Sildenafil increases AAV9 transduction after a systemic administration and enhances AAV9-dystrophin therapeutic effect in mdx mice.

Adeno-associated virus (AAV) vectors have been successfully used to deliver genes for treating rare diseases. However, the systemic administration of high AAV vector doses triggers several adverse effects, including immune response, the asymptomatic elevation of liver transaminase levels, and complement activation. Thus, improving AAV transduction and reducing AAV dosage for treatment is necessary. Recently, we found that a phosphodiesterase-5 inhibitor significantly promoted AAV9 transduction in vitro by regulating the caveolae and macropinocytosis pathways. When AAV9-Gaussian luciferase (AAV9-Gluc) and AAV9-green fluorescent protein (AAV9-GFP) were injected intravenously into mice pre-treated with sildenafil, the expressions of Gluc in the plasma and GFP in muscle tissues significantly increased (P < 0.05). Sildenafil also improved Evans blue permeation in tissues. Additionally, we found that sildenafil promoted Treg proliferation, inhibited B-cell activation, and decreased anti-AAV9 IgG levels (P < 0.05). Furthermore, sildenafil significantly promoted Duchenne muscular dystrophy gene therapy efficacy using AAV9 in mdx mice; it increased micro-dystrophin gene expression, forelimb grip strength, and time spent on the rotarod test, decreased serum creatine kinase levels, and ameliorated histopathology by improving muscle cell morphology and reducing fibrosis (P < 0.05). These results show that sildenafil significantly improved AAV transduction, suppressed the levels of anti-AAV9 IgG, and enhanced the efficacy of gene therapy.

[Study on the Therapeutic Effect of Lenalidomide on Hemophilic Arthropathy].

OBJECTIVE: To explore the effect of lenalidomide on human fibroblast-like synovial cells (HFLS) and the therapeutic efficacy on hemophilic arthropathy in hemophilia A mice model. METHODS: In vitro, to remodel the inflammatory environment of synovial tissue after hemorrhage, ferric citrate and recombinant TNF-α were added into the cell culture medium of HFLS. Cell Counting Kit-8 (CCK-8), Enzyme-linked immunosorbent assay (ELISA), Quantitative Real-time PCR (RT-qPCR) and flow cytometry were employed for detection of the effects of lenalidomide on the proliferation ability, pro-inflammatory cytokines release and apoptosis of HFLS cells. In vivo, hemophilia arthropathy was remodeled in hemophilia A mice by induction of hemarthrosis. A series of doses of lenalidomide (0.1, 0.3 and 1.0 g/kg) was administrated intra-articularly. Tissues of knee joints were collected on the 14th day after administration, and the protective effect of lenalidomide on arthritis in hemophilia A mice were evaluated by RT-qPCR and histological grading. RESULTS: In vitro, compared with the untreated control group, lenalidomide could significantly inhibit the proliferation of HFLS cells (P<0.05), and the effect was the most significant when the concentration was 0.01 µmol/L (P<0.001). Compared with the control group, lenalidomide could significantly inhibit the expression levels of TNF-α, IL-1ß, IL-6 and IFN-γ in HFLS cells (P<0.05). The flow cytometry results showed that lenalidomide could enhance the apoptotis of HFLS cells (P<0.05). The results of RT-qPCR showed that lenalidomide could significantly reduce the mRNA expression levels of TNF-α, IL-1ß, IL-6,MCP-1 and VEGF in the joint tissues (P<0.05). Histological results showed that compared with the injured group, lenalidomide could significantly reduce the pathological sequela after hemarthrosis induction, e.g. synovial thickening and neo-angiogenesis in the synovium. The protection displayed a dose-response pattern roughly. CONCLUSION: In vitro, lenalidomide can inhibit the proliferation of HFLS cells, promote their apoptosis, and inhibit the expression of pro-inflammatory cytokines. In vivo, lenalidomide can significantly decrease the expression of pro-inflammatory cytokines in the joints of mice, and prevent the development of inflammation and neo-angiogenesis. The results provide a theoretical and experimental basis for the clinical application of lenalidomide in the treatment of hemophilic arthropathy.

Treatment of experimental autoimmune encephalomyelitis using AAV gene therapy by blocking T cell costimulatory pathways.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), characterized by inflammation and demyelination. Presently, repeated relapses of MS necessitate long-term immune-regulatory therapy. Blocking the CD28-B7 and CD40-CD40L costimulatory pathways is an effective and synergistic method for the prevention and amelioration of clinical symptoms of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this study, to explore the efficacy and safety of MS gene therapy, we used adeno-associated virus (AAV) as a vector to deliver CTLA4-immunoglobulin (Ig) or CD40-Ig on the EAE induced by myelin oligodendrocyte glycoprotein (MOG). Our results showed that a single administration of AAV8-CTLA4-Ig, either alone or with AAV8-CD40-Ig, protected mice from EAE and reversed disease progression. Decreased CD4+ and CD8+ T cell infiltration, inhibition of MOG antibody response, and downregulation of neuroinflammation were observed in mice receiving AAV, suggesting that autoimmunity was suppressed in EAE pathology. Moreover, no hematological or hepatic toxicity was observed in AAV-treated mice. Thus, compared with treatment with recombinant CTLA4-Ig (belatacept), AAV gene therapy could effectively control clinical symptoms and suppress autoimmunity in the long term. In summary, our study provides a potential therapeutic method for blocking T cell costimulation for the treatment of MS via gene therapy.

Safety and activity of an engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX administered with prophylactic glucocorticoids in patients with haemophilia B: a single-centre, single-arm, phase 1, pilot trial.

BACKGROUND: A novel, engineered, liver-tropic adeno-associated virus vector expressing a hyperactive Padua factor IX (FIX) protein (BBM-H901) has been developed and is promising for haemophilia B gene therapy. We aimed to explore its safety and activity in increasing FIX concentrations and reducing bleeding frequency. METHODS: We did a single-centre, single-arm, phase 1, pilot trial evaluating the safety and activity of a single intravenous infusion of BBM-H901 at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (Tianjin, China). We enrolled adult patients with haemophilia B (aged >18 years) with baseline FIX coagulation activity (FIX:C) of less than 2 IU/dL, no FIX inhibitor, and low titre of neutralising antibodies (≤1:4) against vector capsid. Eligible participants were intravenously infused with a single dose of 5 × 1012 vector genomes (vg)/kg of BBM-H901 after 1 week of prophylactic prednisone treatment (1 mg/kg per day). Primary endpoints were the incidence of treatment-related adverse events, change in alanine aminotransferase (ALT) and aspartate amino transferase (AST), and development of antibodies against vector capsid within 1 year of infusion. We report the results of the prespecified 1-year analysis following complete enrolment. The trial is registered with ClinicalTrials.gov, NCT04135300, and is complete. FINDINGS: Between Oct 16, 2019, and Jan 13, 2021, 12 male participants were assessed, and ten Chinese participants were enrolled and infused with BBM-H901. After a median follow-up of 58 weeks (IQR 51·5-99·5), mean FIX:C reached mean 36·9 IU/dL (SD 20·5). No serious adverse events, no grade 3-4 adverse events were observed. Grade 1-2 adverse events related to BBM-H901 include pyrexia (1 [10%]) and elevation of aminotransferase(1 [10%]). No FIX inhibitors were observed. All participants developed antibodies against vector capsid after infusion. Eight (80%) participants had ALT and AST concentrations below the upper limit of normal throughout the follow-up period. Two (20%) participants had elevation of ALT and AST accompanied with decrease of FIX:C, which remained at 7 IU/dL and 11.8 IU/dL, respectively. INTERPRETATION: This pilot study suggests that liver-tropic BBM-H901 is safe 1 year after infusion. Vector derived FIX:C concentration is sufficiently high to prevent bleeding events and minimise the need for replacement therapy in small populations with haemophilia B. These findings support further study. FUNDING: Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences, National Key Research and Development Program of China, National Natural Science Foundation of China, Tianjin Municipal Science and Technology Commission Grant, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.

Directed evolution of adeno-associated virus 5 capsid enables specific liver tropism.

Impressive achievements in clinical trials to treat hemophilia establish a milestone in the development of gene therapy. It highlights the significance of AAV-mediated gene delivery to liver. AAV5 is a unique serotype featured by low neutralizing antibody prevalence. Nevertheless, its liver infectivity is relatively weak. Consequently, it is vital to exploit novel AAV5 capsid mutants with robust liver tropism. To this aim, we performed AAV5-NNK library and barcode screening in mice, from which we identified one capsid variant, called AAVzk2. AAVzk2 displayed a similar yield but divergent post-translational modification sites compared with wild-type serotypes. Mice intravenously injected with AAVzk2 demonstrated a stronger liver transduction than AAV5, roughly comparable with AAV8 and AAV9, with undetectable transduction of other tissues or organs such as heart, lung, spleen, kidney, brain, and skeletal muscle, indicating a liver-specific tropism. Further studies showed a superior human hepatocellular transduction of AAVzk2 to AAV5, AAV8 and AAV9, whereas the seroreactivity of AAVzk2 was as low as AAV5. Overall, we provide a novel AAV serotype that facilitates a robust and specific liver gene delivery to a large population, especially those unable to be treated by AAV8 and AAV9.

Naive haemophilia mice displayed different pattern of cytokine profiles of cytokine profiles changes might be associated with subclinical bleeding.

Subclinical bleeding is a haemorrhage event not clinically detected in haemophilia, and no reliable method is available for predicting subclinical bleeding. We investigated whether haemophilia mice have subclinical haemorrhage and evaluated potential biomarkers including multiple cytokine changes to predict subclinical haemorrhage. Plasma from naïve FVIII-/- and FIX-/- mice and their wild-type counterparts (FVIII WT and FIX WT, respectively) were measured for prothrombin fragment 1 + 2 (F1 + 2) and multiple cytokines. Haemophilia mice with induced hemarthrosis were used as positive clinical bleeding controls. Naive haemophilia mice that displayed higher levels than positive bleeding control were counted. Univariate and multivariate analyses of cytokines were performed. Compared with wild-type mice (FVIII WT 1.1-6.2 vs. FIX WT 2.7-6.7 pmol/l), F1 + 2 widely varied in both haemophilia mouse strains (FVIII-/- 3.7-25.7 vs. FIX-/- 2.7-15.7 pmol/l). Each cytokine varied widely in both naive haemophilia A and B mice, but not significantly, for most cytokines. In comparison to haemophilia mice with hemarthrosis bleeding challenge, naive FVIII-/- mice had elevated pro-inflammatory cytokines and FIX-/- mice had elevated anti-inflammatory cytokines. In addition, interleukin (IL)-4, followed by IL-1, IL-6, TNF-α and MIP-1α in FVIII-/- mice and MIP-1α, followed by IL-1, IL-10 in FVIII-/- mice exhibited significant differences potentially associated with potential subclinical bleeding. Naive haemophilia mice showed elevated pro-inflammatory cytokines with different patterns, represented by pro-inflammatory cytokine elevation in more naïve FVIII-/- mice and more anti-inflammatory cytokines in FIX-/- mice.

Systemic effect of FHL1 on neuromuscular junction and myotube formation via insulin-like growth factor and myostatin signaling pathways.

Four-and-a-half LIM domain protein 1 (FHL1) is a member of the FHL protein family that serves as a scaffold protein to maintain normal cellular structure and function. Its mutations have been implicated in multiple muscular diseases. These FHL1 related myopathies are characterized by symptoms such as progressive muscle loss, rigid or bent spine, even cardiac or respiratory failure in some patients, which implies pathological problems not only in muscles, but also in the nervous system. Moreover, decreased FHL1 protein level has been found in patients with FHL1 mutations, indicating the protein loss-of-function as a pathological cause of such diseases. These findings suggest the significance of understanding the systemic role of FHL1 in the homeostasis of nervous system and muscle. Here we reported that Fhl1 loss in C2C12 myotubes obscured acetylcholine receptor (AChR) clustering in addition to myotube fusion, which was associated with impaired MuSK phosphorylation. Mechanistically, myostatin-SMAD2/3 signaling was enhanced, whereas IGF-PI3K-AKT signaling was suppressed in Fhl1-/- C2C12 myotubes. Reversion of these molecular alterations rescued AChR clustering and differentiation deficits. These data outline a systemic regulation of AChR clustering and myotube fusion by FHL1, which may offer clues for mechanism study and development of therapeutic strategies to treat FHL1 related myopathies.

Exploring the Potential Feasibility of Intra-Articular Adeno-Associated Virus-Mediated Gene Therapy for Hemophilia Arthropathy.

Hemophilia arthropathy (HA) represents the majority of morbidity in severe hemophilia patients, especially in resource-limited countries. Adeno-associated virus (AAV)-mediated gene therapy is showing promise for managing hemophilia. However, patients with neutralizing antibodies (NAbs) against AAV, and inhibitors to clotting factors, are excluded from such therapy. This study explored the feasibility of AAV-mediated local gene therapy for HA. Factor VIII knockout (FVIII-/-) mice, with or without a FVIII inhibitor, were subjected to hemarthrosis induction and treated with either intravenous (IV) or intraarticular (IA) recombinant human factor VIII (rhFVIII). To investigate whether rhFVIII carried the risk to develop a FVIII inhibitor, FVIII-/- mice were treated with three doses of IV or IA rhFVIII and inhibitor development was measured. In patients with established HA requiring synovial fluid aspiration, plasma, and synovial fluid were collected and measured for anti-AAV capsid IgG (serotypes 1-9 and 843) and NAbs for AAV843. IA rhFVIII provided better protection from synovitis compared with IV rhFVIII, with or without the FVIII inhibitor. While IV rhFVIII led to all FVIII-/- mice developing an FVIII inhibitor (n = 31, median 4.9 Bethesda units [BU]/mL), only 50% of the mice developed a FVIII inhibitor by IA administration, and at a lower titer (median 0.55 BU/mL). In hemophilia patients, total anti-AAV IgG was lowest for AAV4 and AAV5, both in plasma and synovial fluid. Anti-AAV IgGs in synovial fluid for most samples were lower or similar to the plasma levels. These results show that direct IA rhFVIII administration yields better protection against bleeding-induced joint damage, even in the presence of an inhibitor antibody. IA rhFVIII delivery carried a lower risk of FVIII inhibitor formation compared with IV FVIII. The anti-AAV antibody level in synovial fluid was similar or lower than the plasma level, supporting the feasibility of local gene therapy for managing HA.

Timely and large dose of clotting factor IX provides better joint wound healing after hemarthrosis in hemophilia B mice.

Bleeding into the joints represents the major morbidity of severe hemophilia and predisposes it to hemophilic arthropathy (HA). In a reproducible hemarthrosis mouse model, we found distinct changes in thrombin activity in joint tissue homogenate following exposure of the joint to blood in wide type (WT) and hemophilic B mice. Specifically, at early time points (4 h and 24 h) after hemarthrosis, thrombin activity in WT mice quickly peaked at 4 h, and returned to baseline after 1 week. In hemophilia B mice, there was no/minimal thrombin activity in joint tissues at 4 h and 24 h, whereas at 72 h and thereafter, thrombin activity kept rising, and persisted at a higher level. Nevertheless, prothrombin had not decreased in both WT and hemophilia. The pattern was also confirmed by Western blotting and immunostaining. To optimize the protection against development of HA, we tested different treatment regimens by administration of clotting factor IX into hemophilia B mouse after hemarthrosis induction, including a total of 600 IU/kg FIX within the first 24 h or the whole 2-week period. We concluded that timely (in the first 24 h) and sufficient hemostasis correction is critical for a better protection against the development of hemophilic arthropathy.

A detrimental mutation on USP40 unlocks the tumorigenesis in a rare case of lung cancer.

Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer. Previous research has shown heterogeneity in lung cancer, with the parallel existence of multiple subclones characterized by their own specific mutational landscape. The aim of our study was to gain insight into the evolutionary pattern of lung cancer by investigating the genomic heterogeneity between a nodule and its distant tumor. Luckily, we obtained nodule and tumor samples derived from surgery and a blood sample from a single patient. The samples are very unique, for tissues with the same genetic background from nodules to malignant tumors are rarely available and require precise micro-cutting. In this study, we performed whole-genome sequencing of these two samples, to identify novel candidate driver genes associated with LUAD. The nodule and tumor were found to have common significant ubiquitin-specific protease 40 (USP40) mutations, indicating an important driver role for the gene. Moreover, we also observed the two novel candidate driver genes ASCL5 and CAPNS1 in the LUAD sample. In summary, we pinpoint the predominant mutations in LUAD by WES, highlighting the substantial genetic alterations contributing to LUAD tumorigenesis. This may provide a better understanding of the clonal evolution during tumor development.

A Translational Study of TNF-Alpha Antagonists as an Adjunctive Therapy for Preventing Hemophilic Arthropathy.

Repeated intra-articular hemorrhages lead to hemophilic arthropathy in severe hemophilia. Inflammation and pro-inflammatory cytokines (e.g., tumor necrosis factor alpha (TNFα)) might be involved in this pathogenesis. We hypothesized that anti-TNFα may provide adjuvant protection for hemophilic arthropathy management. We measured TNFα in synovial lavage from hemophilia mice subjected to hemarthrosis induction and synovial fluid from patients with hemophilic arthropathy (n = 5). In hemophilia mice, recurrent hemarthroses were induced, anti-TNFα was initiated either from day (D)7 after one hemarthrosis episode or D21 after three hemarthroses episodes (n ≥ 7/treatment group). In patients with hemophilic arthropathy (16 patients with 17 affected joints), a single dose of anti-TNFα was administered intra-articularly. Efficacy, characterized by synovial membrane thickness and vascularity, was determined. Elevated TNFα in synovial lavage was found in the hemophilia mice and patients with hemophilic arthropathy. Hemophilia mice subjected to three hemarthroses developed severe synovitis (Synovitis score of 6.0 ± 1.6). Factor IX (FIX) replacement alone partially improved the pathological changes (Synovitis score of 4.2 ± 0.8). However, anti-TNFα treatment initiated at D7, not D21, significantly provided protection (Synovitis score of 1.8 ± 0.9 vs. 3.9 ± 0.3). In patients with hemophilic arthropathy, intra-articular anti-TNFα significantly decreased synovial thickness and vascularity during the observed period from D7 to D30. Collectively, this preliminary study seems to indicate that TNFα may be associated with the pathogenicity of hemophilic arthropathy and anti-TNFα could provide adjuvant protection against hemophilic arthropathy. Further studies are required to confirm the preliminary results shown in this study.

Effect of TEAD4 on multilineage differentiation of muscle-derived stem cells.

TEAD4 is a member of transcriptional enhancer factor (TEF) family of transcription factors and plays a pivotal role in regulating embryonic development and muscle regeneration. Known previously, dysfunction of TEAD4 in mouse myoblasts impairs myotube development. However, the effects of TEAD4 on multipotency of muscle-derived stem cells (MDSCs) have not been clearly understood. Recently, bovine MDSCs (bMDSCs) were successfully isolated from adult bovine muscle. Our derived bMDSCs could differentiate into mesodermal cells, including myotubes, adipocytes, and osteoid cells. Our results also revealed that bMDSCs had the capacity to develop into ectodermal and endodermal lineages including neuron-like cells and insulin-secreting cells. After TEAD4 knock-down (TEAD4-KD), bMDSCs still kept the original capacity to differentiate into neuron-like cells and insulin-secreting cells, as shown by acquisition of both neuronal and pancreatic markers normally expressed in differentiated cells. However, up-regulation of CAV3 and ßMHC failed during myogenesis of bMDSCs with TEAD4-KD, although TEAD4-KD in bMDSCs did not affect osteoid cells and myotube formation. More interestingly, adipogenic differentiation of TEAD4-KD bMDSCs was significantly suppressed. During adipogenic differentiation, TEAD4-KD systematically impaired upregulation of TEAD1, TEAD2, and TEAD3, as well as the activation of C/EBP2, ADD1, and PPARγ as the key transcription factors for adipogenic differentiation. Finally, TEAD4-KD led to the failure of adipogenesis from bMDSCs. Together, our results support that TEAD4 is essential during adipogenic differentiation of bMDSCs. It has little effect on myogenesis of bMDSCs, and does not affect ostegenesis, neurogenesis, or pancreatic differentiation of bMDSCs. Our findings will be helpful for future study on the roles of the TEAD family during differentiation of MDSCs, and for controlling MDSC differentiation for stem cell applications.