Biodistribution and safety of a single rAAV3B-AAT vector for silencing and replacement of alpha-1 antitrypsin in Cynomolgus macaques.

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkeys. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this nondeficient model, we did not find downregulation of endogenous AAT. However, the dual-function vector did serve as a potentially "liver-sparing" alternative for high-dose liver-mediated AAT gene replacement in the context of underlying liver disease.

Serum Western Blot for the Detection of a c-Myc Protein Tag in Non-human Primates and Mice.

This protocol allows for the detection of a c-Myc tag on alpha-1 antitrypsin (AAT) delivered to species that already have endogenous AAT such as non-human primates allowing reliable and repeatable semi-quantitation of serum levels of AAT.

The roles of N6-methyladenosine and its target regulatory noncoding RNAs in tumors: classification, mechanisms, and potential therapeutic implications.

N6-methyladenosine (m6A) is one of the epigenetic modifications of RNA. The addition of this chemical mark to RNA molecules regulates gene expression by affecting the fate of the RNA molecules. This posttranscriptional RNA modification is reversible and regulated by methyltransferase "writers" and demethylase "erasers". The fate of m6A-modified RNAs depends on the function of different "readers" that recognize and bind to them. Research on m6A methylation modification has recently increased due to its important role in regulating cancer progression. Noncoding RNAs (ncRNAs) are a class of RNA molecules that are transcribed from the genome but whose roles have been overlooked due to their lack of well-defined potential for translation into proteins or peptides. However, this misconception has now been completely overturned. ncRNAs regulate various diseases, especially tumors, and it has been confirmed that they play either tumor-promoting or tumor-suppressing roles in almost all types of tumors. In this review, we discuss the m6A modification of different types of ncRNA and summarize the mechanisms involved. Finally, we discuss the progress of research on clinical treatment and discuss the important significance of the m6A modification of ncRNAs in the clinical treatment of tumors.

The application of single-cell sequencing in pancreatic neoplasm: analysis, diagnosis and treatment.

Pancreatic neoplasms, including pancreatic ductal adenocarcinoma (PDAC), intraductal papillary mucinous neoplasm (IPMN) and pancreatic cystic neoplasms (PCNs), are the most puzzling diseases. Numerous studies have not brought significant improvements in prognosis and diagnosis, especially in PDAC. One important reason is that previous studies only focused on differences between patients and healthy individuals but ignored intratumoral heterogeneity. In recent years, single-cell sequencing techniques, represented by single-cell RNA sequencing (scRNA-seq), have emerged by which researchers can analyse each cell in tumours instead of their average levels. Herein, we summarise the new current knowledge of single-cell sequencing in pancreatic neoplasms with respect to techniques, tumour heterogeneities and treatments.

SARS-CoV-2 Serosurveys: How Antigen, Isotype and Threshold Choices Affect the Outcome.

BACKGROUND: Evaluating the performance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays and clearly articulating the utility of selected antigens, isotypes, and thresholds is crucial to understanding the prevalence of infection within selected communities. METHODS: This cross-sectional study, implemented in 2020, screened PCRconfirmed coronavirus disease 2019 patients (n 86), banked prepandemic and negative samples (n 96), healthcare workers and family members (n 552), and university employees (n 327) for antiSARS-CoV-2 receptor-binding domain, trimeric spike protein, and nucleocapsid protein immunoglobulin (Ig)G and IgA antibodies with a laboratory-developed enzyme-linked immunosorbent assay and tested how antigen, isotype and threshold choices affected the seroprevalence outcomes. The following threshold methods were evaluated: (i) mean 3 standard deviations of the negative controls; (ii) 100 specificity for each antigen-isotype combination; and (iii) the maximal Youden index. RESULTS: We found vastly different seroprevalence estimates depending on selected antigens and isotypes and the applied threshold method, ranging from 0.0 to 85.4. Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3 to 25.9. CONCLUSIONS: This study revealed the importance of evaluating serosurvey tools for antigen-, isotype-, and threshold-specific sensitivity and specificity, to interpret qualitative serosurvey outcomes reliably and consistently across studies.

Comprehensive exploration of tumor immune microenvironment feature and therapeutic response in colorectal cancer based on a novel immune-related long non-coding RNA prognostic signature.

Colorectal cancer (CRC) is one of the most common malignant tumors with a high incidence rate and mortality. LncRNA is an important regulator of the immune system. It is of great significance to study immune-related lncRNAs (IR-lncRNAs) for CRC. In this study, we screened IR-lncRNAs differentially expressed in normal and CRC tissues, and Univariate Cox regression and the Least Absolute Shrinkage and Selection Operator were applied to construct IR-lncRNA prognostic signature in TCGA training dataset, and its predictive capability for the prognosis of CRC patients was verified in GSE39582 validation dataset. The novel signature was identified as an independent predictor of prognosis in CRC patients. In addition, the signature could accurately predict the feature of the immune microenvironment and therapeutic response in CRC patients. The CMap database was adopted to screen for small molecule candidate drugs that can reverse and treat high-risk CRC patients. Finally, the expression of six IR-lncRNAs were verified by qRT-PCR in clinical specimens from our patient cohort. In conclusion, we construct an IR-lncRNA prognostic signature, which is a powerful biomarker of CRC and can accurately predict the prognosis, immune microenvironment feature, and therapeutic response of CRC patients.

A nomogram for clinical estimation of acute biliary pancreatitis risk among patients with symptomatic gallstones: A retrospective case-control study.

Background/Purpose: Currently, there are no effective tools to accurately assess acute biliary pancreatitis (ABP) risk in patients with gallstones. This study aimed to develop an ABP risk nomogram in patients with symptomatic gallstones. Methods: We conducted a retrospective nested case-control study and data on 816 conservatively treated patients with symptomatic gallstones admitted to The First Affiliated Hospital of Harbin Medical University between January 6, 2007 and January 22, 2016 were retrospectively collected. We conducted a propensity-score matched (PSM) analysis based on follow-up time in a ratio of 1:4 between ABP group (n=65) and non-ABP group (n=260). These matched patients were randomly divided into study cohort (n=229) and validation cohort (n=96) according to a ratio of 7:3. In the study cohort, independent risk factors for ABP occurrence identified using Cox regression were included in nomogram. Nomogram performance and discrimination were assessed using the concordance index (C-index), area under the curve (AUC), calibration curve, decision curve analysis (DCA) and clinical impact curve (CIC). The model was also validated in the validation cohort. Results: Nomogram was based on 7 independent risk factors: age, diabetes history, gallbladder wall thickness, gallstone diameter, coexisting common bile duct (CBD) stones, direct bilirubin (DBIL), and white blood cell count (WBC). The C-index of nomogram was 0.888, and the 10-year AUCs of nomogram was 0.955. In the validation cohort, nomogram still had good discrimination (C-index, 0.857; 10-year AUC, 0.814). The calibration curve showed good homogeneity between the prediction by nomogram and the actual observation. DCA and CIC demonstrated that nomogram was clinically useful. Conclusions: The ABP risk nomogram incorporating 7 features is useful to predict ABP risk in symptomatic gallstone patients.

Tumor-derived exosomes in the cancer immune microenvironment and cancer immunotherapy.

Tumor-derived exosomes (TDEs) are key immune regulators in the tumor microenvironment. They have been shown to reshape the immune microenvironment and prevent antitumor immune responses via their immunosuppressive cargo, thereby determining responsiveness to cancer therapy. By delivering suppressive cargo to the immune cells, TDEs directly or indirectly influence the functions and antitumor activities of immune cells. TDE-based therapy is emerging as a cutting-edge and promising strategy for inhibiting tumor progression or enhancing antitumor immunity. Therefore, in this study, we reviewed the mechanism by which TDEs regulate immune cells and their applications in immunotherapy.

Metastases to the Thyroid Gland: What Can We Do?

Metastases to the thyroid gland arise from other malignant tumors such as renal cell carcinoma, colorectal cancer, lung cancer, and breast cancer. In clinical practice, the incidence is low, and the symptoms are not specific, so it is often missed and misdiagnosed. It is finally diagnosed via the comprehensive application of many diagnostic methods, such as ultrasound, fine-needle aspiration biopsy, and immunohistochemistry analysis. Surgery-based comprehensive treatment is often adopted, but because it is usually in the late stage of the primary tumor, the prognosis is poor. In order to better understand the related characteristics of thyroid metastatic cancer and then improve the clinical diagnosis and treatment and the prognosis of patients, in this paper, we systematically summarize the research status of thyroid metastatic cancer.

Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells.

Immune responses to adeno-associated virus (AAV) capsids limit the therapeutic potential of AAV gene therapy. Herein, we model clinical immune responses by generating AAV capsid-specific chimeric antigen receptor (AAV-CAR) T cells. We then modulate immune responses to AAV capsid with AAV-CAR regulatory T cells (Tregs). AAV-CAR Tregs in vitro display phenotypical Treg surface marker expression, and functional suppression of effector T cell proliferation and cytotoxicity. In mouse models, AAV-CAR Tregs mediated continued transgene expression from an immunogenic capsid, despite antibody responses, produced immunosuppressive cytokines, and decreased tissue inflammation. AAV-CAR Tregs are also able to bystander suppress immune responses to immunogenic transgenes similarly mediating continued transgene expression, producing immunosuppressive cytokines, and reducing tissue infiltration. Taken together, AAV-CAR T cells and AAV-CAR Tregs are directed and powerful immunosuppressive tools to model and modulate immune responses to AAV capsids and transgenes in the local environment.

Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus.

A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.

Engraftment of Human Hepatocytes in the PiZ-NSG Mouse Model.

We recently described the generation of a novel mouse strain that efficiently and readily engrafts human primary hepatocytes to create liver xenografts (Borel et al., Mol Ther, 25: 2477-89, 2017). A transgenic mouse strain expressing a human PiZ allele for the SerpinA1 gene was crossed with the NOD-SCID-gamma chain knockout (NSG) strain to create a recipient strain (PiZ-NSG) for human hepatocyte xenotransplantation. In this chapter we provide a description of the methods to achieve these liver xenografts in the PiZ-NSG mouse.

Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study.

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 µg/mL with VLP versus 38 µg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

In Vivo Genome Editing Partially Restores Alpha1-Antitrypsin in a Murine Model of AAT Deficiency.

CRISPR (clustered regularly interspaced short palindromic repeats) genome editing holds promise in the treatment of genetic diseases that currently lack effective long-term therapies. Patients with alpha-1 antitrypsin (AAT) deficiency develop progressive lung disease due to the loss of AAT's antiprotease function and liver disease due to a toxic gain of function of the common mutant allele. However, it remains unknown whether CRISPR-mediated AAT correction in the liver, where AAT is primarily expressed, can correct either or both defects. Here we show that AAV delivery of CRISPR can effectively correct Z-AAT mutation in the liver of a transgenic mouse model. Specifically, we co-injected two AAVs: one expressing Cas9 and another encoding an AAT guide RNA and homology-directed repair template. In both neonatal and adult mice, this treatment partially restored M-AAT in the serum. Furthermore, deep sequencing confirmed both indel mutations and precise gene correction in the liver, permitting careful analysis of gene editing events in vivo. This study demonstrates a proof of concept for the application of CRISPR-Cas9 technology to correct AAT mutations in vivo and validates continued exploration of this approach for the treatment of patients with AAT deficiency.

Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of α-1 Antitrypsin Deficiency.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

Quantification of Total Human Alpha-1 Antitrypsin by Sandwich ELISA.

In this chapter we describe an enzyme-linked immunosorbent assay (ELISA) to quantitatively measure human alpha-1 antitrypsin (AAT) protein levels in serum, other body fluids or liquid media. This assay can be used to measure the expression of the human AAT (hAAT) gene in a variety of gene transfer or gene downregulation experiments.A hAAT-specific capture antibody and a HRP-conjugated anti-AAT detection antibody are used in this assay. The conjugated anti-AAT used in this protocol, instead of the typical sandwich which employs an unconjugated antibody followed by a specifically conjugated IgG, makes the assay simpler and decreases variability. This provides a useful tool to evaluate the AAT levels in clinical and research samples and can allow fairly rapid testing of a large number of samples.

Quantification of Z-AAT by a Z-Specific "Sandwich" ELISA.

This protocol describes an enzyme-linked immunosorbent assay (ELISA) to specifically detect Z-alpha-1 antitrypsin (AAT), the most common protein variant associated with alpha-1 antitrypsin deficiency. This "sandwich" ELISA relies on an anti-Z-AAT specific capture antibody and a HRP-conjugated anti-AAT detection antibody. This method would be of interest to identify and quantify Z-AAT in a variety of samples such as cell culture medium, cell or tissue lysate, animal or patient serum. Because this method is specific and sensitive, it would be particularly valuable for detection of Z-AAT in the presence of background M-AAT, for instance when quantifying silencing of Z-AAT in patients undergoing M-AAT augmentation therapy.

Investigation of an Immunoassay with Broad Specificity to Quinolone Drugs by Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Data Sets and Advanced Quantitative Structure-Activity Relationship Analysis.

A polyclonal antibody against the quinolone drug pazufloxacin (PAZ) but with surprisingly broad specificity was raised to simultaneously detect 24 quinolones (QNs). The developed competitive indirect enzyme-linked immunosorbent assay (ciELISA) exhibited limits of detection (LODs) for the 24 QNs ranging from 0.45 to 15.16 ng/mL, below the maximum residue levels (MRLs). To better understand the obtained broad specificity, a genetic algorithm with linear assignment of hypermolecular alignment of data sets (GALAHAD) was used to generate the desired pharmacophore model and superimpose the QNs, and then advanced comparative molecular field analysis (CoMFA) and advanced comparative molecular similarity indices analysis (CoMSIA) models were employed to study the three-dimensional quantitative structure-activity relationship (3D QSAR) between QNs and the antibody. It was found that the QNs could interact with the antibody with different binding poses, and cross-reactivity was mainly positively correlated with the bulky substructure containing electronegative atom at the 7-position, while it was negatively associated with the large bulky substructure at the 1-position of QNs.

Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors.

Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

A genetic variant (rs17251221) in the calcium-sensing receptor relates to hepatocellular carcinoma susceptibility and clinical outcome treated by transcatheter hepatic arterial chemoembolization (TACE) therapy.

Experimental and epidemiologic studies indicated that calcium-sensing receptor (CaSR) polymorphisms were associated with cancer risk, yet no data are available for candidate gene and hepatocellular carcinoma (HCC) risk. To address this, we evaluated whether CaSR rs17251221 polymorphism is associated with HCC susceptibility, clinicopathological parameters, and prognosis in HCC patients treated by TACE. A TaqMan assay was used to genotype rs17251221 SNP in this case (n = 843)-control (n = 783) study. A significant increased risk of HCC was observed in patients carrying rs17251221 GG (adjusted OR 1.355, 95 % CI 1.024-1.793, P = 0.033), AG/GG genotype (adjusted OR 1.254, 95 % CI 1.007-1.561, P = 0.043), and G allele (adjusted OR 1.163, 95 % CI 1.013-1.335, P = 0.032). Furthermore, a significant association was found between Child-Pugh class, serum BCLC stage, and AFP level and rs17251221 genotypes. More importantly, individuals carrying rs17251221 AG, GG genotype showed significantly longer MST than AA genotype and significant hazard ration (AG: adjusted HR 0.484, 95 % CI 0.406-0.577, P < 0.001; GG: adjusted HR 0.633, 95 % CI 0.575-0.697, P < 0.001, respectively). Meanwhile, we found a favorable HR for AG/GG genotype carriers (adjusted HR 0.645, 95 % CI 0.542-0.768, P < 0.001). These results indicated that CaSR rs17251221 polymorphism is associated with susceptibility to HCC, and rs17251221 G allele genotype showed significant independent better prognosis of HCC patients treated with TACE.