Lyophilization as an effective tool to develop AAV8 gene therapy products for refrigerated storage.

Recombinant adeno-associated virus (rAAV) has emerged as the leading gene delivery platform for treatment of monogenic disorders. Currently, for clinical and commercial products, rAAVs are typically formulated and stored below -65 °C as frozen liquid. Their long-term storage is often far from ideal because it may result in shorter drug product (DP) shelf-life compared to recombinant protein-based biologics, and also presents challenges for supply chain and inventory management. Consequently, there is great interest in developing robust lyophilized AAV DPs that are stable at 2 to 8 °C. In this study, we evaluated formulation excipients required for stable lyophilized AAV8 products including buffers, salts, cryoprotectants/lyoprotectants, surfactants, and bulking agents, and optimized the concentrations and ratios between the excipients. This led to the identification of the lead formulation that demonstrated short-term in-solution stability at 25 °C and, upon lyophilization, sufficient long-term stability at 2 to 8 °C. Our study demonstrated that, in the presence of 110 mM salts, mannitol can serve as an effective bulking agent with the appropriate formulation and lyophilization process design, and the sucrose to mannitol ratio is critical to maintain the stability and cake appearance of the lyophilized AAV8 DP. Thorough characterization of the effect of formulation components on the properties and quality of the lyophilized DP led to an optimized AAV8 lyophilized DP. This approach could be applied to streamline the future development of lyophilized AAV gene therapy products with various target transgenes and capsid serotypes.

A Case of Latent Plasmodium Falciparum Malaria in a Patient With Coexisting Systemic Lupus Erythematosus (SLE) and Neuromyelitis Optica Spectrum Disorder (NMOSD).

Malaria is a global health concern with high morbidity and mortality. It is often attributed to the Plasmodium (P.) falciparum species, particularly in sub-Saharan Africa, and it normally has an incubation period of seven to 14 days. Dormant disease secondary to P. vivax and P. ovale is well-reported, yet only a handful of cases report dormant malaria secondary to P. falciparum. Even though malaria is significantly less common in the United States in comparison to other parts of the world, it is still a growing concern given international travel from endemic regions and a growing immunocompromised population. Here, we present a case of Plasmodium falciparum malaria in a patient with systemic lupus erythematosus (SLE) with neuromyelitis optica spectrum disorder (NMOSD) and renal transplant without travel to sub-Saharan Africa in 10 years.

"Diffusion of innovations": a feasibility study on the pericapsular nerve group block in the emergency department for hip fractures.

OBJECTIVE: Hip fractures are associated with significant morbidity and mortality. Ultrasound-guided peripheral nerve blocks are a safe method to manage pain and decrease opioid usage. The pericapsular nerve group (PENG) block is a novel, potentially superior block because of its motor-sparing effects. Through training, simulation, and supervision, we aim to determine whether it is feasible to perform the PENG block in the emergency department. METHODS: Phase 1 consisted of emergency physicians attending a workshop to demonstrate ultrasound proficiency, anatomical understanding, and procedural competency using a low-fidelity model. Phase 2 consisted of a prospective, observational, feasibility study of 10 patients with hip fractures. Pain scores, side effects, and opioid usage data were collected. RESULTS: The median pain score at time 0 (time of block) was 9 (interquartile range [IQR], 6.5-9). The median pain score at 30 minutes was 4 (IQR, 2.0-6.8) and 3.5 (IQR, 1.0-4.8) at 4 hours. All 10 patients required narcotics prior to the initiation of the PENG block with a median dosage of 6.25 morphine milligram equivalents (MME; IQR, 4.25-7.38 MME). After the PENG block, only 30% of the patients required further narcotics with a median dosage of 0 MME (IQR, 0-0.6 MME) until operative fixation. CONCLUSION: In this feasibility study, PENG blocks were safely administered by trained emergency physicians under supervision. We demonstrated data suggesting a trend of pain relief and decreased opiate requirements, and further investigation is necessary to measure efficacy.

Severity of Sleep Apnea and COVID-19 Illness.

OBJECTIVE: To characterize the relationship between severity of sleep apnea and coronavirus disease 2019 (COVID-19) hospitalization and severe illness. STUDY DESIGN: Retrospective cohort study. SETTING: Montefiore Health System in the Bronx, New York. METHODS: The data set consisted of adult patients with an active diagnosis of obstructive sleep apnea in the past 2 years and a positive severe acute respiratory syndrome coronavirus 2 quantitative polymerase chain reaction test at our institution between March 16, 2020, and May 26, 2020. Sleep apnea severity and continuous positive airway pressure compliance data were abstracted from the electronic medical record. The International Classification of Diseases, 10th Revision was used to classify comorbidities. RESULTS: A total of 461 patients with sleep apnea tested positive for COVID-19, of whom 149 were excluded for missing data in the electronic medical record. Patients with moderate and severe sleep apnea had higher rates of COVID-19 hospitalization compared to those with mild sleep apnea (P = .003). This association was reduced when accounting for confounders, most notably the Charlson Comorbidity Index, a measure of comorbid illness burden. Moderate and severe sleep apnea were associated with increased Charlson Comorbidity Indices, compared to mild sleep apnea (P = .01). Sleep apnea severity was not associated with a composite outcome of mechanical ventilation, intensive care unit admission, and death. CONCLUSION: Sleep apnea severity was associated with the Charlson Comorbidity Index and may be a risk factor for COVID-19 hospitalization. We found no evidence that sleep apnea severity among hospitalized patients was associated with a composite outcome of mechanical ventilation, intensive care unit admission, and death.

Implementation of the telephone montreal cognitive assessment in a telemedicine based pre-admission testing clinic during COVID-19.

Introduction: Postoperative delirium (POD) affects 10-70% of patients 60 years or older and has been linked to increasing length of hospitalization, mortality, and morbidity. Pre-existing cognitive impairment is a predictor of POD. COVID-19 restricted use of in-person cognitive screens. The Telephone Montreal Cognitive Assessment (T-MoCA) can screen for cognitive dysfunction remotely. We evaluated the feasibility of administering T-MoCA in a multiethnic population during pre-operative testing televisits. Methods: Patients scheduled for surgery between July 2020 and August 2020 were asked to participate in the T-MoCA at the end of their preadmission testing (PAT) televisit. A retrospective chart review was conducted to collect patient comorbidities and demographics. Patients were stratified by negative (T-MoCA≥19) or positive (T-MoCA<19) for mild cognitive impairment (MCI) and compared using 2-tailed χ2-tests. Univariate logistic regression was used to identify associations between patient characteristics and positive T-MoCA result. Results: Fifty out of 65 (77%) patients who consented to the T-MoCA completed the test. The average time to complete the assessment was 10.5 mins. Twenty two (44%) had a negative score and 28 (56%) had a positive score. Patients who had a positive T-MoCA were older (70.04±7.61 yrs) compared to those with a negative T-MoCA (67.68±4.69 yrs, p=0.007), although the distribution of patients above and below age 65 was not different (p=0.243). The two groups did not vary by gender, race/ethnicity, obesity, surgery type, or medical co-morbidities. When we examined our population for predictors of a positive T-MoCA, we found a trend toward men being less likely to score positive on T-MoCA (OR=0.33, 95% CI: 0.10-1.10, p=0.07) compared to women; and that patients with Hispanic race/ethnicity were more likely to test positive on the T-MoCA (OR=4.13, 95% CI: 0.84-20.28, p=0.08) compared to Non-Hispanic Whites. Conclusions: Implementation of the T-MoCA in a telemedicine-based PAT setting is feasible. In our cohort, most people who consented to the assessment completed it, and more than half scored positively, which may have important implications on the surgical plan and post-operative recovery. There may be limitations in using T-MoCA in certain populations, such as non-English preferred language, hearing difficulties, lack of focus, and use of external aids, which would need to be explored in a larger sample size.

Mutational Analysis of ZFY in Sporadic Parathyroid Adenomas.

CONTEXT: The molecular pathogenesis of sporadic parathyroid adenomas is incompletely understood, with alterations in cyclin D1/PRAD1 and MEN1 most firmly established as genetic drivers. The gene encoding the X-linked zinc finger protein (ZFX) has recently been implicated in the pathogenesis of a subset of parathyroid adenomas after recurrent, hotspot-focused somatic mutations were identified. ZFX escapes X inactivation and is transcribed from both alleles in women, and a highly homologous gene encoding the Y-linked zinc finger protein (ZFY) provides dosage compensation in males. OBJECTIVE: We sought to investigate the role of ZFY mutation in sporadic parathyroid adenoma. INTERVENTION: Polymerase chain reaction and Sanger sequencing were used to examine DNA from typically presenting, sporadic (nonfamilial, nonsyndromic) parathyroid adenomas from male patients for mutations within the ZFY gene. RESULTS: No mutations were identified among 117 adenomas. CONCLUSIONS: The absence of ZFY mutations in this series suggests that ZFY rarely, if ever, acts as a driver oncogene in sporadic parathyroid adenomas. The apparent differences in tumorigenic capabilities between the closely related zinc finger proteins ZFX and ZFY suggest that structure-function studies could represent an opportunity to gain insight into neoplastic processes in the parathyroid glands.

A placental growth factor is silenced in mouse embryos by the zinc finger protein ZFP568.

Insulin-like growth factor 2 (IGF2) is the major fetal growth hormone in mammals. We identify zinc finger protein 568 (ZFP568), a member of the rapidly evolving Kruppel-associated box-zinc finger protein (KRAB-ZFP) family linked primarily to silencing of endogenous retroelements, as a direct repressor of a placental-specific Igf2 transcript (designated Igf2-P0) in mice. Loss of Zfp568, which causes gastrulation failure, or mutation of the ZFP568-binding site at the Igf2-P0 promoter causes inappropriate Igf2-P0 activation. Deletion of Igf2 can completely rescue Zfp568 gastrulation phenotypes through late gestation. Our data highlight the exquisite selectivity with which members of the KRAB-ZFP family repress their targets and identify an additional layer of transcriptional control of a key growth factor regulating fetal and placental development.