Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors.

Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients' cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.

Assessment of mesenchymal stem/stromal cell-based therapy in K/BxN serum transfer-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and cartilage/bone destruction with systemic comorbidities. Despite advances in understanding the aetiology of RA and novel biologic drugs, a substantial number of individuals with RA remain intolerant or resistant to these therapies. In this context, mesenchymal stem/stromal cell (MSC)-based therapy has emerged as an innovative therapeutic alternative to address unresolved treatment issues for patients with RA thanks to the immunomodulatory properties of these cells. The majority of preclinical studies in MSC-based therapy have been conducted using the well-known collagen-induced arthritis (CIA) mouse model however due to its low incidence, the mouse strain restriction and the prolonged induction phase of collagen-induced arthritis, alternative experimental models of RA have been developed such as K/BxN serum transfer-induced arthritis (STIA), which mimics many of human RA features. In this study, we evaluate whether the K/BxN STIA model could be used as an alternative model to study the immunomodulatory potential of MSC-based therapy. Unexpectedly, our data suggest that adipose-derived MSC-based therapy is unsuitable for modulating the progression of K/BxN serum-transfer arthritis in mice despite the various experimental parameters tested. Based on the differences in the immune status and monocytic/macrophage balance among the different arthritic models, these results could help to identify the cellular targets of the MSCs and, most importantly to predict the RA patients that will respond positively to MSC-based therapy.

Mechanism of Immunoregulatory Properties of Vasoactive Intestinal Peptide in the K/BxN Mice Model of Autoimmune Arthritis.

The K/BxN mouse model of rheumatoid arthritis (RA) closely resembles the human disease. In this model, arthritis results from activation of autoreactive KRN T cells recognizing the glycolytic enzyme glucose-6-phosphate isomerase (GPI) autoantigen, which provides help to GPI-specific B cells, resulting in the production of pathogenic anti-GPI antibodies that ultimately leads to arthritis symptoms from 4 weeks of age. Vasoactive intestinal peptide (VIP) is a neuropeptide broadly distributed in the central and peripheral nervous system that is also expressed in lymphocytes and other immune cell types. VIP is a modulator of innate and adaptive immunity, showing anti-inflammatory and immunoregulatory properties. Basically, this neuropeptide promotes a shift in the Th1/Th2 balance and enhances dedifferentiation of T regulatory cells (Treg). It has demonstrated its therapeutic effects on the collagen-induced arthritis (CIA) mouse model of RA. In the present hypothesis and theory article, we propose that the immunoregulatory properties of VIP may be due likely to the inhibition of T cell plasticity toward non-classic Th1 cells and an enhanced follicular regulatory T cells (Tfr) activity. The consequences of these regulatory properties are the reduction of systemic pathogenic antibody titers.

Mesenchymal stem/stromal cell-based therapy for the treatment of rheumatoid arthritis: An update on preclinical studies.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction and is a primary cause of disability worldwide. Despite the existence of numerous anti-rheumatic drugs, a significant number of patients with RA do not respond or are intolerant to current treatments. Mesenchymal stem/stromal cell (MSCs) therapy represents a promising therapeutic tool to treat RA, mainly attributable to the immunomodulatory effects of these cells. This review comprises a comprehensive analysis of the scientific literature related to preclinical studies of MSC-based therapy in RA to analyse key aspects of current protocols as well as novel approaches which aim to improve the efficacy of MSC-based therapy.

Mesenchymal Stem/Stromal Cells for Rheumatoid Arthritis Treatment: An Update on Clinical Applications.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects the lining of the synovial joints leading to stiffness, pain, inflammation, loss of mobility, and erosion of joints. Its pathogenesis is related to aberrant immune responses against the synovium. Dysfunction of innate and adaptive immunity, including dysregulated cytokine networks and immune complex-mediated complement activation, are involved in the progression of RA. At present, drug treatments, including corticosteroids, antirheumatic drugs, and biological agents, are used in order to modulate the altered immune responses. Chronic use of these drugs may cause adverse effects to a significant number of RA patients. Additionally, some RA patients are resistant to these therapies. In recent years, mesenchymal stem/stromal cell (MSCs)-based therapies have been largely proposed as a novel and promising stem cell therapeutic approach in the treatment of RA. MSCs are multipotent progenitor cells that have immunomodulatory properties and can be obtained and expanded easily. Today, nearly one hundred studies in preclinical models of RA have shown promising trends for clinical application. Proof-of-concept clinical studies have demonstrated satisfactory safety profile of MSC therapy in RA patients. The present review discusses MSC-based therapy approaches with a focus on published clinical data, as well as on clinical trials, for treatment of RA that are currently underway.

Comparative Analysis between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis.

Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the "responder" mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.

BMP4 Induces M2 Macrophage Polarization and Favors Tumor Progression in Bladder Cancer.

Purpose: Bladder cancer is a current clinical and social problem. At diagnosis, most patients present with nonmuscle-invasive tumors, characterized by a high recurrence rate, which could progress to muscle-invasive disease and metastasis. Bone morphogenetic protein (BMP)-dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in bladder cancer is still unclear.Experimental Design: Tumor and normal tissue from 68 patients with urothelial cancer were prospectively collected and analyzed for expression of BMP and macrophage markers. The mechanism of action was assessed in vitro by experiments with bladder cancer cell lines and peripheral blood monocyte-derived macrophages.Results: We observed BMP4 expression is associated and favored type II macrophage differentiation. In vitro experiments showed that both recombinant BMP4 and BMP4-containing conditioned media from bladder cancer cell lines favored monocyte/macrophage polarization toward M2 phenotype macrophages, as shown by the expression and secretion of IL10. Using a series of human bladder cancer patient samples, we also observed increased expression of BMP4 in advanced and undifferentiated tumors in close correlation with epithelial-mesenchymal transition (EMT). However, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced, due to the increased miR-21 expression leading to reduced BMPR2 expression.Conclusions: These findings suggest that BMP4 secretion by bladder cancer cells provides the M2 signal necessary for a protumoral immune environment. In addition, the repression of BMPR2 by miR-21 makes the tumor cells refractory to the prodifferentiating actions mediated by BMP ligands, favoring tumor growth. Clin Cancer Res; 23(23); 7388-99. ©2017 AACR.

Biodistribution and Efficacy of Human Adipose-Derived Mesenchymal Stem Cells Following Intranodal Administration in Experimental Colitis.

Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.

Adipose-derived mesenchymal stromal cells modulate experimental autoimmune arthritis by inducing an early regulatory innate cell signature.

Modulation of innate immune responses in rheumatoid arthritis and other immune-mediated disorders is of critical importance in the clinic since a growing body of information has shown the key contribution of dysregulated innate responses in the progression of the disease. Mesenchymal stromal cells (MSCs) are the focus of intensive efforts worldwide due to their key role in tissue regeneration and modulation of inflammation. In this study, we define innate immune responses occurring during the early course of treatment with a single dose of expanded adipose-derived MSCs (eASCs) in established collagen-induced arthritis. eASCs delay the progression of the disease during the early phase of the disease. This is accompanied by a transient induction of Ly6C+ monocytes that differentiate into IL10+F4/80+ cells in arthritic mice. Strikingly, the induced IL10+F4/80+ myeloid cells preferentially accumulated in the draining lymph nodes. This effect was accompanied with a concomitant declining of their frequencies in the spleens. Our results show that eASCs attenuate the arthritic process by inducing an early innate cell signature that involves a transient induction of Ly6C+ monocytes in periphery that differentiate into IL10+F4/80+ macrophages. Our findings demonstrate that early regulatory innate cell responses, involving the monocyte compartment, are targeted by the eASCs during the onset of collagen-induced inflammation.

Human Adipose-Derived Mesenchymal Stem Cells Modulate Experimental Autoimmune Arthritis by Modifying Early Adaptive T Cell Responses.

Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunosuppressive properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Recent data have identified that GM-CSF-expressing CD4 T cells and Th17 cells have critical roles in the pathogenesis of arthritis and other inflammatory diseases. Although many studies have demonstrated that MSCs can either prevent or suppress inflammation, no studies have addressed their modulation on GM-CSF-expressing CD4 T cells and on the plasticity of Th17 cells. To address this, a single dose of human expanded adipose-derived mesenchymal stem cells (eASCs) was administered to mice with established collagen-induced arthritis. A beneficial effect was observed soon after the infusion of the eASCs as shown by a significant decrease in the severity of arthritis. This was accompanied by reduced number of pathogenic GM-CSF(+) CD4(+) T cells in the spleen and peripheral blood and by an increase in the number of different subsets of regulatory T cells like FOXP3(+) CD4(+) T cells and IL10(+) IL17(-) CD4(+) T cells in the draining lymph nodes (LNs). Interestingly, increased numbers of Th17 cells coexpressing IL10 were also found in draining LNs. These results demonstrate that eASCs ameliorated arthritis after the onset of the disease by reducing the total number of pathogenic GM-CSF(+) CD4(+) T and by increasing the number of different subsets of regulatory T cells in draining LNs, including Th17 cells expressing IL10. All these cellular responses, ultimately, lead to the reestablishment of the regulatory/inflammatory balance in the draining LNs.

Survival and biodistribution of xenogenic adipose mesenchymal stem cells is not affected by the degree of inflammation in arthritis.

BACKGROUND: Application of mesenchymal stem/stromal cells (MSCs) in treating different disorders, in particular osteo-articular diseases, is currently under investigation. We have already documented the safety of administrating human adipose tissue-derived stromal MSCs (hASCs) in immunodeficient mice. In the present study, we investigated whether the persistence of MSC is affected by the degree of inflammation and related to the therapeutic effect in two inflammatory models of arthritis. METHODOLOGY/PRINCIPAL FINDINGS: We used C57BL/6 or DBA/1 mice to develop collagenase-induced osteoarthritis (CIOA) or collagen-induced arthritis (CIA), respectively. Normal and diseased mice were administered 2.5×10(5) hASCs in the knee joints (i.a.) or 10(6) in the tail vein (i.v.). For CIA, clinical scores were monitored during the time course of the disease while for CIOA, OA scores were assessed by histology at euthanasia. Thirteen tissues were recovered at different time points and processed for real-time PCR and Alu sequence detection. Immunological analyses were performed at euthanasia. After i.v. infusion, no significant difference in the percentage of hASCs was quantified in the lungs of normal and CIA mice at day 1 while no cell was detected at day 10 taking into account the sensitivity of the assay, indicating that a high level of inflammation did not affect the persistence of cells. In CIOA mice, we reported the therapeutic efficacy of hASCs at reducing OA clinical scores at day 42 when hASCs were not detected in the joints. However, the percentage and distribution of hASCs were similar in osteoarthritic and normal mice at day 1 and 10 after implantation indicating that moderate inflammation does not alter hASC persistence in vivo. CONCLUSIONS/SIGNIFICANCE: While inflammatory signals are required for the immunosuppressive function of MSCs, they do not enhance their capacity to survive in vivo, as evaluated in two xenogeneic inflammatory pre-clinical models of arthritis.

Functional characterisation of the WW minimal domain for delivering therapeutic proteins by adenovirus dodecahedron.

Protein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron) has recently been shown to deliver proteins fused to the tandem WW(2-3-4) structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW(3) and its mutant form WW(3)_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application.

In vivo delivery of antigens by adenovirus dodecahedron induces cellular and humoral immune responses to elicit antitumor immunity.

Cancer vaccines based on virus-like particles (VLPs) vectors may offer many advantages over other antigen-delivery systems and represent an alternative to the ex vivo cell therapy approach. In this study, we describe the use of penton-dodecahedron (Pt-Dd) VLPs from human adenovirus type 3 (Ad3) as cancer vaccine vehicle for specific antigens, based on its unique cellular internalization properties. WW domains from the ubiquitin ligase Nedd4 serve as an adapter to bind the antigen to Pt-Dd. By engineering fusion partners of WW with the model antigen ovalbumin (OVA), Pt-Dd can efficiently deliver WW-OVA in vitro and the Pt-Dd/WW complex can be readily internalized by dendritic cells (DCs). Immunization with WW-OVA/Pt-Dd results in 90% protection against B16-OVA melanoma implantation in syngeneic mice. This high level of protection correlates with the development of OVA-specific CD8(+) T cells. Moreover, vaccination with WW-OVA Pt-Dd induces robust humoral responses in mice as shown by the high levels of anti-OVA antibodies (Abs) detected in serum. Importantly, treatment of mice bearing B16-OVA tumors with WW-OVA/Pt-Dd results in complete tumor regression in 100% of cases. Thus, our data supports a dual role of Pt-Dd as antigen-delivery vector and natural adjuvant, able to generate integrated cellular and humoral responses of broad immunogenic complexity to elicit specific antitumor immunity. Antigen delivery by Pt-Dd vector is a promising novel strategy for development of cancer vaccines with important clinical applications.

Development and application of quantitative real time PCR and RT-PCR assays that discriminate between the full-length and truncated herpes simplex virus thymidine kinase gene.

Allogeneic donor T lymphocytes manipulated genetically to express the herpes simplex virus thymidine kinase (HSV-TK) gene have emerged as promising tools to alter the balance between graft versus host disease and graft versus leukemia after allogeneic stem cell transplantation, since they can be eliminated selectively in vivo with ganciclovir. Recently, it was reported that in SFCMM-3, an HSV-TK-encoding retroviral vector, two cryptic splice sites in the HSV-TK sequence led to the generation of an HSV-TK splice variant (deltaHSV-TK) that encodes a ganciclovir-resistant gene product. In order to quantify wtHSV-TK and deltaHSV-TK RNA levels we have developed two real time Taqman PCR assays. We demonstrate that the sensitivity of both PCR assays is 10(-4). It was found that the splice variant is generated in the packaging cell line and results in approximately 4.8+/-1.9% of virions that contain deltaHSV-TK RNA. After transduction of human T cells no significant increase in deltaHSV-TK RNA could be detected. Thus, at maximum 4.2+/-1.2% of T cells transduced with SFCMM-3 will be resistant to ganciclovir due to this mechanism only. Together, these assays provide a powerful method to monitor patients in future clinical trials.