AAV9:PKP2 improves heart function and survival in a Pkp2-deficient mouse model of arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need. Mutations in Plakophilin-2 (PKP2), encoding a desmosomal protein, account for approximately 40% of ARVC cases and result in reduced gene expression. METHODS: Our goal is to examine the feasibility and the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2. RESULTS: We show that a single dose of AAV9:PKP2 gene delivery prevents disease development before the onset of cardiomyopathy and attenuates disease progression after overt cardiomyopathy. Restoration of PKP2 expression leads to a significant extension of lifespan by restoring cellular structures of desmosomes and gap junctions, preventing or halting decline in left ventricular ejection fraction, preventing or reversing dilation of the right ventricle, ameliorating ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling. RNA sequencing analyses show that restoration of PKP2 expression leads to highly coordinated and durable correction of PKP2-associated transcriptional networks beyond desmosomes, revealing a broad spectrum of biological perturbances behind ARVC disease etiology. CONCLUSIONS: We identify fundamental mechanisms of PKP2-associated ARVC beyond disruption of desmosome function. The observed PKP2 dose-function relationship indicates that cardiac-selective AAV9:PKP2 gene therapy may be a promising therapeutic approach to treat ARVC patients with PKP2 mutations.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heart disease that leads to abnormal heartbeats and a higher risk of sudden cardiac death. ARVC is often caused by changes in a gene called PKP2, that then makes less PKP2 protein. PKP2 protein is important for the normal structure and function of the heart. Human ARVC characteristics can be mimicked in a mouse model missing this gene. Given no therapeutic option, our goal was to test if adding a working copy of PKP2 gene in the heart of this mouse model, using a technique called gene therapy that can deliver genes to cells, could improve heart function. Here, we show that a single dose of PKP2 gene therapy can improve heart function and heartbeats as well as extend lifespan in mice. PKP2 gene therapy may be a promising approach to treat ARVC patients with PKP2 mutations.

Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

BACKGROUND: Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS: Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS: We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSIONS: Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Platelet-Derived Growth Factor-D Fusion-Positive Dermatofibrosarcoma Protuberans: Case Report of an Atypical Breast Mass and Literature Review.

Dermatofibrosarcoma protuberans (DFSP) is a rare, CD34+ mesenchymal neoplasm that classically involves the dermis. A COL1A1::PDGFB t(17;22) translocation is present in 91.4% to 96% of cases, resulting in aberrant proliferation due to tyrosine kinase hyperactivity. Here, we present a postmenopausal woman with a CD34-positive spindle cell neoplasm of the breast without cutaneous involvement, lacking muscle marker expression, STAT6 expression, and 13q14 deletion by fluorescence in situ hybridization (FISH). Although the classic PDGFB translocation was not detected by FISH, the overall features were highly suspicious for DFSP. Subsequent RNA-based next-generation sequencing revealed an EMILIN2::PDGFD fusion. A literature review showed that PDGFD fusions can be detected in up to 55% PDGFB FISH negative cases, with EMILIN2::PDGFD fusion highly associated with fibrosarcomatous transformation. This holds important diagnostic and prognostic information as fibrosarcomatous-DFSP is associated with higher recurrence and metastatic potential. The tumor was completely resected with clear margins, showed no fibrosarcomatous areas, and no evidence of recurrence is documented 2 years since resection. This review and case report adds to the literature regarding PDGFD-translocation positive DFSP as a differential diagnosis of CD34-positive spindle cell tumors of the breast, while emphasizing the prognostic importance of EMILIN2::PDGFD fusions.

Comparison of Ocular Biometric Parameters Between Hispanic and Non-Hispanic Ethnicities in White Adults Undergoing Cataract Surgery.

OBJECTIVES: To compare ocular biometric parameters between Hispanic and non-Hispanic White adult patients undergoing cataract surgery. METHODS: We included 433 adult patients undergoing surgery for senile cataract. Only patients with race and ethnicities of Hispanic and non-Hispanic White were included. The following parameters measured by the IOLMaster 700 were compared between Hispanic and non-Hispanic patients: mean keratometry, corneal astigmatism, anterior chamber depth (ACD), lens thickness, vitreous length, axial length, white-to-white diameter, and emmetropic intraocular lens power. RESULTS: There were 219 Hispanic patients and 214 non-Hispanic patients with a mean age of 70.1±7.7 years (range, 50-88 years), and 66.7% were women. Although sex distribution was similar between the two groups, Hispanic patients had a lower age compared with non-Hispanic patients (69.3±8.3 vs. 70.9±6.9 years, P=0.02). In biometric values, ACD was significantly lower in Hispanic patients (3.07±0.40 mm) than in non-Hispanic patients (3.16±0.37 mm, P=0.01). Such statistically significant difference persisted after adjustment for age and sex (P=0.01). No other significant differences were found in other ocular parameters measured. CONCLUSIONS: Anterior chamber depth is significantly shorter in Hispanic patients compared with non-Hispanic patients. Such ethnic difference should be considered when performing cataract and corneal surgeries because this ethnic difference may be associated with a higher risk of corneal endothelial injury.

Bioprocess optimization for cell culture based influenza vaccine production.

Uncertainties and shortcomings associated with the current influenza vaccine production processes demand attention and exploration of new vaccine manufacture technologies. Based on a newly developed mammalian cell culture-based production process we investigated selected process parameters and describe three factors that are shown to impact productivity, process robustness and development time. They are time of infection, harvest time and virus input, or multiplicity of infection (MOI). By defining the time of infection as 4-5 days post cell seeding and harvest time as 2-3 days post-infection and comparing their effect on virus production, MOI is subsequently identified as the most impactful process parameter for live attenuated influenza vaccine (LAIV) manufacture. Infection at very low MOI (between 10(-4) and 10(-6) FFU/cell) resulted in high titer virus production (up to 30-fold productivity improvement) compared to higher MOI infections (10(-3) to 10(-2) FFU/cell). Application of these findings has allowed us to develop a platform process that can reduce the development time to approximately three weeks for an influenza vaccine manufacture process for new strains.