Development of cellular and animal models of coagulation factors deficiencies for the assessment of innovative therapeutic approaches acting on transcriptional and post-transcriptional regulation

In the last decades, enormous efforts have been pushed toward the development of molecular therapeutic approaches for human genetic diseases, and the research all over the world has obtained remarkable achievements, especially in gene therapy field. Notwithstanding, the intense research also led to potential therapeutic strategies based on the correction of the specific disease-causing defects, which might circumvent some limitations of gene replacement therapy. These approaches are of great interest for patients with coagulation deficiencies, since they would benefit even from small increase in functional protein levels. This work propose the development of in-vitro and in–vivo models of rare bleeding disorders, in order to explore corrective molecular approaches acting on the specific disease-causing defects, both at transcriptional and post-transcriptional level. The first part of this work deals with the usage of engineered transcription factors (eTFs) as potential therapeutic strategy for factor VII (F7) deficiency caused by two severe promoter mutations. Through the expression of gene reporter plasmids we created a cellular model for the two F7 promoter variants. Then, we assembled four eTFs (TF1-4) designed to target different regions on the F7 proximal promoter in order to test their efficacy in stimulating transcriptional activity on the target gene. The treatment with the different eTFs demonstrated that TF4, targeting a sequence between the mutations, induced a robust increase of gene transcription in the presence of the defective promoter. Interestingly, TF4 appreciably increased the endogenous F7 transcription and mRNA levels in HepG2 cells and induced F7 expression in Hek293 cells that do not virtually express factor VII. The second part describes the exploitation of the Sleeping Beauty Transposon System (SBTS) to develop cellular and mouse model of haemophilia B (HB) caused by splicing mutations, in order to assess the efficacy of an RNA-based therapeutic approach. In the last years, modified small nuclear RNAs U1 (U1snRNAs) have been exploited to correct splicing mutations causing severe coagulation factor VII deficiency and HB, but only in minigenes assays. Therefore, the evaluation of the U1snRNA-mediated correction strategy in–vivo implies the creation of proper mouse models for each specific splicing-variant, not yet available. Here we used the SBTS to develop cellular/mouse models of HB caused by the factor IX ex5-2C splicing variant. We have generated Hek293 stable clones expressing the normal or mutated human splicing-competent factor IX cassettes integrated into the genome as a result of the transposase activity. These preliminary studies provided us with optimized experimental protocol to create cellular models of human disease caused by splicing mutations. This also provides with the rationale for the creation of mouse models through hydrodynamic injection of the transposon plasmids and of the transposase in wt mice, for the assessment of the modified U1 snRNAs-mediated rescue in–vivo in a genomic expression context instead of a transient episomal system

Development of cellular and animal models of coagulation factors deficiencies for the assessment of innovative therapeutic approaches acting on transcriptional and post-transcriptional regulation

In the last decades, enormous efforts have been pushed toward the development of molecular therapeutic approaches for human genetic diseases, and the research all over the world has obtained remarkable achievements, especially in gene therapy field. Notwithstanding, the intense research also led to potential therapeutic strategies based on the correction of the specific disease-causing defects, which might circumvent some limitations of gene replacement therapy. These approaches are of great interest for patients with coagulation deficiencies, since they would benefit even from small increase in functional protein levels. This work propose the development of in-vitro and in–vivo models of rare bleeding disorders, in order to explore corrective molecular approaches acting on the specific disease-causing defects, both at transcriptional and post-transcriptional level. The first part of this work deals with the usage of engineered transcription factors (eTFs) as potential therapeutic strategy for factor VII (F7) deficiency caused by two severe promoter mutations. Through the expression of gene reporter plasmids we created a cellular model for the two F7 promoter variants. Then, we assembled four eTFs (TF1-4) designed to target different regions on the F7 proximal promoter in order to test their efficacy in stimulating transcriptional activity on the target gene. The treatment with the different eTFs demonstrated that TF4, targeting a sequence between the mutations, induced a robust increase of gene transcription in the presence of the defective promoter. Interestingly, TF4 appreciably increased the endogenous F7 transcription and mRNA levels in HepG2 cells and induced F7 expression in Hek293 cells that do not virtually express factor VII. The second part describes the exploitation of the Sleeping Beauty Transposon System (SBTS) to develop cellular and mouse model of haemophilia B (HB) caused by splicing mutations, in order to assess the efficacy of an RNA-based therapeutic approach. In the last years, modified small nuclear RNAs U1 (U1snRNAs) have been exploited to correct splicing mutations causing severe coagulation factor VII deficiency and HB, but only in minigenes assays. Therefore, the evaluation of the U1snRNA-mediated correction strategy in–vivo implies the creation of proper mouse models for each specific splicing-variant, not yet available. Here we used the SBTS to develop cellular/mouse models of HB caused by the factor IX ex5-2C splicing variant. We have generated Hek293 stable clones expressing the normal or mutated human splicing-competent factor IX cassettes integrated into the genome as a result of the transposase activity. These preliminary studies provided us with optimized experimental protocol to create cellular models of human disease caused by splicing mutations. This also provides with the rationale for the creation of mouse models through hydrodynamic injection of the transposon plasmids and of the transposase in wt mice, for the assessment of the modified U1 snRNAs-mediated rescue in–vivo in a genomic expression context instead of a transient episomal system

Tissue-engineered grafts from human decellularized extracellular matrices: A systematic review and future perspectives

Tissue engineering and regenerative medicine involve many different artificial and biologic materials, frequently integrated in composite scaffolds, which can be repopulated with various cell types. One of the most promising scaffolds is decellularized allogeneic extracellular matrix (ECM) then recellularized by autologous or stem cells, in order to develop fully personalized clinical approaches. Decellularization protocols have to efficiently remove immunogenic cellular materials, maintaining the nonimmunogenic ECM, which is endowed with specific inductive/differentiating actions due to its architecture and bioactive factors. In the present paper, we review the available literature about the development of grafts from decellularized human tissues/organs. Human tissues may be obtained not only from surgery but also from cadavers, suggesting possible development of Human Tissue BioBanks from body donation programs. Many human tissues/organs have been decellularized for tissue engineering purposes, such as cartilage, bone, skeletal muscle, tendons, adipose tissue, heart, vessels, lung, dental pulp, intestine, liver, pancreas, kidney, gonads, uterus, childbirth products, cornea, and peripheral nerves. In vitro recellularizations have been reported with various cell types and procedures (seeding, injection, and perfusion). Conversely, studies about in vivo behaviour are poorly represented. Actually, the future challenge will be the development of human grafts to be implanted fully restored in all their structural/functional aspects

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes which developed a myopathic phenotype consistent with that of human myofibrillar myopathy including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets

Proteasome system dysregulation and treatment resistance mechanisms in major depressive disorder

Several studies have demonstrated that allelic variants related to inflammation and the immune system may increase the risk for major depressive disorder (MDD) and reduce patient responsiveness to antidepressant treatment. Proteasomes are fundamental complexes that contribute to the regulation of T-cell function. Only one study has shown a putative role of proteasomal PSMA7, PSMD9 and PSMD13 genes in the susceptibility to an antidepressant response, and sparse data are available regarding the potential alterations in proteasome expression in psychiatric disorders such as MDD. The aim of this study was to clarify the role of these genes in the mechanisms underlying the response/resistance to MDD treatment. We performed a case-control association study on 621 MDD patients, of whom 390 were classified as treatment-resistant depression (TRD), and we collected peripheral blood cells and fibroblasts for mRNA expression analyses. The analyses showed that subjects carrying the homozygous GG genotype of PSMD13 rs3817629 had a twofold greater risk of developing TRD and exhibited a lower PSMD13 mRNA level in fibroblasts than subjects carrying the A allele. In addition, we found a positive association between PSMD9 rs1043307 and the presence of anxiety disorders in comorbidity with MDD, although this result was not significant following correction for multiple comparisons. In conclusion, by confirming the involvement of PSMD13 in the MDD treatment response, our data corroborate the hypothesis that the dysregulation of the complex responsible for the degradation of intracellular proteins and potentially controlling autoimmunity- and immune tolerance–related processes may be involved in several phenotypes, including the TRD

In vitro assessment of a novel composite scaffold for articular cartilage restoration

Articular cartilage (AC) lesions are a particular challenge for regenerative medicine due to cartilage low self-ability repair in case of damage. Hence, a significant goal of musculoskeletal tissue engineering is the development of suitable structures in virtue of their matrix composition and biomechanical properties [1]. The objective of our study was to design in vitro a supporting structure for cartilage chondrocytes to treat focal articular joint defects. We realized a bio-hybrid composite scaffold combining decellularized Wharton’s jelly (W’s J) with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). The hydrogel itself and the more specific decellularized cartilage matrix were used as controls. Immunohistochemical analysis highlighted a similar histomorphology for W’s J and AC matrices. Human chondrocytes were isolated from articular cartilage by collagenase II digestion and then characterized by flow-cytometry and RT-PCR to assess the expression of specific markers. CD44+/CD73+/CD151+ chondrocytes were seeded on PVA, PVA/AC and PVA/W’s J scaffolds to test their ability to support cell colonization. According to SEM micrographs and MTT proliferation assay, PVA/W’s J revealed a singular attitude to sustain cell proliferation despite its aspecific origin. Our preliminary evidences highlighted the chance of using Wharton’s jelly in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration

In vitro and in vivo study of a novel biodegradable synthetic conduit for injured peripheral nerves

In case of peripheral nerve injury (PNI) with wide substance-loss, surgical reconstruction is still a challenge. Bridging the gap by autologous sensory nerves as grafts is the current standard; nevertheless, the related issues have prompted the research towards the development of effective artificial synthetic/biological nerve conduits (NCs). Here, we manufactured a novel NC using oxidized polyvinyl alcohol (OxPVA) that is a biodegradable cryogel recently patented by our group [1]. Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) NCs through in vitro/in vivo analysis. Considering in vitro studies, a morphological characterization was performed by Scanning Electron Microscopy (SEM). Thereafter, cell adhesion and proliferation of a Schwann-cell line (SH-SY5Y) were evaluated by SEM and MTT assay. Regarding in vivo tests, the NCs were implanted into the surgical injured sciatic nerve (gap: 5 mm) of Sprague-Dawley rats, and the functional recovery was assessed after 12-weeks. The NCs were then processed for histological, immunohistochemical (anti-CD3; -β-tubulin; -S100) and Transmission Electron Microscopy (TEM) analyses. In particular, morphometric analyses (section area, total number and density of nerve fibers) were performed at the level of proximal, central and distal portions with respect to NC. In vitro results by SEM showed that PVA and SF supports have a smoother surface than OxPVA scaffolds. Moreover, unlike SF scaffolds, PVA-based ones do not support SH-SY5Y adhesion and proliferation. Regarding the in vivo study, all animals showed a functional recovery with normal walk, even though only animals implanted with PVA and SF NCs sometimes showed spasms while walking. On the contrary, animals implanted with OxPVA NCs exhibited a normal movement. Anti-CD3 immunohistochemistry assessed the absence of severe inflammatory reactions in all the grafts. A strong positive immunoreaction for β-tubulin and S100 demonstrated the good regeneration of nervous fibers. TEM highlighted regeneration of myelinated/un-myelinated axons and Schwann cells in all the grafts. However, morphometric analysis demonstrated that OxPVA assure a better outcome in nerve regeneration in terms of total number of nerve fibers. Our results sustain the potential of OxPVA for the development of NCs useful for PNI with substance loss with the advantage of biodegradation

Using Line Profiles to Test the Fraternity of Type Ia Supernovae at High and Low Redshifts

Using archival data of low-redshift (z < 0.01) Type Ia supernovae (SN Ia) and recent observations of high-redshift (0.16 < z <0.64; Matheson et al. 2005) SN Ia, we study the "uniformity'' of the spectroscopic properties of nearby and distant SN Ia. We find no difference in the measures we describe here. In this paper, we base our analysis solely on line-profile morphology, focusing on measurements of the velocity location of maximum absorption (vabs) and peak emission (vpeak). We find that the evolution of vabs and vpeak for our sample lines (Ca II 3945, Si II 6355, and S II 5454, 5640) is similar for both the low- and high-redshift samples. We find that vabs for the weak S II 5454, 5640 lines, and vpeak for S II 5454, can be used to identify fast-declining [dm15 > 1.7] SN Ia, which are also subluminous. In addition, we give the first direct evidence in two high-z SN Ia spectra of a double-absorption feature in Ca II 3945, an event also observed, though infrequently, in low-redshift SN Ia spectra (6/22 SN Ia in our local sample). We report for the first time the unambiguous and systematic intrinsic blueshift of peak emission of optical P-Cygni line profiles in Type Ia spectra, by as much as 8000 km/s. All the high-z SN Ia analyzed in this paper were discovered and followed up by the ESSENCE collaboration, and are now publicly available.Comment: 28 pages (emulateapj), 15 figures; accepted for publication in A

Short Bowel Syndrome and Tissue Engineering: a preliminary study towards the development of a new regenerative approach in paediatric patients

Pediatric Short Bowel Syndrome (SBS) is a malabsorption state following massive surgical resections of the small intestine. The current therapeutic options issues (i.e. parental nutrition, surgical lengthening, transplantation) have prompt the research in Tissue Engineering. Thus, our aim was to preliminary investigate in vitro/in vivo two composite scaffolds for engineering the small intestine without resorting to autologous intestinal epithelial organoid units which, to date, are the cell source mainly considered for this purpose. In particular, we developed composite supports consisting of a novel biocompatible/resorbable cryogel that is oxidized polyvinyl alcohol (OxPVA) [1] crosslinked with intestinal mucosa whole (wIM/OxPVA) or homogenized (hIM/OxPVA). After evaluating the scaffolds by histology and Scanning Electron Microscopy (SEM), we assessed their interaction with adipose mesenchymal stem cells. Thereafter, the in vivo behavior of scaffolds was studied implanting them in the omentum of Sprague Dawley rats. At 4 weeks, explants were processed by histology and immunohistochemistry (CD3; F4/80; Ki-67; desmin; α-SMA; MNF116). Considering the in vitro evidence, both histological and SEM results proved the effectiveness of the decellularization, and allowed to appreciate the preservation of intestinal villi of the wIM as well as the characteristic features of the hIM. At 7 days from cell seeding, MTT assay showed that hIM/OxPVA scaffolds could support cell adhesion/proliferation even if the wIM/OxPVA ones seem to significantly increase cell growth (

Characterization of novel autologous leukocyte fibrin platelet membranes for tissue engineering applications

Autologous hemocomponents have recently emerged as potential biologic tools for regenerative purpose, consisting mainly of platelet concentrates which locally release growth factors (GFs) to enhance the tissue healing process. Despite two decades of clinical studies, the therapeutic efficacy of platelet concentrates is still controversial. This work represents a first characterization of a novel autologous leukocyte fibrin platelet membrane (LFPm), which is prepared by the Department of Immunohematology of Belluno Hospital according to a well standardized protocol. The quantification of their specific content showed that LFPms are enriched not only with platelets, but also with monocytes/macrophages, fibrinogen and CD34+ cells. Mechanical properties of LFPms were investigated by tensile tests, revealing that the specific elasticity of membranes was maintained over time. Furthermore, the release kinetics of Platelet Derived Growth Factor, Vascular Endothelial Growth Factor, Tumor Necrosis Factor alpha and Interleukin-10 was assessed by ELISA, demonstrating that LFPms act as GF delivery systems which sustain the local release of bioactive molecules. For in vitro biodegradation analysis, LFPm samples were incubated into PBS solution for 4, 7, 14, 21 days. SEM micrographs showed a progressive loss in cellular elements associated to a simultaneous exposure of the fibrin scaffold, also confirmed by histological and immunohistochemical investigations. In parallel, LFPm disks were implanted into a subcutaneous dorsal pouch of healthy nude rats and explanted after 4, 7, 14, 21 days for in vivo biodegradation study. SEM, histological and immunohistochemical analysis revealed that the typical LFPm fibrin structure was maintained until day 7, with a contemporary loss of cellular elements. From day 14, the morphology and texture of samples became less and less recognizable, confirming that a progressive biodegradation occurred. Overall, collected evidences could support the rationale for the clinical use of LFPms, shading some light on the regenerative effect they may exert after the autologous implant on a defect site