Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in 24 patients out of 34 (71%) was predicted by the type of mutations. Preexisting Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8.

Recently, development of Adeno-associated virus (AAV) vectors has been focusing on expanding the genetic diversity of vectors from existing sequences via directed evolution or epitope remapping. Apart from intelligent design, AAV isolation from natural sources remains an important source of new AAVs with unique biological features. In this study, several new AAV sequences were isolated from porcine tissues (AAVpo2.1, -po4, -po5, and -po6), which aligned in divergent new clades. Viral particles generated from these sequences displayed tissue tropism and transduction efficiency profile specific to each porcine-derived AAV. When delivered systemically, AAVpo2.1 targeted the heart, kidney, and muscle, AAVpo5 performed poorly but was able to transduce muscle fibers when injected intramuscularly, whereas AAVpo4 and -po6 efficiently transduced all the major organs sampled, contending with 'gold-standard' AAVs. When delivered systemically, AAVpo4 and -po6 were detected by polymerase chain reaction (PCR) and histochemical staining of the transgene product in adult mouse brain, suggesting that these vectors can pass through the blood-brain barrier with efficiencies that may be useful for the development of therapeutic approaches. Porcine tissues are antigenically similar to human tissues and by inference, porcine AAVs may provide fresh tools to contribute to the development of gene therapy-based solutions to human diseases.

Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.

Background: We investigate the role of family history of cancer (FHC) and diagnosis of metachronous and/or synchronous multiple neoplasms (MN), during anti-PD-1/PD-L1 immunotherapy. Design: This was a multicenter retrospective study of advanced cancer patients treated with anti-PD-1/PD-L1 immunotherapy. FHC was collected in lineal and collateral lines, and patients were categorized as follows: FHC-high (in case of cancer diagnoses in both the lineal and collateral family lines), FHC-low (in case of cancer diagnoses in only one family line), and FHC-negative. Patients were also categorized according to the diagnosis of MN as follows: MN-high (>2 malignancies), MN-low (two malignancies), and MN-negative. Objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and incidence of immune-related adverse events (irAEs) of any grade were evaluated. Results: 822 consecutive patients were evaluated. 458 patients (55.7%) were FHC-negative, 289 (35.2%) were FHC-low, and 75 (9.1%) FHC-high, respectively. 29 (3.5%) had a diagnosis of synchronous MN and 94 (11.4%) of metachronous MN. 108 (13.2%) and 15 (1.8%) patients were MN-low and MN-high, respectively. The median follow-up was 15.6 months. No significant differences were found regarding ORR among subgroups. FHC-high patients had a significantly longer PFS (hazard ratio [HR] = 0.69 [95% CI: 0.48-0.97], p = .0379) and OS (HR = 0.61 [95% CI: 0.39-0.93], p = .0210), when compared to FHC-negative patients. FHC-high was confirmed as an independent predictor for PFS and OS at multivariate analysis. No significant differences were found according to MN categories. FHC-high patients had a significantly higher incidence of irAEs of any grade, compared to FHC-negative patients (p = .0012). Conclusions: FHC-high patients seem to benefit more than FHC-negative patients from anti-PD-1/PD-L1 checkpoint inhibitors.

Stem cells offer the basis for the promotion of robust new therapeutic approaches for a variety of human disorders. There are still many limitations to be overcome before clinical therapeutic application, including a better understanding of the mechanism by which stem cell therapies may lead to enhanced recovery. In vitro investigations are necessary to dissect the mechanisms involved and to support the potential development in stem cell-based therapies. In spite of growing interest in human amniotic fluid stem cells, not much is known about the characteristics of their secretome and regarding the potential neuroprotective mechanism in different pathologies, including stroke. To get more insight on amniotic fluid cells therapeutic potential, signal transduction pathways activated by human amniotic fluid stem cells (hAFSCs)-derived secretome in a stroke in vitro model (ischemia/reperfusion [I/R] model) were investigated by Western blot. Moreover, miRNA expression in the exosomal fraction of the conditioned medium was analyzed. hAFSCs-derived secretome was able to activate pro-survival and anti-apoptotic pathways. MicroRNA analysis in the exosomal component revealed a panel of 16 overexpressed miRNAs involved in the regulation of coherent signaling pathways. In particular, the pathways of relevance in ischemia/reperfusion, such as neurotrophin signaling, and those related to neuroprotection and neuronal cell death, were analyzed. The results obtained strongly point toward the neuroprotective effects of the hAFSCs-conditioned medium in the in vitro stroke model here analyzed. This can be achieved by the modulation and activation of pro-survival processes, at least in part, due to the activity of secreted miRNAs.

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.