Lentiviral gene therapy with IGF2-tagged GAA normalizes the skeletal muscle proteome in murine Pompe disease.

Pompe disease is a lysosomal storage disorder caused by deficiency of acid alpha-glucosidase (GAA), resulting in glycogen accumulation with profound pathology in skeletal muscle. We recently developed an optimized form of lentiviral gene therapy for Pompe disease in which a codon-optimized version of the GAA transgene (LV-GAAco) was fused to an insulin-like growth factor 2 (IGF2) peptide (LV-IGF2.GAAco), to promote cellular uptake via the cation-independent mannose-6-phosphate/IGF2 receptor. Lentiviral gene therapy with LV-IGF2.GAAco showed superior efficacy in heart, skeletal muscle, and brain of Gaa -/- mice compared to gene therapy with untagged LV-GAAco. Here, we used quantitative mass spectrometry using TMT labeling to analyze the muscle proteome and the response to gene therapy in Gaa -/- mice. We found that muscle of Gaa -/- mice displayed altered levels of proteins including those with functions in the CLEAR signaling pathway, autophagy, cytoplasmic glycogen metabolism, calcium homeostasis, redox signaling, mitochondrial function, fatty acid transport, muscle contraction, cytoskeletal organization, phagosome maturation, and inflammation. Gene therapy with LV-GAAco resulted in partial correction of the muscle proteome, while gene therapy with LV-IGF2.GAAco resulted in a near-complete restoration to wild type levels without inducing extra proteomic changes, supporting clinical development of lentiviral gene therapy for Pompe disease. SIGNIFICANCE: Lysosomal glycogen accumulation is the primary cause of Pompe disease, and leads to a cascade of pathological events in cardiac and skeletal muscle and in the central nervous system. In this study, we identified the proteomic changes that are caused by Pompe disease in skeletal muscle of a mouse model. We showed that lentiviral gene therapy with LV-IGF2.GAAco nearly completely corrects disease-associated proteomic changes. This study supports the future clinical development of lentiviral gene therapy with LV-IGF2.GAAco as a new treatment option for Pompe disease.

IGF2-tagging of GAA promotes full correction of murine Pompe disease at a clinically relevant dosage of lentiviral gene therapy.

Pompe disease is caused by deficiency of acid α-glucosidase (GAA), resulting in glycogen accumulation in various tissues, including cardiac and skeletal muscles and the central nervous system (CNS). Enzyme replacement therapy (ERT) improves cardiac, motor, and respiratory functions but is limited by poor cellular uptake and its inability to cross the blood-brain barrier. Previously, we showed that hematopoietic stem cell (HSPC)-mediated lentiviral gene therapy (LVGT) with codon-optimized GAA (LV-GAAco) caused glycogen reduction in heart, skeletal muscles, and partially in the brain at high vector copy number (VCN). Here, we fused insulin-like growth factor 2 (IGF2) to a codon-optimized version of GAA (LV-IGF2.GAAco) to improve cellular uptake by the cation-independent mannose 6-phosphate/IGF2 (CI-M6P/IGF2) receptor. In contrast to LV-GAAco, LV-IGF2.GAAco was able to completely normalize glycogen levels, pathology, and impaired autophagy at a clinically relevant VCN of 3 in heart and skeletal muscles. LV-IGF2.GAAco was particularly effective in treating the CNS, as normalization of glycogen levels and neuroinflammation was achieved at a VCN between 0.5 and 3, doses at which LV-GAAco was largely ineffective. These results identify IGF2.GAA as a candidate transgene for future clinical development of HSPC-LVGT for Pompe disease.

Advanced 3D-Printing Bioinks for Articular Cartilage Repair.

Chondral lesions caused by stressors, such as injury or inflammation, lead to osteoarthritis (OA). OA is a degenerative joint disease that has become a challenge worldwide. As the articular cartilage is incapable of self-regeneration due to the absence of vessels and nerves, novel cartilage repair techniques are urgently needed. Three-dimensional (3D) bioprinting, which allows the precise control of internal architecture and geometry of printed scaffolds, has stepped up to be a promising strategy in cartilage restoration. With regards to 3D bioprinting, bioinks with proper chemical and mechanical properties play one of the most critical roles in designing successful cartilage tissue constructs. In particular, hydrogels as 3D hydrophilic cross-linked polymer networks are highly recommended as bioinks because of their fine biocompatibility, easy fabrication, and tunable mechanical strength. Herein, we highlight the widely used polymers for hydrogel preparation and further provide a non-exhaustive overview of various functional modified additives (such as cells, drugs, bioactive factors and ceramic) to exploit the unique properties suitable for bioprinted cartilage. Finally, a prospective on future development for 3D-bioprinting in cartilage repair is elucidated in this review.

Lentiviral gene therapy prevents anti-human acid α-glucosidase antibody formation in murine Pompe disease.

Enzyme replacement therapy (ERT) is the current standard treatment for Pompe disease, a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). ERT has shown to be lifesaving in patients with classic infantile Pompe disease. However, a major drawback is the development of neutralizing antibodies against ERT. Hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) provides a novel, potential lifelong therapy with a single intervention and may induce immune tolerance. Here, we investigated whether ERT can be safely applied as additional or alternative therapy following HSPC-LVGT in a murine model of Pompe disease. We found that lentiviral expression at subtherapeutic dose was sufficient to induce tolerance to the transgene product, as well as to subsequently administered ERT. Immune tolerance was established within 4-6 weeks after gene therapy. The mice tolerated ERT doses up to 100 mg/kg, allowing ERT to eliminate glycogen accumulation in cardiac and skeletal muscle and normalizing locomotor function. The presence of HSPC-derived cells expressing GAA in the thymus suggested the establishment of central immune tolerance. These findings demonstrate that lentiviral gene therapy in murine Pompe disease induced robust and long-term immune tolerance to GAA either expressed by a transgene or supplied as ERT.

Research on Damage Characteristics of Ultrasonic Vibration-Assisted Grinding of a C/SIC Composite Material.

C/SiC composites are the preferred materials for high temperature resistant (usually above 1500 °C) structural parts in aerospace, aviation, shipbuilding, and other industries. When this kind of material component is processed efficiently by grinding, the damage forms of fiber step brittle fracture and fiber pulling out are often produced on the machined surface/subsurface. The existence of these damage forms deteriorates the quality of the machine surface and may reduce the bending strength of materials to a certain extent. Therefore, it is very important to study the mechanism and the damage law of ordinary grinding and ultrasonic vibration-assisted grinding and take reasonable measures to restrain the machining damage. In this paper, the typical damage forms of C/SiC composites during the end and side grinding are explored. The surface and subsurface damage degree of C/SiC composites during grinding and ultrasonic vibration-assisted grinding were compared. The effects of different process parameters on material damage were compared and analyzed. The results show that the damage forms of ordinary grinding and ultrasonic grinding are basically the same. Compared with ordinary grinding, ultrasonic-assisted grinding can reduce surface damage to a certain extent and subsurface damage significantly.

Effect of voluntary breathing exercises on stable coronary artery disease in heart rate variability and rate-pressure product: a study protocol for a single-blind, prospective, randomized controlled trial.

BACKGROUND: At present, China has more than 11 million patients with stable coronary heart disease and this is becoming a major public health problem. The pathological changes of coronary heart disease can lead to dysfunction of the cardiac autonomic nervous system, which increases the risk of complications such as malignant arrhythmia (ventricular flutter, ventricular fibrillation, etc.), heart rate, systolic blood pressure, and rate-pressure product (RPP), which is highly correlated with myocardial oxygen consumption and indirectly reflects myocardial blood supply and oxygen consumption. Although the guidelines recommend that such patients take drugs to reduce heart rate and myocardial oxygen consumption, the clinical control of heart rate is still not ideal. Thus, in this trial, we will use voluntary breathing exercises as the strategy of exercise rehabilitation for patients with stable coronary artery disease (SCAD), in order to increase the vagus nerve activity and/or reduce the sympathetic nervous activity, help maintain or rebuild the balance of plant nerve system, improve the time-domain index of heart rate variability, reduce the burden on the heart, and relieve patients' anxiety and other negative emotions. METHODS: This is a 6-month single-blind, randomized controlled clinical trial that will be conducted in the First Affiliated Hospital of Soochow University. A total of 140 patients who fill out the Informed Consent Form are registered and randomized 1:1 into the Voluntary Breathing Exercises (VBE)-based clinical trial monitoring group (n = 70) or the Routine follow-up group (n = 70). The VBE-based clinical trial monitoring group is given VBE training on the basis of conventional treatment and health education, while the control group received conventional health education and follow-up. The primary outcomes will be measured heart rate variability and RPP. Secondary outcomes will include changes in Self-rating Anxiety Scale, total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, weight, and body mass index. DISCUSSION: This trial will carry out scientific respiratory exercise for patients with SCAD, which belongs to the category of active secondary prevention for patients, and changes from remedial to pre-protective. VBE is easy to operate and is not limited by time and place. It is important and meaningful to carry out VBE for patients with SCAD. This study will provide considerable evidence for further large-scale trials and alternative strategies for the rehabilitation nursing of patients with SCAD. TRIAL REGISTRATION: Chinese Clinical Trials Registry, 1900024043 . Registered on 23 June 2019.

Interfacial Microstructure in W/2024Al Composite and Inhibition of W-Al Direct Reaction by CeO2 Doping: Formation and Crystallization of Al-Ce-Cu-W Amorphous Layers.

In this work, interfacial microstructure in W/2024Al composite and inhibition of the W-Al direct reaction by CeO2 doping were investigated. The composites were prepared through powder sintering, and after preparation the composites were treated by annealing at 823 K. For the prepared W/2024Al composite, a multi-phase thin layer composed of WAl12 and WAl5 compounds were formed at the interface due to the W-Al direct reaction. While doping CeO2 in the composite, Al-Ce-Cu-W amorphous substituting of W-Al compounds were formed at the interfacial reaction layer. In an annealed state, the composite with CeO2 doping shows a significant inhibitory effect on W-Al compounds, which was attributed to the crystallized layer that evolved from Al-Ce-Cu-W amorphous as an interfacial obstacle. The crystallization product for Al-Ce-Cu-W amorphous layer was identified as bcc-structure Al-Ce-Cu-W phase without any binary/ternary Ce-containing phases. Therefore, by doping CeO2 in W/2024Al composite, W-Al direct reaction was markedly inhibited during both preparation and annealing.

Silane-Treated Basalt Fiber⁻Reinforced Poly(butylene succinate) Biocomposites: Interfacial Crystallization and Tensile Properties.

In this work, an economical modifier silane agent-KH550-was used for surface treatment of basalt fiber. Then, a biodegradable poly(butylene succinate) (PBS)/modified basalt fiber (MBF) biocomposite was successfully developed. The effects of silane treatment and fiber mass content on crystalline structure, isothermal crystallization process and mechanical performance of composites were evaluated. The interfacial crystallization of PBS on the surface of MBF was investigated by using a polarized optical microscope (POM). The transcrystalline (TC) structure could be clearly observed and it grew perpendicular to the surface of MBF, which boosted the nucleation ability on PBS crystallization and the strong interfacial interaction between PBS and silane-treated basalt fiber. Under isothermal crystallization kinetics, the incorporation of basalt fiber enhanced the crystallization rate and reduced the crystallization half-time values of composites compared with that of neat PBS due to a heterogeneous nucleation effect. Furthermore, tensile results confirmed that the presence of MBF could greatly improve the tensile strength and modulus. The predicted interfacial shear strength (IFSS) suggested that an enhancement of interfacial bonding could be realized via interfacial crystallization, which was also verified by SEM images. The PBS/MBF biocomposites can be applied in many fields as a low-cost, lightweight, and biodegradable composite material.