Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2.

Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.

Safety and Immunogenicity of Betuvax-CoV-2, an RBD-Fc-Based SARS-CoV-2 Recombinant Vaccine: Preliminary Results of the First-in-Human, Randomized, Double-Blind, Placebo-Controlled Phase I/II Clinical Trial.

COVID-19, being a life-threatening infection that evolves rapidly, remains a major public health concern calling for the development of vaccines with broad protection against different pathogenic strains and high immunogenicity. Aside from this, other concerns in mass immunization settings are also the scalability of production and relative affordability of the technology. In that regard, adjuvanted protein vaccines with particles mimicking the virus stand out among known vaccine technologies. The "Betuvax-CoV-2" vaccine, developed on the basis of a recombinant protein and an adjuvant, has already been tested in preclinical studies and has advanced to clinical evaluation. Open, double-blinded, placebo-controlled, randomized phase I/II clinical trial of the "Betuvax-CoV-2," recombinant protein subunit vaccine based on the S-protein RBD fused with the Fc-fragment of IgG, was conducted to evaluate safety and immunogenicity in response to the vaccination. METHODS: In the phase I/II clinical trial, 116 healthy adult men and women, ages 18-58, were enrolled: 20 in Stage I, and 96 in Stage II. In Stage I, 20 µg of the vaccine was administered intramuscularly on day 2, and either 5 µg (group 1) or 20 µg (group 2) on day 30. In Stage II, 20 µg of the vaccine was administered intramuscularly on day 2, and either 5 µg (group 3) or 20 µg (group 4) on day 30. In group 5, both injections were replaced with placebo. The primary outcome measures were safety (number of participants with adverse events throughout the study) and antigen-specific humoral immunity (SARS-CoV-2-specific antibodies measured by ELISA and CMIA). Antigen-specific cell-mediated immunity and changes in neutralizing antibodies (detected with a SARS-CoV-2 neutralization assay) were measured as a secondary outcome. The trial is registered with ClinicalTrials.gov (Study Identifier: NCT05270954). FINDINGS: Both vaccine formulations (20 µg + 5 µg and 20 µg + 20 µg) were safe and well tolerated. Most adverse events were mild, and no serious adverse events were detected. On day 51,anti-SARS-CoV-2 total and IgG antibody titers and anti-SARS-CoV-2 neutralizing antibodies were significantly higher in the vaccine groups (both formulations) than in the placebo. A more pronounced CD4+-mediated immune response was observed in the group of volunteers administered with the 20 + 20 µg vaccine formulation. INTERPRETATIONS: RBD-Fc-based COVID-19 "Betuvax-CoV-2" vaccine in doses (20 + 5 µg and 20 + 20 µg) demonstrated an excellent safety profile and induced a strong humoral response. Further research on the protective effectiveness of the "Betuvax-CoV-2" vaccine for the prevention of COVID-19 is on its way.

Correlation of the regenerative potential of dermal fibroblasts in 2D culture with the biological properties of fibroblast-derived tissue spheroids.

In situ 3D bioprinting is a new emerging therapeutic modality for treating human skin diseases. The tissue spheroids have been previously suggested as a powerful tool in rapidly expanding bioprinting technology. It has been demonstrated that the regenerative potential of human dermal fibroblasts could be quantitatively evaluated in 2D cell culture and confirmed after implantation in vivo. However, the development of unbiassed quantitative criteria of the regenerative potential of 3D tissue spheroids in vitro before their in situ bioprinting remains to be investigated. Here it has been demonstrated for the first time that specific correlations exist between the regenerative potential of human dermal fibroblasts cultured in vitro as 2D cell monolayer with biological properties of 3D tissue spheroids fabricated from these fibroblasts. In vitro assessment of biological properties included diameter, spreading and fusion kinetics, and biomechanical properties of 3D tissue spheroids. This comprehensive characterization could be used to predict tissue spheroids' regenerative potential in vivo.

SARS-CoV-2 Subunit Virus-like Vaccine Demonstrates High Safety Profile and Protective Efficacy: Preclinical Study.

Public health threat coming from a rapidly developing COVID-19 pandemic calls for developing safe and effective vaccines with innovative designs. This paper presents preclinical trial results of "Betuvax-CoV-2", a vaccine developed as a subunit vaccine containing a recombinant RBD-Fc fusion protein and betulin-based spherical virus-like nanoparticles as an adjuvant ("Betuspheres"). The study aimed to demonstrate vaccine safety in mice, rats, and Chinchilla rabbits through acute, subchronic, and reproductive toxicity studies. Along with safety, the vaccine demonstrated protective efficacy through SARS-CoV-2-neutralizing antibody production in mice, rats, hamsters, rabbits, and primates (rhesus macaque), and lung damage and infection protection in hamsters and rhesus macaque model. Eventually, "Betuvax-CoV-2" was proved to confer superior efficacy and protection against the SARS-CoV-2 in preclinical studies. Based on the above results, the vaccine was enabled to enter clinical trials that are currently underway.

Design and Immunological Properties of the Novel Subunit Virus-like Vaccine against SARS-CoV-2.

The COVID-19 pandemic is ongoing, and the need for safe and effective vaccines to prevent infection and to control spread of the virus remains urgent. Here, we report the development of a SARS-CoV-2 subunit vaccine candidate (Betuvax-CoV-2) based on RBD and SD1 domains of the spike (S) protein fused to a human IgG1 Fc fragment. The antigen is adsorbed on betulin adjuvant, forming spherical particles with a size of 100-180 nm, mimicking the size of viral particles. Here we confirm the potent immunostimulatory activity of betulin adjuvant, and demonstrate that two immunizations of mice with Betuvax-CoV-2 elicited high titers of RBD-specific antibodies. The candidate vaccine was also effective in stimulating a neutralizing antibody response and T cell immunity. The results indicate that Betuvax-CoV-2 has good potential for further development as an effective vaccine against SARS-CoV-2.

Bringing a Gene-Activated Bone Substitute Into Clinical Practice: From Bench to Bedside.

Bone grafting and reconstruction are still challenging in clinical practice because of the limitations of bone autografts and the drawbacks of currently approved bone substitutes. We thus developed a gene-activated bone substitute based on octacalcium phosphate and naked plasmid DNA carrying the vascular endothelial growth factor gene. This advanced combined therapy medicinal product had no cytotoxic effects in vitro, slightly decreased bone marrow mesenchymal stromal cell (MSC) doubling time, and was characterized by a prolonged level of gene construct delivery in vivo in a luciferase bioimaging assay. In the model of critically sized cranial bone defects in rabbits, the gene-activated matrix increased bone tissue formation through angiogenesis induction. After preclinical studies, we conducted an open-label non-randomized clinical trial (NCT03076138). The primary study outcome was the proportion of patients with newly formed bone tissue within the surgical area as measured by computed tomography within 6 months after surgery. The main secondary outcomes included frequencies of adverse events (AEs) and serious adverse events (SAEs) as well as the surgical failure rate. After completing the clinical trial, the patients had dental implants placed in the bone grafting area, and trephine biopsy samples were collected. In total, 20 patients with alveolar ridge atrophy (n = 16) and jaw bone defects (n = 4) were enrolled in the study. There were no AEs or SAEs during the clinical trial or the follow-up period (30 months). In all patients, newly formed tissues with a bone density of 908.13 ± 114.40 HU were detected within the zone of bone grafting. There were no significant differences between the subgroups of patients with atrophy and bone defects: 915.28 ± 125.85 and 879.56 ± 48.36 HU, respectively (p = 0.60). Histological analysis showed that the bone grafting area comprised newly formed bone tissue with some fragments of the gene-activated bone substitute partially resorbed and integrated with bone, without fibrous tissue in between. The preclinical data and clinical trial results proved the feasibility, safety, and efficacy of the investigated material for jaw bone grafting, allowing us to bring the world's first gene-activated bone substitute from bench to bedside.

Magnetic resonance imaging pattern variability in dysferlinopathy.

The widespread use of magnetic resonance imaging (MRI) in the diagnosis of myopathies has made it possible to clarify the typical MRI pattern of dysferlinopathy. However, sufficient attention has not been given to the variability of MRI patterns in dysferlinopathy. MATERIALS AND METHODS: Twenty-five patients with the clinical manifestations of dysferlinopathy were examined. For all patients, creatine phosphokinase levels were measured and molecular genetics were examined. In two patients, immunohistochemical examinations of muscle biopsies were performed. MRI scanning was included T2 multi-slice multi-echo, T1 weighted, T2 weighted and Short Tau Inversion Recovery T2 weighted sequences. Quantitative and semi-quantitative evaluations of fatty replacement and swelling of the muscles were undertaken. RESULTS: Variability in the MRI patterns was lowest in the pelvis and leg muscles and highest in the thigh muscles. Three main types of MRI patterns were distinguished: posterior-dominant (80%), anterior-dominant (16%), and diffuse (4%). Among patients with the anterior-dominant pattern, the collagen-like variant (4%), proximal variant (4%) and pseudo-myositis (8%) were separately distinguished. CONCLUSIONS: Awareness of atypical MRI patterns in dysferlinopathy is important for increasing the efficiency of routine diagnostics and optimizing the search for causative gene mutations.

Intrafamilial Phenotypic Variability of Collagen VI-Related Myopathy Due to a New Mutation in the COL6A1 Gene.

A family of five male siblings (three survivors at 48, 53 and 58 years old; two deceased at 8 months old and 2.5 years old) demonstrating significant phenotypic variability ranging from intermediate to the myosclerotic like Bethlem myopathy is presented. Whole-exome sequencing (WES) identified a new homozygous missense mutation chr21:47402679 T > C in the canonical splice donor site of the second intron (c.227 + 2T>C) in the COL6A1 gene. mRNA analysis confirmed skipping of exon 2 encoding 925 amino-acids in 94-95% of resulting transcripts. Three sibs presented with intermediate phenotype of collagen VI-related dystrophies (48, 53 and 2.5 years old) while the fourth sibling (58 years old) was classified as Bethlem myopathy with spine rigidity. The two older siblings with the moderate progressive phenotype (48 and 53 years old) lost their ability to maintain a vertical posture caused by pronounced contractures of large joints, but continued to ambulate throughout life on fully bent legs without auxiliary means of support. Immunofluorescence analysis of dermal fibroblasts demonstrated that no type VI collagen was secreted in any of the siblings' cells, regardless of clinical manifestations severity while fibroblast proliferation and colony formation ability was decreased. The detailed genetic and long term clinical data contribute to broadening the genotypic and phenotypic spectrum of COL6A1 related disease.

In Vitro Angiogenic Properties of Plasmid DNA Encoding SDF-1α and VEGF165 Genes.

The stromal-derived factor-1 alpha (SDF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and exert a significant trophic function. SDF-1α is a chemoattractant for endothelial progenitor cells derived from bone marrow and promotes new blood vessel formation. VEGF regulates all types of vascular growth, stimulates angiogenesis, and is involved in the induction of lymphangiogenesis. The possibility of using these growth factors for regenerative medicine is currently under investigation. The angiogenic potential of a pBud-SDF-1α-VEGF165 bicistronic plasmid construct which simultaneously encodes VEGF165 and SDF-1α genes cDNA was evaluated in this study. The conditioned medium collected from HEK293T cells transfected with the pBud-SDF-1α-VEGF165 plasmid was shown to stimulate the formation of capillary-like structures by human umbilical vein-derived endothelial cells (HUVEC) on Matrigel and to increase the proliferative activity of these cells in vitro. Thus, the pBud-SDF-1α-VEGF165 plasmid exhibits angiogenic properties in cell cultures in vitro. As interest in the development of non-viral techniques for regenerative medicine increases, this plasmid which simultaneously expresses VEGF165 and SDF-1α may provide a platform for advanced methods of stimulating therapeutic angiogenesis.

Glu20Ter Variant in PLEC 1f Isoform Causes Limb-Girdle Muscle Dystrophy with Lung Injury.

Plectinopathies are orphan diseases caused by PLEC gene mutations. PLEC is encoding the protein plectin, playing a role in linking cytoskeleton components in various tissues. In this study, we describe the clinical case of a 26-year-old patient with an early onset plectinopathy variant "limb-girdle muscle dystrophy type 2Q," report histopathological and ultrastructural findings in m. vastus lateralis biopsy and a novel homozygous likely pathogenic variant (NM_201378.3:c.58G>T, NP_958780.1:p.Glu20Ter) in isoform 1f of the gene PLEC. The patient had an early childhood onset with retarded physical development, moderate weakness in pelvic girdle muscles, progressive weakening of limb-girdle muscles after the age of 21, pronounced atrophy of axial muscles, and hypertrophy of the gastrocnemius, deltoid, and triceps muscles, intermittent dyspnea, and no skin involvement. Findings included: non-infectious bronchiolitis and atelectasis signs, biopsy revealed myodystrophal pattern without macrophage infiltration, muscle fiber cytoskeleton disorganization resulted from the plectin loss, incomplete reparative rhabdomyogenesis, and moderate endomysial fibrosis. We have determined a novel likely pathogenic variant in PLEC 1f isoform that causes limb-girdle muscle dystrophy type 2Q and described the third case concerning an isolated myodystrophic phenotype of LGMD2Q with the likely pathogenic variant in PLEC 1f isoform. In addition, we have demonstrated the presence of severe lung injury in a patient and his siblings with the same myodystrophic phenotype and discussed the possible role of plectin deficiency in its pathogenesis.

Corrigendum: Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan.

[This corrects the article on p. 77 in vol. 8, PMID: 28337173.].

Twenty-Year Clinical Progression of Dysferlinopathy in Patients from Dagestan.

To date, over 30 genes with mutations causing limb-girdle muscle dystrophy have been described. Dysferlinopathies are a form of limb-girdle muscle dystrophy type 2B with an incidence ranging from 1:1,300 to 1:200,000 in different populations. In 1996, Dr. S. N. Illarioshkin described a family from the Botlikhsky district of Dagestan, where limb-girdle muscle dystrophy type 2B and Miyoshi myopathy were diagnosed in 12 members from three generations of a large Avar family. In 2000, a previously undescribed mutation in the DYSF gene (c.TG573/574AT; p. Val67Asp) was detected in the affected members of this family. Twenty years later, in this work, we re-examine five known and seven newly affected family members previously diagnosed with dysferlinopathy. We observed disease progression in family members who were previously diagnosed and noted obvious clinical polymorphism of the disease. A typical clinical case is provided.

pCMV-vegf165 Intramuscular Gene Transfer is an Effective Method of Treatment for Patients With Chronic Lower Limb Ischemia.

Effective treatment of chronic lower limb ischemia is one of the most challenging issues confronting vascular surgeons. There are a number of choices available to the vascular surgeon. Open or endovascular revascularization is the treatment of choice when applicable. Current pharmacological therapies play an auxiliary role and cannot prevent disease progression. Therefore, new methods of treatment are needed. We conducted a phase 2b/3 multicenter randomized controlled clinical trial of the intramuscular transfer of a plasmid DNA encoding vascular endothelial growth factor (VEGF) 165 with cytomegalovirus promotor (CMV) in patients with atherosclerotic lower limb ischemia. A total of 100 patients were enrolled in the study, that is, 75 patients were randomized into the test group and received 2 intramuscular injections of 1.2 mg of pCMV-vegf165, 14 days apart together with standard pharmacological treatment. In all, 25 patients were randomized into the control group and received standard treatment only. The following end points were evaluated within the first 6 months of the study and during a 1.5-year additional follow-up period: pain-free walking distance (PWD), ankle-brachial index (ABI), and blood flow velocity (BFV). The pCMV-vegf165 therapy appeared to be significantly more effective than standard treatment. The PWD increased in the test group by 110.4%, 167.2%, and 190.8% at 6 months, 1 year, and 2 years after treatment, respectively. The pCMV-vegf165 intramuscular transfer caused a statistically significant increase in ABI and BFV. There were no positive results in the control group. Thus, pCMV-vegf165 intramuscular gene transfer is an effective method of treatment of moderate to severe claudication due to chronic lower limb ischemia.