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The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the major disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1 knock-out (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn (GNA), motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into two adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at eight weeks post-treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year, and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primate, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

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Recombinant adeno-associated virus (rAAV)-based gene therapy is entering clinical and commercial stages at an unprecedented pace. Triple transfection of HEK293 cells is currently the most widely used platform for rAAV manufacturing. Here, we develop low-cis triple transfection that decreases transgene plasmid use by 10- to 100-fold and overcomes several major limitations associated with standard triple transfection. This new method improves packaging of yield-inhibiting transgenes by up to 10-fold, and generates rAAV batches with reduced plasmid backbone contamination that otherwise cannot be eliminated in downstream processing. When tested in mice and compared with rAAV produced by standard triple transfection, low-cis rAAV shows comparable or superior potency and results in diminished plasmid backbone DNA and RNA persistence in tissue. Mechanistically, low-cis triple transfection relies on the extensive replication of transgene cassette (i.e., inverted terminal repeat-flanked vector DNA) in HEK293 cells during production phase. This cost-effective method can be easily implemented and is widely applicable to producing rAAV of high quantity, purity, and potency.

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Gene therapy using recombinant adeno-associated virus (rAAV) relies on safe, efficient, and precise in vivo gene delivery that is largely dependent on the AAV capsid. The proteinaceous capsid is highly amenable to engineering using a variety of approaches, and most resulting capsids carry substitutions or insertions comprised of natural amino acids. Here, we incorporated a non-canonical amino acid (ncAA), Nε-2-azideoethyloxycarbonyl-L-lysine (also known as NAEK), into the AAV5 capsid using genetic code expansion, and serendipitously found that several NAEK-AAV5 vectors transduced various cell lines more efficiently than the parental rAAV5. Furthermore, one NAEK-AAV5 vector showed lung-specific transduction enhancement following systemic or intranasal delivery in mice. Structural modeling suggests that the long side chain of NAEK may impact on the 3-fold protrusion on the capsid surface that plays a key role in tropism, thereby modulating vector transduction. Recent advances in genetic code expansion have generated synthetic proteins carrying an increasing number of ncAAs that possess diverse biological properties. Our study suggests that ncAA incorporation into the AAV capsid may confer novel vector properties, opening a new and complementary avenue to gene therapy vector discovery.

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Age-associated B cells (ABC) accumulate with age and are associated with autoimmunity and chronic infection. However, their contributions to acute infection in the aged and their developmental pathways are unclear. We find that the response against influenza A virus infection in aged mice is dominated by a Fas+ GL7- effector B cell population we call infection-induced ABC (iABC). Most iABC express IgM and include antibody-secreting cells in the spleen, lung, and bone marrow. We find that in response to influenza, IgD+ CD21- CD23- ABC are the precursors of iABC and become memory B cells. These IgD+ ABC develop in germ-free mice, so are independent of foreign antigen recognition. The response of ABC to influenza infection, resulting in iABC, is T cell independent and requires both extrinsic TLR7 and TLR9 signals. In response to influenza infection, IgD+ ABC can induce a faster recovery of weight and higher total anti-influenza IgG and IgM titers that can neutralize virus. Immunization with whole inactivated virus also generates iABC in aged mice. Thus, in unimmunized aged mice, whose other B and T cell responses have waned, IgD+ ABC are likely the naive B cells with the potential to become Ab-secreting cells and to provide protection from infection in the aged.

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Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

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Previously, we discovered that influenza-generated CD4 effectors must recognize cognate Ag at a defined effector checkpoint to become memory cells. Ag recognition was also required for efficient protection against lethal influenza infection. To extend these findings, we investigated if vaccine-generated effectors would have the same requirement. We compared live infection with influenza to an inactivated whole influenza vaccine. Live infection provided strong, long-lasting Ag presentation that persisted through the effector phase. It stimulated effector generation, long-lived CD4 memory generation, and robust generation of Ab-producing B cells. In contrast, immunization with an inactivated virus vaccine, even when enhanced by additional Ag-pulsed APC, presented Ag for 3 d or less and generated few CD4 memory cells or long-lived Ab-producing B cells. To test if checkpoint Ag addition would enhance this vaccine response, we immunized mice with inactivated vaccine and injected Ag-pulsed activated APC at the predicted effector checkpoint to provide Ag presentation to the effector CD4 T cells. This enhanced generation of CD4 memory, especially tissue-resident memory in the lung, long-lived bone marrow Ab-secreting cells, and influenza-specific IgG Ab. All responses increased as we increased the density of peptide Ag on the APC to high levels. This suggests that CD4 effectors induced by inactivated vaccine require high levels of cognate Ag recognition at the effector checkpoint to most efficiently become memory cells. Thus, we suggest that nonlive vaccines will need to provide high levels of Ag recognition throughout the effector checkpoint to optimize CD4 memory generation.

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OBJECTIVE: To evaluate the effectiveness of the Traditional Chinese Medicine tonifying-kidney and regulating-liver therapy on diminished ovarian reserve (DOR). METHODS: The literature was comprehensively searched up to August 2019 using four Chinese and three English electronic databases to extract randomized clinical trials (RCTs) comparing Traditional Chinese Medicine tonifying-kidney and regulating-liver prescriptions (combined with hormone therapy or not) with Western Medicine. Data quality evaluation was conducted using the Cochrane risk of bias tool. Meta-analysis was conducted using Revman 5.3 software with effect estimates presented as mean difference (MD), risk ratio (RR), and 95% confidence interval (CI). RESULTS: A total of nine RCTs with 512 participants were extracted and eligible for Meta-analysis. There were no significant differences between Chinese medicine and Western Medicine on basal serum follicle-stimulating hormone (FSH) level (MD 0.11, 95% CI －0.52 to 0.74, 392 participants, seven trials), anti-Müllerian hormone level (MD 0.48, 95% CI －0.62 to 1.58, 95 participants, two trials), and the FSH and luteinizing hormone ratio (MD 0.01, 95% CI －0.95 to 0.96, 115 participants, two trials). Chinese medicine was more effective at improving Traditional Chinese Medicine symptom scores (TCMSS) (MD －2.39, 95% CI －3.83 to －0.94, 160 participants, three trials), effective rate of TCMSS (RR 1.18, 95% CI 1.02 to 1.36, 160 participants, three trials), antral follicle count (AFC) (MD 0.55, 95% CI 0.05 to 1.04, 155 participants, three trials), and FSH levels at 3 months post-treatment (MD -4.77, 95% CI －6.09 to －3.45, 137 participants, two trials). CONCLUSION: Compared with Western Medicine, tonifying-kidney and regulating-liver therapy is more effective at relieving symptoms and improving AFC and FSH at 3 months post-treatment.

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