Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy.

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders.

Bioengineering of viral vectors for therapeutic gene delivery is a pivotal strategy to reduce doses, facilitate manufacturing, and improve efficacy and patient safety. Here, we engineered myotropic adeno-associated viral (AAV) vectors via a semirational, combinatorial approach that merges AAV capsid and peptide library screens. We first identified shuffled AAVs with increased specificity in the murine skeletal muscle, diaphragm, and heart, concurrent with liver detargeting. Next, we boosted muscle specificity by displaying a myotropic peptide on the capsid surface. In a mouse model of X-linked myotubular myopathy, the best vectors-AAVMYO2 and AAVMYO3-prolonged survival, corrected growth, restored strength, and ameliorated muscle fiber size and centronucleation. In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function. Our pipeline is compatible with complementary AAV genome bioengineering strategies, as demonstrated here with two promoters, and could benefit many clinical applications beyond muscle gene therapy.

Transcriptomic profiling revealed FZD10 as a novel biomarker for nasopharyngeal carcinoma recurrence.

Background: Nasopharyngeal carcinoma (NPC) is a type of cancers that develops in the nasopharynx, the very upper part of the throat behind the nose. NPC is typically diagnosed in later stages of the disease and has a high rate of recurrence due to the location of the tumor growth site. In this study, we compared the gene expression profiles of NPC tissues from patients with and without recurrence to identify potential molecular biomarkers of NPC recurrence. Methods: Microarrays were used to analyze the expression of genes in 15 NPC tissues taken at the time of diagnosis and at the site of recurrence following therapeutic treatment. Pathway enrichment analysis was used to examine the biological interactions between the major differentially expressed genes. The target identified was then validated using immunohistochemistry on 86 NPC tissue samples. Results: Our data showed that the Wnt signaling pathway was enhanced in NPC tissues with recurrence. FZD10, a component of the Wnt signaling pathway, was significantly expressed in NPC tissues, and was significantly associated with NPC recurrence. Conclusion: Our study provides new insights into the pathogenesis of NPC and identifies FZD10 as a potential molecular biomarker for NPC recurrence. FZD10 may be a promising candidate for NPC recurrence and a potential therapeutic target.

Limited Sensitivity of a Rapid SARS-CoV-2 Antigen Detection Assay for Surveillance of Asymptomatic Individuals in Thailand.

COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is a global pandemic. Therefore, rapid and accurate tests for SARS-CoV-2 screening are urgently needed to expedite disease prevention and control especially in community transmission. Since late December 2020, Thailand has faced a new wave of COVID-19 outbreaks. The Thai National Disease Control program at the Ministry of Public Health has identified suitable measure for mass screening. A SARS-CoV-2 antigen-based assay is a surveillance option for active cases. Here, we evaluated the feasibility and test performance of a rapid SARS-CoV-2 antigen test during our field activities in 1,100 asymptomatic individuals in Samut Sakhon, Thailand, during the second wave COVID-19 outbreak (December 26-30, 2020). The results showed that the rapid antigen test had a sensitivity of 47.97% (95% CI: 36.10-59.96%) and a specificity of 99.71% (95% CI: 99.15-99.94%) versus standard reverse-transcriptase polymerase chain reaction. The rapid test performed better in cases with higher viral loads determined by the cycle threshold value. In real-world setting, the test performance can be compromised by several factors including viral loads, logistic chains, temperature, technical expertise of the operators, validity, and accuracy of the testing itself. Our study highlights a prerequisite for reevaluation of any given testing before implementing it at the national level.

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

Validation of miR-20a as a Tumor Suppressor Gene in Liver Carcinoma Using Hepatocyte-Specific Hyperactive piggyBac Transposons.

We established a semi-high-throughput in vivo screening platform using hyperactive piggyBac (hyPB) transposons (designated as PB-miR) to identify microRNAs (miRs) that inhibit hepatocellular carcinoma (HCC) development in vivo, following miR overexpression in hepatocytes. PB-miRs encoding six different miRs from the miR-17-92 cluster and nine miRs from outside this cluster were transfected into mouse livers that were chemically induced to develop HCC. In this slow-onset HCC model, miR-20a significantly inhibited HCC. Next, we developed a more aggressive HCC model by overexpression of oncogenic Harvey rat sarcoma viral oncogene homolog (HRASG12V) and c-MYC oncogenes that accelerated HCC development after only 6 weeks. The tumor suppressor effect of miR-20a could be demonstrated even in this rapid-onset HRASG12V/c-MYC HCC model, consistent with significantly prolonged survival and decreased HCC tumor burden. Comprehensive RNA expression profiling of 95 selected genes typically associated with HCC development revealed differentially expressed genes and functional pathways that were associated with miR-20a-mediated HCC suppression. To our knowledge, this is the first study establishing a direct causal relationship between miR-20a overexpression and liver cancer inhibition in vivo. Moreover, these results demonstrate that hepatocyte-specific hyPB transposons are an efficient platform to screen and identify miRs that affect overall survival and HCC tumor regression.

Distinct transduction of muscle tissue in mice after systemic delivery of AAVpo1 vectors.

The human musculature is a promising and pivotal target for human gene therapy, owing to numerous diseases that affect this tissue and that are often monogenic, making them amenable to treatment and potentially cure on the genetic level. Particularly attractive would be the possibility to deliver clinically relevant DNA to muscle tissue from a minimally invasive, intravenous vector delivery. To date, this aim has been approximated by the use of Adeno-associated viruses (AAV) of different serotypes (rh.74, 8, 9) that are effective, but unfortunately not specific to the muscle and hence not ideal for use in patients. Here, we have thus studied the muscle tropism and activity of another AAV serotype, AAVpo1, that was previously isolated from pigs and found to efficiently transduce muscle following direct intramuscular injection in mice. The new data reported here substantiate the usefulness of AAVpo1 for muscle gene therapies by showing, for the first time, its ability to robustly transduce all major muscle tissues, including heart and diaphragm, from peripheral infusion. Importantly, in stark contrast to AAV9 that forms the basis for ongoing clinical gene therapy trials in the muscle, AAVpo1 is nearly completely detargeted from the liver, making it a very attractive and potentially safer option.

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

Transcriptome meta-analysis reveals dysregulated pathways in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant cancer arising from the epithelial surface of the nasopharynx that mostly appears in advanced stages of the disease, leading to a poor prognosis. To date, a number of mRNA profiling investigations on NPC have been reported in order to identify suitable biomarkers for early detection. However, the results may be specific to each study with distinct sample types. In this study, an integrative meta-analysis of NPC transcriptome data was performed to determine dysregulated pathways, potentially leading to identification of molecular markers. Ten independent NPC gene expression profiling microarray datasets, including 135 samples from NPC cell lines, primary cell lines, and tissues were assimilated into a meta-analysis and cross-validation to identify a cohort of genes that were significantly dysregulated in NPC. Bioinformatics analyses of these genes revealed the significant pathways and individual players involving in cellular metabolism, cell cycle regulation, DNA repair, as well as ErbB pathway. Altogether, we propose that dysregulation of these molecular pathways in NPC might play a role in the NPC pathogenesis, providing clues, which could eventually translate into diagnostic and therapeutic approaches.

ERBB3 as an independent prognostic marker for nasopharyngeal carcinoma.

AIM: Although the ERBB proteins have been shown to be associated in many types of human tumours and serve as important cancer therapeutic targets, however, data regarding the expression and clinical relevance of ERBBs in nasopharyngeal carcinoma (NPC) are still conflicting. The aim of this study is to investigate the expression pattern of all ERBB members simultaneously in NPC tissues using immunohistochemistry and determine their clinical relevance. METHODS: The expression of all members of ERBB proteins was evaluated using immunohistochemistry on 82 NPC tissue samples. Relationship between the ERBB protein expression, clinicopathological parameters and patient outcome was assessed using univariate and multivariate analyses. RESULTS: We found that ERBB1, ERBB2 and ERBB3 were strongly expressed in the normal nasopharyngeal epithelial cells. A marked reduction of ERBB1 and ERBB2 expression in NPC was observed compared with the non-cancerous tissues. 76 of 82 (92.7%) cases were ERBB3-positive, while ERBB4 was not expressed in both normal and NPC. The univariate log-rank analysis showed that regional lymph node metastasis, systemic metastasis, recurrence and ERBB3 expression were associated with patient survival. The ERBB3 expression was not correlated to other clinicopathological factors. Furthermore, multivariate analysis revealed that ERBB3 expression was an independent prognostic factor influencing patient survival. CONCLUSIONS: Our results suggested that the expression of ERBB3 is associated with patient survival and could serve as a novel and valuable predictor for prognostic evaluation of patients with NPC.

Nasopharyngeal carcinoma signaling pathway: an update on molecular biomarkers.

Nasopharyngeal carcinoma (NPC) is an uncommon cancer, which has a distinctive ethnic and geographic distribution. Etiology of NPC is considered to be related with a complex interaction of environmental and genetic factors as well as Epstein-Barr virus infection. Since NPC is located in the silent painless area, the disease is usually therefore diagnosed at the advanced stages; hence early detection of NPC is difficult. Furthermore, understanding in molecular pathogenesis is still lacking, pondering the identification of effective prognostic and diagnostic biomarkers. Dysregulation of signaling molecules in intracellular signal transduction, which regulate cell proliferation, apoptosis, and adhesion, underlines the basis of NPC pathogenesis. In this paper, the molecular signaling pathways in the NPC are discussed for the holistic view of NPC development and progression. The important insights toward NPC pathogenesis may offer strategies for identification of novel biomarkers for diagnosis and prognosis.

Prolactin alters the mRNA expression of osteoblast-derived osteoclastogenic factors in osteoblast-like UMR106 cells.

Prolactin (PRL) is known to participate in the lactation-induced maternal bone loss, presumably by inducing the release of receptor activator of nuclear factor-κB ligand (RANKL), a potent osteoclastogenic factor from osteoblasts. Since maternal bone resorption was too massive to be solely explained by RANKL and osteoclasts did not express PRL receptors (PRLR), the involvement of some other osteoblast-derived osteoclastogenic modulators was anticipated. Herein, the authors used quantitative real-time PCR to investigate the mRNA expressions of various osteoclastogenic factors in osteoblast-like UMR106 cells directly exposed to PRL for 48 h. These cells were found to express PRLR and respond to 300 ng/ml PRL by increasing RANKL mRNA expression. This PRL concentration (comparable to plasma PRL levels in lactation) also induced the upregulation of monocyte chemoattractant protein (MCP)-1, cyclooxygenase (Cox)-2, and ephrin-B1, whereas a higher concentration (500 ng/ml) was required to upregulate tumor necrosis factor (TNF)-α and interleukin (IL)-1. However, 100-500 ng/ml PRL affected neither the cell proliferation, the cell viability nor the mRNA expressions of macrophage colony-stimulating factor, IL-6, ephrin type-B receptor 4 and ephrin-B2. In conclusion, besides RANKL overexpression, PRL upregulated the expressions of other osteoclastogenic modulators, i.e., MCP-1, Cox-2, TNF-α, IL-1, and ephrin-B1, thus, further explaining how PRL induced bone loss in lactating mothers.