Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone

The bone marrow-derived multipotent mesenchymal cells (MSCs) have captured scientific interest due to their multi-purpose features and clinical applications. The operational dimension of MSCs is not limited to the bone marrow reservoir, which exerts bone-building and niche anabolic tasks; they also meet the needs of quenching inflammation and restoring inflamed tissues. Thus, the range of MSC activities extends to conditions such as neurodegenerative diseases, immune disorders and various forms of osteopenia. Steering these cells towards becoming an effective therapeutic tool has become mandatory. Many laboratories have employed distinct strategies to improve the plasticity and secretome of MSCs. We aimed to present how photobiomodulation therapy (PBM-t) can manipulate MSCs to render them an extraordinary anti-inflammatory and osteogenic instrument. Moreover, we discuss the outcomes of different PBM-t protocols on MSCs, concluding with some perplexities and complexities of PBM-t in vivo but encouraging and feasible in vitro solutions

Somatic Mutation Tracking in Hematopoietic Stem Cell Gene Therapy Reveals Absence of Clonal Hematopoiesis

In hematopoietic stem cell Gene Therapy (GT), patients’ Hematopoietic Stem and Progenitor Cells (HSPCs) are genetically corrected ex vivo and reinfused to reconstitute the entire hematopoietic system and provide therapeutic benefit. During this process, HSPCs are subjected to tremendous pressures to sustain high levels of proliferation until the hematopoietic reconstitution is complete. So far it is unknown if this likely stressful condition may result in the accumulation of somatic mutations which may trigger decay of the hematopoietic functions and increased risk of oncogenesis. The impact of prolonged and heightened HSPCs proliferation rates on cellular fitness and safety remains an open question in GT. Indeed, HSPCs are not comprehensively geno-protected from DNA damage accumulation in HSPCs during aging and/or in specific disease conditions as it has been observed in sickle cell disease and Fanconi Anemia.Here we performed an analysis of somatic mutations in exons of 40 genes involved in clonal hematopoiesis and myeloid cancer in 23 GT patients treated for metachromatic leukodystrophy (MLD, 15 30 years old). We used genomic DNA from HSPCs cells prior infusion and peripheral blood mononuclear cells harvested at 2 years after GT and at the last available time point (2.5-7.5 years after GT). Sequencing reads correctly aligned on the targeted exon panel resulted in an average coverage of 4,400 and 4,300 reads/base in β-Thal and MLD patients respectively.The average mutation rate in the adult β-Thal patients was >2 fold higher than the rate measured in the pediatric patients (11.3 ± 11 vs 5.6 ± 3.5). Moreover, the average somatic mutation rates in adult and pediatric β-Thal patients were both significantly higher than in MLD patients (1.6 ± 0.72, p-value=0.0136 vs adult β-Thal, p-value=0.012 vs pediatric β-Thal, by Kruskal-Wallis test). None of the mutations were pathological or likely pathological. Most somatic mutations (85 out of 96) exhibited a Variant Allele Frequency of less than 2%. The average number of mutations in both the clinical trials remained consistent across all time points, showing no statistically significant variations. Considering that the sequenced genomic interval corresponds to 76,715 bps and that we analyzed a total of 8,100 equivalent genomes per patient, the resulting mutation rate in β-Thal patients was 1.21x10-8 mutations/bp, while MLD patients resulted in a mutation rate of 2.6x10-9 mutations/bp. Five mutations (4 in β-Thal and 1 in MLD) were found at more than one time point, but none showed a progressive increase in abundance, suggesting that these mutations did not confer a selective advantage to the mutated cell clones.Our work revealed that no somatic mutations known to drive clonal hematopoiesis or myeloid cancer, nor accumulation of somatic mutations, were found in our GT patients, indicating that the GT treatment was neutral in these conditions. However, the underlying disease in β-Thal patients resulted in a significantly higher mutation burden than MLD patients, a finding worth of deeper analysis and that supports long-term follow-up assessments of the clonal composition of the hematopoietic system in GT patients

Molecular and Functional Characterization of Three Different Postzygotic Mutations in PIK3CA-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

BACKGROUND PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. METHODS: We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. RESULTS AND CONCLUSION: Our data indicate that patients' cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients

miRNA-126 Orchestrates an Oncogenic Program in B Cell Precursor Acute Lymphoblastic Leukemia

MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden

Recommendations for pre-symptomatic genetic testing for hereditary transthyretin amyloidosis in the era of effective therapy: a multicenter Italian consensus

Hereditary transthyretin amyloidosis (ATTRv, v for variant) is a late-onset, autosomal dominant disease caused by progressive extracellular deposition of transthyretin amyloid fibrils, leading to organ damage and death. For other late-onset fatal diseases, as Huntington's disease, protocols for pre-symptomatic genetic testing (PST) are available since decades. For ATTRv, limited experience has been reported to date, mostly gathered before the availability of approved therapies. We aimed at developing recommendations for a safe and feasible PST protocol in ATTRv in the era of emerging treatments, taking also into account Italian patients' characteristics and healthcare system rules. After an initial survey on ongoing approaches to PST for ATTRv in Italy, two roundtable meetings were attended by 24 experts from 16 Italian centers involved in the diagnosis and care of this disease. Minimal requirements for PST offer and potential critical issues were highlighted. By November 2019, 457 families affected by ATTRv with 209 molecularly confirmed pre-symptomatic carriers were counted. The median age at PST was 41.3years of age, regardless of the specific mutation. Half of the Italian centers had a multidisciplinary team, including a neurologist, an internist, a cardiologist, a medical geneticist and a psychologist, although in most cases not all the specialists were available in the same center. A variable number of visits was performed at each site. Experts agreed that PST should be offered only in the context of genetic counselling to at risk individuals aged 18 or older. Advertised commercial options for DNA testing should be avoided. The protocol should consist of several steps, including a preliminary clinical examination, a pre-test information session, an interval time, the genetic test and a post-test session with the disclosure of the test results, in the context of an experienced multidisciplinary team. Recommendations for best timing were also defined. Protocols for PST in the context of ATTRv can be refined to offer at risk individuals the best chance for early diagnosis and timely treatment start, while respecting autonomous decisions and promoting safe psychological adjustment to the genetic result

MKS3/TMEM67 mutations are a major cause of COACH syndrome, a joubert syndrome related disorder with liver involvement

The acronym COACH defines an autosomal recessive condition of Cerebellar vermis hypo/ aplasia, Oligophrenia, congenital Ataxia, Coloboma and Hepatic fibrosis. Patients present the “molar tooth sign”, a midbrain-hindbrain malformation pathognomonic for Joubert Syndrome (JS) and Related Disorders (JSRDs). The main feature of COACH is congenital hepatic fibrosis (CHF), resulting from malformation of the embryonic ductal plate. CHF is invariably found also in Meckel syndrome (MS), a lethal ciliopathy already found to be allelic with JSRDs at the CEP290 and RPGRIP1L genes. Recently, mutations in the MKS3 gene (approved symbol TMEM67), causative of about 7% MS cases, have been detected in few Meckel-like and pure JS patients. Analysis of MKS3 in 14 COACH families identified mutations in 8 (57%). Features such as colobomas and nephronophthisis were found only in a subset of mutated cases. These data confirm COACH as a distinct JSRD subgroup with core features of JS plus CHF, which major gene is MKS3, and further strengthen gene-phenotype correlates in JSRDs