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.

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.

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.

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.

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.

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.

Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy.

Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 10(12) vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD(-/-) mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD(-/-) mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD(-/-) mice maintained euglycemia, whereas untreated VLCAD(-/-) mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

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.

[Research on EGCG improving the degenerative changes of the brain in AD model mice induced with chemical drugs].

OBJECTIVE: To study the effect and mechanism of (-)-Epigallocatechin-3-gallate (EGCG) on the degeneretive changes of the brain in Alzheimer's disease (AD) model mice induced with chemical drugs. METHODS: AD model mice were established by subcutaneously injecting with 3% D-gal at the dose of 150 mg/kg body weight once daily for 6 weeks. From the third week, the mice of D-gal + V(E) 280 U/kg group, D-gal + EGCG 2 mg/(kg x d) group and D-gal + EGCG 6 mg/(kg x d) group were intragastricly given with 5.6% V(E) at the dose of 280 IU/kg and EGCG at the dose of 2 mg/kg x d or 6 mg/kg x d respectively after injection of D-gal. The mice of control group, D-gal + dd H2O group and D-gal + oil group were administered with same volume vehicle distilled water and soybean oil respectively. The pathological changes of the brain in AD model mice were observed by HE staining analysis, the immunohistochemical analysis of beta-amyloid (Abeta) and evaluating the expression of amyloid precursor protein (APP) in the hippocampus of mice by Western blot analysis. RESULTS: EGCG 2 mg/(kg x d) or 6 mg/(kg x d) 4 weeks, ig evidently released neuronal injury in the hippocampus of the AD mice induced by D-gal, and significantly reduced the express of Abeta and APP in the hippocampus of AD model mice induced by D-gal (P < 0.01). CONCLUSION: EGCG has a protective effect on AD model mice induced by D-gal by decreasing the expression of APP and beta-Amyloid in the hippocampus of mice.

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.

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.

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.

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.

Modulation of exaggerated-IgE allergic responses by gene transfer-mediated antagonism of IL-13 and IL-17e.

Asthma and allergic rhinitis are almost invariable accompanied by elevated levels of immunoglobin E (IgE), and more importantly a genetic link between IgE levels and airway hyper-responsiveness has been established. We hypothesized that expression of soluble receptors directed against interleukin (IL)-13 and IL-17e would prevent the cytokines from engaging the cell-bound receptors and therefore help to attenuate allergic responses in a Cftr(-/-)-dependent mouse model of exaggerated-IgE responses. Cftr(-/-) mice were injected with recombinant adeno-associated virus 1 (rAAV1) intramuscularly expressing soluble receptors to IL-17e (IL-17Rh1fc) or IL-13 (IL-13Ralpha2Fc). Total IgE levels, in mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapy, were lower than in the control group. Interestingly Aspergillus fumigatus (Af)-specific IgE levels were undetectable in both the mice receiving the IL-17Rh1fc and IL-13Ralpha2Fc therapies. Further flow cytometry analysis of intracellular gene expression suggests that blocking IL-17e may be interfering with signaling upstream of CD4+ and CD11b+ cells and reducing IgE levels by affecting signaling on these cell populations. In contrast it appears that IL-13 blockade acts downstream to reduce IgE levels probably by directly affecting B-cell maturation. These studies demonstrate the feasibility of targeting T helper 2 (Th2) cytokines with rAAV-delivered fusion proteins as a means to treat aberrant immune responses.

[The expression changes of N-methyl-D-aspartic acid receptor in hippocampus of offspring from female rats exposed to aluminum in the pregnancy and lactation].

OBJECTIVE: To investigate the effects of aluminum on learning and memory and the expression of N-methyl-D-aspartic acid receptor (NMDAR) of hippocampus in offspring from female rats exposed to Al in the pregnancy or lactation, and to explore the mechanism of toxic effects of Al on central nervous system (CNS) during development. METHODS: The pregnant Wistar rats were randomly divided into 3 groups based on their body weight, i.e. control group was exposed to distilled water, low exposure group (0.2%AlCl3) and high exposure group (0.4%AlCl3) were exposed orally to AlCl3 in pregnancy and lactation for 6 weeks, 10 rats each group. Aluminum content in blood and brains was determined by atomic absorption spectrophotometry (AAS). Platform experiment was used to detect the abilities of learning and memory. The expression levels of NMDARs were detected by western blot assay. RESULTS: The Al content in blood and brains of rats in exposure groups increased significantly with Al dose, as compared with the control group (P < 0.05). In platform experiment, the incubation periods of rats in low and high exposure groups were (202.71 ± 81.99) and (19.67 ± 8.44) s respectively, which were significantly lower than that [(300.00 ± 0.00) s] in control group (P < 0.01), but the mistake times of rats in low and high exposure groups were 1.43 ± 0.85 and 2.47 ± 0.99 respectively, which were significantly higher than that (0.00 ± 0.00) in control group (P < 0.01). The Al exposure could change the proportion of NMDAR subtypes, the expression levels of NR1 and NR2B in hippocampus of newborn rats in low and high exposure groups were 25.22 ± 0.68, 81.23 ± 15.37 and 24.75 ± 0.71, 56.63 ± 7.82, respectively, which were significantly lower than those (31.69 ± 3.44, 107.61 ± 9.05) in control group (P < 0.05). CONCLUSION: Aluminum exposure in pregnancy and lactation could reduce the abilities of learning and memory in newborn rats, and change the proportion of NMDAR subtypes. The reduced NR1 and NR2B expression levels may be one of important mechanisms to influence the abilities of learning and memory in offspring.

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.

Recombinant AAV serotype and capsid mutant comparison for pulmonary gene transfer of alpha-1-antitrypsin using invasive and noninvasive delivery.

Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 10(10) vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT ("long serpin") indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency-related emphysema.

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.

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.