vitamin B cocktail did not significantly affect any of these parameters. The positive effects of B vitamins on functional and behavioral disorders of diabetic rats suggest a potential for use in treating painful diabetic neuropathy.

Ingestion of superwarfarin leading to coagulopathy: a case report and review of the literature.

Superwarfarins are found in many pesticides, including D-con, Prufe I and II, Ramik, Talon-G, Ratak, and Contrac. Ingestion of can lead to significant morbidity and even mortality. Physicians need to consider this diagnosis in any patient presenting with coagulopathy of unclear etiology. We present a patient with superwarfarin-induced coagulopathy and review previous cases in adults in the literature. The patient is a 60-year-old man who presented to our medical center with painless hematuria. Laboratory studies revealed an elevated prothrombin time (PT) (42.5 seconds), partial thromboplastin time (PTT) (64.6 seconds), and international normalized ratio (INR) of 7. Liver-associated enzymes were normal, and complete blood cell count (CBC) showed no evidence of disseminated intravascular coagulation. Subsequent work-up included the absence of an inhibitor by mixing study and deficiencies of vitamin K-dependent coagulation factors. The patient's warfarin level was negative. A brodifacoum level was positive, confirming superwarfarin-induced coagulopathy. The patient is currently doing well with normal coagulation studies after receiving high doses of vitamin K for several weeks. The cause of his exposure to superwarfarin remains uncertain. Physicians need to be cognizant of this unusual cause of coagulopathy in adults. The appropriate diagnostic work-up and unique features of therapy are discussed.