Switching of mesodermal and endodermal properties in hTERT-modified and expanded fetal human pancreatic progenitor cells

INTRODUCTION: The ability to expand organ-specific stem/progenitor cells is critical for translational applications, although uncertainties often arise in identifying the lineage of expanded cells. Therefore, superior insights into lineage maintenance mechanisms will be helpful for cell/gene therapy. METHODS: We studied epithelial cells isolated from fetal human pancreas to assess their proliferation potential, changes in lineage markers during culture, and capacity for generating insulin-expressing beta cells. Cells were isolated by immunomagnetic sorting for epithelial cell adhesion molecule (EpCAM), and characterized for islet-associated transcription factors, hormones, and ductal markers. Further studies were performed after modification of cells with the catalytic subunit of human telomerase reverse transcriptase (hTERT). RESULTS: Fetal pancreatic progenitor cells efficiently formed primary cultures, although their replication capacity was limited. This was overcome by introduction and expression of hTERT with a retroviral vector, which greatly enhanced cellular replication in vitro. However, we found that during culture hTERT-modified pancreatic progenitor cells switched their phenotype with gain of additional mesodermal properties. This phenotypic switching was inhibited when a pancreas-duodenal homeobox (Pdx)-1 transgene was expressed in hTERT-modified cells with a lentiviral vector, along with inductive signaling through activin A and serum deprivation. This restored endocrine properties of hTERT-modified cells in vitro. Moreover, transplantation studies in immunodeficient mice verified the capacity of these cells for expressing insulin in vivo. CONCLUSIONS: Limited replication capacity of pancreatic endocrine progenitor cells was overcome by the hTERT mechanism, which should facilitate further studies of such cells, although mechanisms regulating switches between meso-endodermal fates of expanded cells will need to be controlled for developing specific applications. The availability of hTERT-expanded fetal pancreatic endocrine progenitor cells will be helpful for studying and recapitulating stage-specific beta lineage advancement in pluripotent stem cells

In Atp7b-/- Mice Modeling Wilson's Disease Liver Repopulation with Bone Marrowderived Myofibroblasts or Inflammatory Cells and not Hepatocytes is Deleterious

Background: In Wilson's disease, ATP7B mutations impair copper excretion with liver or brain damage. Healthy transplanted hepatocytes repopulate liver, excrete copper and reverse hepatic damage in animal models of Wilson's disease. In Fah-/- mice with tyrosinemia and alpha-1 antitrypsin mutant mice, liver disease is resolved by expansions of healthy hepatocytes derived from transplanted healthy bone marrow stem cells. This potential of stem cells has not been defined for Wilson's disease. Methods: In diseased Atp7b-/- mice we reconstituted bone marrow with donor cells expressing green fluorescent protein reporter from healthy transgenic mice. Mature hepatocytes originating from donor bone marrow were identified by immunostaining for green fluorescence protein and bile canalicular marker, dipeptidylpeptidase-4. Mesenchymal and inflammatory cell markers were used for other cells from donor bone marrow cells. Gene expression, liver tests and tissues were analyzed for outcomes in Atp7b-/- mice. Results: After bone marrow transplantation in Atp7b-/- mice, donor-derived hepatocytes containing bile canaliculi appeared within weeks. Despite this maturity, donor-derived hepatocytes neither divided nor expanded. Liver of Atp7b-/- mice was not repopulated by donor-derived hepatocytes: Atp7b mRNA remained undetectable; liver tests, copper content and fibrosis actually worsened. Restriction of proliferation in hepatocytes accompanied oxidative DNA damage. By contrast, donor-derived mesenchymal and inflammatory cells extensively proliferated. These contributed to fibrogenesis through greater expression of inflammatory cytokines. Conclusion: In Wilson's disease, donor bone marrow-derived cells underwent different fates: hepatocytes failed to proliferate; inflammatory cells proliferated to worsen disease outcomes. This knowledge will help guide stem cell therapies for conditions with pro-inflammatory or pro-fibrogenic microenvironments

522. Targeting FVIII-Expression To Liver Sinusoidal Cells By Lentiviral Vectors Corrects the Bleeding Phenotype in Hemophilia A Overcoming Immunological Responses

Hemophilia A (HA) is an X-linked bleeding disorder due to mutations in clotting factor (F) VIII gene. To date the treatment for preventing major bleeding episodes is represented by replacement therapy with recombinant or plasma-derived FVIII. The two major concerns are high cost and development of FVIII neutralizing antibodies in 20-30% of patients.Several studies on gene transfer by direct injection of LV for HA have been recently published. Many efforts were focused on the improvement of LV, to obtain a selective targeting of transgene expression, or on the production of several bioengineered FVIII, in order to overcome some of the issues related to FVIII expression in HA animal models. However, in most cases, the immune responses associated with FVIII remain the major obstacle.We prepared LVs containing the B-domain deleted (BDD) hFVIII under the control of PGK, VEC or CD11b promoters with or without the addition of the miRTs used for initial GFP expression studies, and we then injected HA mice with 109 TU/mouse of these LVs (3 mice for LV PGK-hFVIII ±42; 4-9 mice for the other vectors) and assessed FVIII activity by aPTT assay.All mice injected with LV-VEC-hFVIII ± miRTs and LV-CD11b-hFVIII ± miRTs showed a FVIII activity between 3.5 and 5% one week after injection, while HA mice injected with LV-PGK-hFVIII± 42 showed a FVIII activity £1%. Moreover, starting from 2 weeks after LVs injection we evaluated the presence of anti-FVIII antibodies by a direct ELISA. We detected the presence of anti-FVIII antibodies in the plasma of mice injected with LV-PGK-hFVIII±miRT-142 1 month after LV injection. Interestingly, the antibody titer was significantly lower in mice injected with LV-PGK-hFVIII-miRT-142-3p. In all mice injected with LV-VEC-hFVIII±miRT-122-142-3pwe detected hFVIII activity by aPTT assay up to 52 weeks after injection without production of anti-FVIII antibodies. HA mice injected LV-CD11b-hFVIII±miRT-126 showed hFVIII activity up to 52 w as well; interestingly, 60% of mice injected with LV-CD11b-hFVIII produced anti-FVIII antibodies 10-16 weeks after LV injection, while no anti-FVIII antibodies were detected in plasma of injected mice with LV-CD11b-hFVIII-miRT-126.Genomic analysis on liver samples from mice 24 w after injection of LV-VEC-hFVIII±miRT-122-142-3p and LV-CD11b-hFVIII±miRT-126 demonstrated the presence of LV sequence integrated in the genome of injected mice. Immunofluorescence on liver sections showed that LSECs and KCs were positive for hFVIII. Next, to assess whether EC, in particular LSECs, are able to induce immunotolerance, we immunized mice with Refacto. Mice producing anti-FVIII Ab were then injected with 109 TU of LV-VEC-hFVIII-miRT-122-142-3p. We detected hFVIII activity in all injected mice and, noteworthy, antibody titer decreased over time in the plasma of these mice.In conclusion, LV expressing FVIII under the control of VEC or CD11b promoters combined with miRTs combinations were able to overcome FVIII off-target expression limiting immune responses and providing phenotypic correction in treated HA mice with FVIII expression by sinusoidal cells

DNA base editing corrects common Hemophilia A mutations and restores factor VIII expression in vitro and ex-vivo models

Background: Replacement and non-replacement therapies effectively control bleedings in Hemophilia A (HA) but imply lifelong interventions. The authorized gene addition therapy could provide a cure but still poses questions on durability. F8 gene correction would definitively restore factor VIII (FVIII) production, as shown in animal models through nucleases mediating homologous recombination (HR). However, low efficiency and potential off-target double-strand break (DSB) still limit HR translatability. Objectives: To correct common model single point mutations leading to severe HA through the recently developed DSB/HR-independent base (BE) and prime (PE) editing approaches. Methods: Screening for efficacy of BE/PE systems in HEK293T transiently expressing FVIII variants and validation at DNA (sequencing) and protein (ELISA; aPTT) level in stable clones. Evaluation of rescue in engineered blood outgrowth endothelial cells (BOEC) by lentiviral-mediated delivery of BE. Results and conclusions: Transient assays identified the best-performing BE/PE systems for each variant, with the highest rescue of FVIII expression (up to 25% of rFVIIIwt) for the p.R2166* and p.R2228Q mutations. In stable clones we demonstrated that the mutation reversion on DNA (∼24%) was consistent with the rescue of FVIII secretion and activity 20-30%). The lentiviral-mediated delivery of the selected BE systems was attempted in engineered BOEC harboring the p.R2166* and p.R2228Q variants, which led to an appreciable and dose-dependent rescue of secreted functional FVIII. Overall data provide the first proof-of-concept for effective BE/PE-mediated correction of HA-causing mutations, which encourage studies in mouse models to develop a personalized cure for large cohorts of patients though a single intervention

Human lipoaspirate as autologous injectable active scaffold for one-step repair of cartilage defects

Research on mesenchymal stem cells from adipose tissue shows promising results for cell-based therapy in cartilage lesions. In these studies, cells have been isolated, expanded, and differentiated in vitro before transplantation into the damaged cartilage or onto materials used as scaffolds to deliver cells to the impaired area. The present study employed in vitro assays to investigate the potential of intra-articular injection of microfragmented lipoaspirate as a one-step repair strategy; it aimed to determine whether adipose tissue can act as a scaffold for cells naturally present at their anatomical site. Cultured clusters of lipoaspirate showed a spontaneous outgrowth of cells with a mesenchymal phenotype and with multilineage differentiation potential. Transduction of lipoaspirate clusters by lentiviral vectors expressing GFP evidenced the propensity of the outgrown cells to repopulate fragments of damaged cartilage. On the basis of the results, which showed an induction of proliferation and ECM production of human primary chondrocytes, it was hypothesized that lipoaspirate may play a paracrine role. Moreover, the structure of a floating culture of lipoaspirate, treated for 3 weeks with chondrogenic growth factors, changed: tissue with a high fat component was replaced by a tissue with a lower fat component and connective tissue rich in GAG and in collagen type I, increasing the mechanical strength of the tissue. From these promising in vitro results, it may be speculated that an injectable autologous biologically active scaffold (lipoaspirate), employed intra-articularly, may 1) become a fibrous tissue that provides mechanical support for the load on the damaged cartilage; 2) induce host chondrocytes to proliferate and produce ECM; and 3) provide cells at the site of injury, which could regenerate or repair the damaged or missing cartilage

Efficient Tet-dependent expression of human factor IX in vivo by a new self-regulating lentiviral vector.

Regulation of gene expression represents a long-sought goal of gene therapy. However, most viral vectors pose constraints on the incorporation of drug-dependent transcriptional regulatory systems. Here, by optimizing the design of self-regulating lentiviral vectors based on the tetracycline system, we have been able to overcome the limitations of previously reported constructs and to reach both robust expression and efficient regulation from a single vector. The improved performance allows us to report for the first time effective long-term in vivo regulation of a human clotting Factor IX (hF.IX) transgene upon systemic administration of a single vector to SCID mice. We showed that hF.IX expression in the plasma could be expressed to therapeutically significant concentrations, adjusted to different set levels by varying the tetracycline dose, rapidly turned off and on, and completely recovered after each treatment cycle. The new vector design was versatile, as it successfully incorporated a tissue-specific promoter that selectively targeted regulated expression to hepatocytes. Robust transgene expression in the systemic circulation coupled to the ability to switch off and even adjust the expression level may open the way to safer gene-based delivery of therapeutics

Axons mediate the distribution of arylsulfatase A within the mouse hippocampus upon gene delivery.

Axonal transport of the lysosomal enzyme arylsulfatase A (ARSA) may be an additional mechanism of enzyme distribution after in vivo brain gene transfer in an animal model of metachromatic leukodystrophy (MLD). Direct molecular demonstration of the movement of this lysosomal enzyme within axonal networks was missing. We generated lentiviral vectors carrying the ARSA cDNA tagged with hemagglutinin or the green fluorescent protein and examined the subcellular localization and anatomical distribution of the tagged enzymes within the MLD hippocampus after in vivo lentiviral gene transfer. The use of tagged ARSA allowed direct real-time observation and tracking of axon–dendritic transport of the enzyme after lentiviral gene therapy. Tagged ARSA was expressed in transduced pyramidal, granule, and hilar neurons within the lentiviral-injected side and was robustly contained in vesicles within ipsilateral axon–dendritic processes as well as in vesicles associated with contralateral axons and commissural axons of the ventral hippocampal commissure. Axonal transport of tagged ARSA led to the correction of hippocampal defects in long-term treated MLD mice, which was accompanied by enzyme uptake in nontransduced contralateral neurons, enzyme accumulation within the lysosomal compartment, and clearance of sulfatide storage deposits in this region of the MLD brain. These results contribute to the understanding of the mechanisms of distribution of lysosomal enzymes within the mammalian brain after direct gene therapy, demonstrating the use of neural processes for enzyme transport

Tumor targeting by monoclonal antibody functionalized magnetic nanoparticles

Tumor-targeted drug-loaded nanocarriers represent innovative and attractive tools for cancer therapy. Several magnetic nanoparticles (MNPs) were analyzed as potential tumor-targeted drug-loaded nanocarriers after functionalization with anti-Met oncogene (anti-Met/HGFR) monoclonal antibody (mAb) and doxorubicin (DOXO). Their cytocompatibility, stability, immunocompetence (immunoprecipitation), and their interactions with cancer cells in vitro (Perl's staining, confocal microscopy, cytotoxic assays: MTT, real time toxicity) and with tumors in vivo (Perl's staining) were evaluated. The simplest silica- and calcium-free mAb-loaded MNPs were the most cytocompatible, the most stable, and showed the best immunocompetence and specificity. These mAb-functionalized MNPs specifically interacted with the surface of Met/HGFR-positive cells, and not with Met/HGFR-negative cells; they were not internalized, but they discharged in the targeted cells DOXO, which reached the nucleus, exerting cytotoxicity. The presence of mAbs on DOXO-MNPs significantly increased their cytotoxicity on Met/HGFR-positive cells, while no such effect was detectable on Met/HGFR-negative cells. Bare MNPs were biocompatible in vivo; mAb presence on MNPs induced a better dispersion within the tumor mass when injected in situ in Met/HGFR-positive xenotumors in NOD/SCID-γnull mice. These MNPs may represent a new and promising carrier for in vivo targeted drug delivery, in which applied gradient and alternating magnetic fields can enhance targeting and induce hyperthermia respectively

Cell Penetrating Peptide Adsorption on Magnetite and Silica Surfaces: A Computational Investigation

Magnetic nanoparticles (MNPs) represent one of the most promising materials as they can act as a versatile platform in the field of bionanotechnology for enhanced imaging, diagnosis, and treatment of various diseases. Silica is the most common compound for preparing coated iron oxide NPs since it improves colloidal stability and the binding affinity for various organic molecules. Biomolecules such as cell penetrating peptides (CPPs) might be employed to decorate MNPs, combining their promising physicochemical properties with a cell penetrating ability. In this work, a computational investigation on adsorption of Antennapedia homeodomain-derived penetrating peptide (pAntp) on silica and magnetite (MAG) surfaces is presented. By employing umbrella sampling molecular dynamics, we provided a quantitative estimation of the pAntp-surface adsorption free energy to highlight the influence of surface hydroxylation state on the adsorption mechanism. The interaction between peptide and surface has shown to be mainly driven by electrostatics. In case of MAG surface, also an important contribution of van der Waals (VdW) attraction was observed. Our data suggest that a competitive mechanism between MNPs and cell membrane might partially inhibit the CPP to carry out its membrane penetrating function