Producing high-quantity and high-quality recombinant adeno-associated virus by low-cis triple transfection.

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.

Improved gene therapy for spinal muscular atrophy in mice using codon-optimized hSMN1 transgene and hSMN1 gene-derived promotor.

Physiological regulation of transgene expression is a major challenge in gene therapy. Onasemnogene abeparvovec (Zolgensma®) is an approved adeno-associated virus (AAV) vector gene therapy for infants with spinal muscular atrophy (SMA), however, adverse events have been observed in both animals and patients following treatment. The construct contains a native human survival motor neuron 1 (hSMN1) transgene driven by a strong, cytomegalovirus enhancer/chicken ß-actin (CMVen/CB) promoter providing high, ubiquitous tissue expression of SMN. We developed a second-generation AAV9 gene therapy expressing a codon-optimized hSMN1 transgene driven by a promoter derived from the native hSMN1 gene. This vector restored SMN expression close to physiological levels in the central nervous system and major systemic organs of a severe SMA mouse model. In a head-to-head comparison between the second-generation vector and a benchmark vector, identical in design to onasemnogene abeparvovec, the 2nd-generation vector showed better safety and improved efficacy in SMA mouse model.

Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice.

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.

Base editing rescue of spinal muscular atrophy in cells and in mice.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9-mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA.

Biodegradable porous polymeric drug as a drug delivery system: alleviation of doxorubicin-induced cardiotoxicity via passive targeted release.

Doxorubicin (DOX) is an effective chemotherapeutic drug developed against a broad range of cancers, and its clinical applications are greatly restricted by the side effects of severe cardiotoxicity during tumour treatment. Herein, the DOX-loaded biodegradable porous polymeric drug, namely, Fc-Ma-DOX, which was stable in the circulation, but easy to compose in the acidic medium, was used as the drug delivery system avoiding the indiscriminate release of DOX. Fc-Ma was constructed via the copolymerization of 1,1'-ferrocenecarbaldehyde with d-mannitol (Ma) through the pH-sensitive acetal bonds. Echocardiography, biochemical parameters, pathological examination, and western blot results showed that DOX treatment caused increased myocardial injury and oxidative stress damage. In contrast, treatment with Fc-Ma-DOX significantly reduced myocardial injury and oxidative stress by DOX treatment. Notably, in the Fc-Ma-DOX treatment group, we observed a significant decrease in the uptake of DOX by H9C2 cells and a significant decrease in reactive oxygen species (ROS) production.

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells.

BACKGROUND: Gene therapy is a promising approach for the treatment of various cancers. However, most viral vectors used for this purpose carry risks, including potential integration into the host genome. OBJECTIVE: We addressed this issue in the present study by constructing an episomal lentiviral vector using the .-interferon matrix attachment region to express the microRNA -145(miR-145), and examining the effect of miR-145 overexpression on human esophageal carcinomas (EC) cells. Some recent relevant patents are also discussed. METHOD: Expression levels of miR-145 and the marker protein enhanced green fluorescent protein (EGFP) in infected ECA109 and EC9706 human esophageal carcinoma cells were detected by quantitative PCR and flow cytometry, respectively. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry, respectively. Plasmid rescue experiments and fluorescence in situ hybridization were used to determine the episomal status of the transfected vector. RESULTS: We found that EGFP and miR-145 were highly expressed in EC cells, and miR-145 overexpression inhibited cell proliferation and induced apoptosis. Moreover, the lentiviral vector did not integrate into the host genome, but was maintained episomally at lower copy numbers. CONCLUSION: Taken together, our results demonstrate that miR-145-expressing episomal lentiviral vectors are a promising tool for gene therapy in the treatment of EC.

Neuroprotective effect of AG490 in experimental traumatic brain injury of rats.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability in children and young adults worldwide. Therefore, we investigated the role of AG490 in regulating brain oedema, expression of CD40 and neurological function after TBI. METHODS: Sprague Dawley rats (n = 240) were randomly divided into a sham operation group, TBI+saline group and TBI+AG490 (JAK/STAT inhibitor) group. Members of each group were euthanized at 6, 12, 24 or 72 hours after injury. Neurological severity score (NSS) was used to evaluate the severity of neurological damage. Brain water was quantitated by wet/dry weight method. The expression of CD40 was assessed by flow cytometry. RESULTS: In both the TBI+saline group and the TBI+AG490 group, the brain water content was elevated after TBI, reached a peak at 24-hour and remained high for the rest of the period investigated; the expression of CD40 reached a peak 24 hours after TBI; the NSS was elevated after TBI and then decreased after 6 hours. Elevations in the level of CD40, degree of brain edema and NSS after TBI were significantly reduced in TBI+AG490 group. CONCLUSION: Inhibition of the JAK/STAT signalling pathway reduces brain oedema, decreases the expression of CD40 and exerts neuroprotective effects after TBI.

[Analysis of volatile oil of garlic by gas chromatography-mass spectrometry].

The volatile oil of garlic was extracted by hydrodistillation method and gas chromatography-mass spectrometry was applied to analyse the compounds in the oil. The best extraction conditions for high-content, effective components were obtained through optimization. The capillary column was an HP-5MS column (25 mm x 0.25 mm i.d. x 0.25 microm); oven temperature increased with a rate of 5 degrees C /min from 80 to 300 degrees C, and then maintained for 20 min; sample size of 1 microL; split ratio of 100:1; carrier gas of helium (1 mL/min). Mass spectra were obtained at 70 eV. The temperatures of injector base, ionization source were maintained at 270 degrees C, 230 degrees C respectively. Under these conditions, twenty compounds in the volatile oil of garlic were isolated and identified, and the content of each was determined. Sulfur-containing compounds were found to be the principal components, of which the major compound was diallyl trisulfide with the content of more than 30%, which is higher than the others in the literature. The experimental results also indicated that hydrodistillation method is an effective method for officinal component extraction. In addition, it was also demonstrated that the garlic volatile oil was stable when stored at 0 degrees C for 6 months.