We present here the results of a first-in-man, first-in-child trial for patients with relapsed/refractory solid tumors using Celyvir, an Advanced Therapy Medicine that combines autologous mesenchymal stem cells carrying an oncolytic adenovirus. Celyvir was manufactured from a bone marrow aspirate and then given intravenously. Patients received weekly infusions for 6 weeks at a dose of 2x106 cells/Kg (children) or 0,5-1x106 cells/Kg (adults), 2x104 viral particles per cell. Fifteen pediatric and 19 adult patients were recruited but 18 were screen failures, mainly because rapid disease progression before Celyvir was available. No grade 2-5 toxicities were reported. Adenoviral replication detected by PCR was found in all but 2 pediatric patient and in none of the adult ones. Absolute numbers of circulating leukocytes suffered minor changes along therapy but some subsets showed differences comparing the pediatric versus the adult cohorts. Two patients with neuroblastoma showed disease stabilization, one of them continued on treatment for up to 6 additional weeks. Celyvir, the combination of MSCs and oncolytic adenovirus is safe and warrants further evaluation in phase 2 setting. The use of MSCs may be a strategy to increase the amount of oncolytic virus administered to patients, minimizing toxicities and avoiding direct tumor injections.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting infants, caused by alterations of the survival motor neuron gene, which results in progressive degeneration of motor neurons (MNs). Although an effective treatment for SMA patients has been recently developed, the molecular pathway involved in selective MNs degeneration has not been yet elucidated. In particular, miRNA-206 has been demonstrated to play a relevant role in the regeneration of neuromuscular junction in several MNs diseases and particularly it is up-regulated in the quadriceps, tibialis anterior, spinal cord and serum of SMA mice. In the present paper, we demonstrated that miRNA-206 was transiently up-regulated also in the brain stem of a mouse model of SMA, in the early phase of the disease, paralleling MNs degeneration. In order to prevent miRNA-206 down-regulation occurring in the symptomatic phase of the disease, we intracerebroventricularly injected miR-206 in SMA pups, demonstrating that miR-206 reduced the severity of SMA pathology, slowing down disease progression, increasing survival rate and improving behavioral performance of mice. Interestingly, miRNA-206 up-regulation induced a reduction of the predicted target sodium calcium exchanger isoform 2, NCX2, one of the main regulators of intracellular [Ca2+] and [Na+]. Therefore, we hypothesized that miR-206 might exert part of its neuroprotective effect modulating NCX2 expression in SMA disease.

Age-related macular degeneration (AMD) is a universal leading cause for irreversible blindness in the elderly population. Dedifferentiation of retinal pigment epithelium (RPE) cells initiates early pathological events in atrophic AMD. Herein, we aim to investigate effects of a circular RNA derived from the NR3C1 gene (circNR3C1) on regulating RPE function and AMD pathogenesis. CircNR3C1 expression was consistently up-regulated along with RPE differentiation, and was down-regulated in dysfunctional RPE and blood serum of AMD patients. Silencing of circNR3C1 reduced RPE characteristic transcripts and proteins, interrupted phagocytosis, accelerated intracellular ROS generation, and promoted RPE proliferation in vitro. CircN3C1 silencing also decreased expressions of RPE characteristic markers and disturbed the ultrastructure of RPE in vivo, as shown by a thickened RPE with twisted basal infoldings and outer segments. Mechanistically, circNR3C1 acted as an endogenous miR-382-5p sponge to sequester its activity, which increased PTEN expression and inhibited the AKT/mTOR pathway. miR-382-5p overexpression and PTEN silencing mimicked effects of circNR3C1 silencing on RPE phenotypes in vivo and in vitro. In conclusion, circNR3C1 prevents AMD progression and protects RPE by directly sponging miR-382-5p to block its interaction with PTEN, and subsequently blocks the AKT/mTOR pathway. Pharmacological circNR3C1 supplementations are promising therapeutic options for atrophic AMD.

Diabetic nephropathy (DN) is an increasing threat to human health and regarded as an important public issue. The pathophysiologic mechanisms of DN are complicated. The initiating molecular events triggering the loss function in mesangial cells in DN are not well-known. In this cross-disciplinary study, transcriptome analysis of high glucose (HG)-treated mouse mesangial cells (MMCs) using next-generation sequencing and systematic bioinformatics analyses indicated that miR-15b-5p and its downstream target B-cell lymphoma 2 (BCL-2) contribute to HG-induced apoptosis in MMCs. HG elevated miR-15b-5p expression, which in turn decreased the translation of BCL-2, leading to MMC apoptosis under HG. Apoptosis of MCs was enhanced in the presence of extracellular vesicles isolated from the urine of type 2 diabetic patients with high levels of miR-15b-5p. Furthermore, increased levels of urinary miR-15b-5p were found in db/db mice and type 2 diabetic patients, and it correlated with low baseline kidney function and rapid decline in kidney function during a mean of follow-up period of 2.4 ± 0.1 year. Therefore, miR-15b-5p induced mesangial cells apoptosis by targeting BCL-2 under HG. miR-15b-5p has the potential to predict kidney injury in DN. Blocking miR-15b-5p epigenetic regulatory network could be a potential therapeutic strategy to prevent mesangial apoptosis in DN.

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients, and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 IgG-based bispecific antibody (7370) with high affinity for FLT3 and long half-life to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal disorder that can be caused by mutations in the superoxide dismutase 1 (SOD1) gene. Though ALS is currently incurable, CRISPR base editors hold the potential to treat the disease through their ability to create nonsense mutations that can permanently disable the expression of the mutant SOD1 gene. However, the restrictive carrying capacity of adeno-associated virus (AAV) vectors has limited their therapeutic application. Here we establish an intein-mediated trans-splicing system that enables in vivo delivery of cytidine base editors (CBEs) consisting of the widely used Cas9 protein from Streptococcus pyogenes. We show that intrathecal injection of dual AAV particles encoding a split-intein CBE engineered to trans-splice and introduce a nonsense-coding substitution into a mutant SOD1 gene prolonged survival and markedly slowed the progression of disease in the G93A-SOD1 mouse model of ALS. Adult animals treated by this split-intein CRISPR base editor had a reduced rate of muscle atrophy, decreased muscle denervation, improved neuromuscular function and up to 40% fewer SOD1 immunoreactive inclusions at end-stage compared to control mice. This work expands the capabilities of single-base editors and demonstrates their potential for gene therapy.

With the FDA and EMA approvals for Zolgensma®, Luxturna® and Glybera®, recombinant adeno-associated viruses (rAAV) are considered efficient tools for gene transfer. However, studies in animals and humans demonstrate that intramuscular (IM) AAV delivery can trigger immune responses to AAV capsid and/or transgene. IM delivery of rAAV1 in humans has also been described to induce tolerance to rAAV characterized by the presence of capsid-specific regulatory T cells (Tregs) in periphery. To understand mechanisms responsible for tolerance and parameters involved, we tested 3 muscle-directed administration routes in rhesus monkeys: IM, venous limb perfusion and the intra-arterial push and dwell method. These 3 methods were well tolerated and led to transgene expression. Interestingly, gene transfer in muscle led to Tregs and exhausted T cell infiltrates in situ at both Day 21 and Day 60 post injection. In human samples, an in-depth analysis of the functionality of these cells demonstrates that capsid-specific exhausted T cells are detected after at least 5 years post vector delivery and that the exhaustion can be reversed by blocking the checkpoint pathway. Overall, our study shows that persisting transgene expression after gene transfer in muscle is mediated by Tregs and exhausted T cells.

Loss of dystrophin leads to Duchenne muscular dystrophy (DMD). A pathogenic feature of DMD is the significant elevation of cytosolic calcium. Supra-physiological calcium triggers protein degradation, membrane damage, and eventually muscle death and dysfunction. Sarco/endoplasmic reticulum (SR) calcium ATPase (SERCA) is a calcium pump that transports cytosolic calcium to the SR during excitation-contraction coupling. We hypothesize that a single systemic delivery of SERCA2a with adeno-associated virus (AAV) may improve calcium recycling and provide long-lasting benefits in DMD. To test this, we injected an AAV9 human SERCA2a vector (6x1012 viral genome particles/mouse) intravenously to 3-month-old mdx mice, the most commonly used DMD model. Immunostaining and western blot showed robust human SERCA2a expression in the heart and skeletal muscle for 18-months. Concomitantly, SR calcium uptake was significantly improved in these tissues. SERCA2a therapy significantly enhanced grip force and treadmill performance, completely prevented myocardial fibrosis, and normalized ECG. Cardiac catheterization showed normalization of multiple systolic and diastolic hemodynamic parameters in treated mice. Importantly, chamber dilation was completely prevented and ejection fraction was restored to the wild-type level. Our results suggest that a single systemic AAV9 SERCA2a therapy has the potential to provide long-lasting benefits for DMD.

Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and humans that received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward.

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared to control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg, P < 0.001, n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared to untreated eyes (25 ± 5/mm2, P < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.

Integration of new DNA into a cellular chromosome can alter the activity of nearby genes, sometimes affecting subsequent cell growth. A potent form of insertional mutagenesis involves integration of retroviral DNA produced by reverse transcription, a required step in the replication of retroviruses. In recent years retroviral replication has been adapted to allow new gene addition by retroviral vectors. Early in the history of retrovirus research, analysis of insertional mutagenesis in laboratory animals was found at times to result in transformation, leading to the discovery of cellular proto-oncogenes. In-depth analysis of the genetic consequences showed that integration of retroviral DNA could alter the gene activity in a variety of ways. Mechanisms of retroviral insertional mutagenesis in humans are much less well documented. However, recent work from the gene therapy and HIV fields now specify four genetic mechanisms of retroviral insertional mutagenesis in humans: 1) gene activation by integration of an enhancer sequence encoded in a retroviral vector (enhancer insertion), 2) gene activation by promoter insertion, 3) gene inactivation by insertional disruption, and 4) gene activation by mRNA 3’ end substitution. In each example, integration in patients was associated with clonal expansion or frank transformation.

The use of gene therapy may allow replacement of the defective gene. Minigenes, such as cDNAs, are often used. However, these may not express normal physiological genetic profiles due to lack of crucial endogenous regulatory elements. We constructed DNA nanoparticles (NPs) that contain either the mouse or human full-length rhodopsin genomic locus, including endogenous promoters, all introns, and flanking regulatory sequences of the 15-16 kb genomic rhodopsin DNA inserts. We transduced the NPs into primary retinal cell cultures from the rhodopsin knockout (RKO) mouse in vitro and into the RKO mouse in vivo and compared the effects on different functions to plasmid cDNA NP counterparts that were driven by ubiquitous promoters. Our results demonstrate that genomic DNA vectors resulted in long-term high levels of physiological transgene expression over a period of 5 months. In contrast, the cDNA counterparts exhibited low levels of expression with sensitivity to the endoplasmic reticulum (ER) stress mechanism using the same transgene copy number both in vitro and in vivo. This study demonstrates for the first time the transducing of the rhodopsin genomic locus using compacted DNA NPs.

Despite extensive usage of gene therapy medicinal products (GTMPs) in clinical studies and recent approval of CAR T cell therapy, little information has been made available on the precise molecular characterization and possible variations in terms of insert integrity and vector copy numbers of different GTMPs during the complete production chain. Within this context we characterize αβT cells Engineered to express a defined γδT cell receptor (TEGs) currently used in a first-in-human clinical study (NTR6541). Utilizing Targeted Locus Amplification in combination with Next Generation Sequencing for the vector producer clone and TEG001 products we report on 5 single nucleotide variants and 9 intact vector copies integrated in the producer clone. The vector copy number in TEG001 cells was on average a factor 0.72 (SD 0.11) below that of the producer cell clone. All nucleotide variants were transferred to TEG001, without having an effect on cellular proliferation during extensive in vitro culture. Based on an environmental risk assessment of the 5 nucleotide variants present in the non-coding viral region of the TEG001 insert, there was no altered environmental impact of TEG001 cells. We conclude that TEG001 cells do not have an increased risk for malignant transformation in vivo.

The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a TGF-β receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T-cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T-cell approach. Here, we describe the development and evaluation of a CAR that targets MISIIR. T-cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T-cells also recognized a panel of human ovarian and endometrial cancer cell lines, and lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.

Pumilio (PUM) proteins are members of a highly conserved RNA-binding protein family that posttranscriptionally regulate gene expression in many organisms. However, their roles in the placenta are unclear. In the present study, we report the requirement for the PUM homolog 1 (PUM1) gene in preeclampsia (PE). Immunofluorescence and immunohistochemical data showed that PUM1 was highly expressed in human placental villi from women with PE compared to healthy controls (HCs). Further, PUM1 overexpression repressed, and knockdown enhanced, the invasion and proliferation of trophoblasts. Interestingly, PUM1 knockdown promoted trophoblast invasion in a villous explant culture model, while PUM1 overexpression repressed these effects. Furthermore, lncRNAs transcriptome sequencing coupled with RNA immunoprecipitation (RIP) revealed that PUM1 inhibits trophoblast invasion in PE by downregulating the expression of lncRNA HOTAIR. Moreover, PUM1 regulates HOTAIR expression via a posttranscriptional mechanism. Using RNA-protein pull-down and mRNA stability assays, we identified PUM1 as a specific binding partner that decreased the half-life of HOTAIR and lowered the steady-state level of HOTAIR expression, suggesting a novel posttranscriptional regulatory mechanism. Collectively, these findings identified a novel RNA regulatory mechanism, revealing a new pathway governing the regulation of PUM1/HOTAIR in trophoblast invasion in the pathogenesis of PE.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which is governed by Wnt signaling cascade. To promote BMSCs differentiation towards osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers canonical Wnt pathway) and Foxc2 (that elicits non-canonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14 days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis in vitro. Furthermore, the robust and prolonged co-activation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

The recycling activity of cytidine deaminase within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. On the other hand, CDA also can deaminate cytidine and deoxycytidine analogue-based therapies, such as gemcitabine. Thus, CDA over-expression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.

Traditional CRISPR/Cas9-based gene knockouts are generated by introducing DNA double-strand breaks (DSBs), but this may cause excessive DNA damage or cell death. CRISPR-based cytosine base editors (CBEs) and adenine base editors (ABEs) can facilitate C-to-T or A-to-G exchanges, respectively, without DSBs. CBEs have been employed in a gene knockout strategy: CRISPR-STOP or i-STOP changes single nucleotides to induce in-frame stop codons. However, this strategy is not applicable for some genes, and the unwanted mutations in CBE systems have recently been reported to be surprisingly significant. As a variant, the ABE systems mediate precise editing and has only rare unwanted mutations. In this study, we implemented a new strategy to induce gene silencing (i-Silence) with an ABE-mediated start codon mutation from ATG to GTG or ACG. Using both in vitro and in vivo model systems, we showed that the i-Silence approach is efficient and precise for producing a gene knockout. In addition, the i-Silence strategy can be employed to analyze ∼17,804 human genes and can be used to mimic 147 kinds of pathogenic diseases caused by start codon mutations. Altogether, compared to other methods, the ABE-based i-Silence method is a safer gene KO strategy, and it has promising application potential.

Emerging evidence has indicated that abnormal methylation of DNA contributes to hepatocarcinogenesis. However, the regulatory mechanisms are not well known. Here, we revealed miR-639 expression is downregulated in liver cancer tissues and cells. The repression of miR-639 expression was attributed to hypermethylation in its promoter region, and DNMT3A was found to mediate this hypermethylation. Repression of miR-639 expression promoted cell growth and migration/invasion in vitro and the growth of tumors in xenograft mouse models. Furthermore, miR-639 bound to the 3’UTR of both MYST2 and ZEB1 and suppressed their expression. MYST2 promoted the growth of liver cancer cells and ZEB1 facilitated the migration/invasion of liver cancer cells. Ectopic expression of MYST2 and ZEB1 counteracted the repression of malignancy induced by miR-639, which coincided with the reciprocal correlation between miR-639 and MYST2 and ZEB1 expression in clinical HCC tissues. Thus, DNMT3A-mediated hypermethylation suppressed miR-639 expression, derepressing the expression of MSYT2 and ZEB1, which promoted tumorigenesis of liver cancer. These findings may shed light on the mechanism of abnormal expression of miRNAs involved in the malignancy of liver cancer and provide new biomarkers for liver cancer.

Transfer of genes by adeno-associated virus (AAV) vectors is benefiting patients with particular genetic defects. Challenges remain by rejection of AAV-transduced cells, which may be caused by CD8+ T lymphocytes directed to AAV capsid antigens. That reducing numbers of CpG motifs from the genome of AAV vectors reduces expansion of naïve T cells directed against an epitope within the capsid. In contrast, AAV capsid-specific memory CD8+ T cells respond more vigorously to AAV vectors lacking CpG motifs than to those with CpG motifs presumably reflecting dampening of T cell expansion by cytokines from the innate immune system. Depending on the purification method, AAV vector preparations can contain substantial amounts of empty AAV particles that failed to package the genome. Others have used empty particles as decoys to AAV neutralizing antibodies. We tested if empty AAV vectors given alone or mixed with genome-containing AAV vectors induce proliferation of naïve or memory CD8+ T cells directed to an antigen within AAV capsid. Naïve CD8+ T cells failed to respond to empty AAV vectors, which in contrast induced expansion of AAV-specific memory CD8+ T cells.

This phase I study investigated the safety and activity of lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin (CART-meso) in patients with malignant pleural mesothelioma, ovarian carcinoma, and pancreatic ductal adenocarcinoma. Fifteen patients with chemotherapy-refractory cancer (n = 5 per indication) were treated with a single CART-meso cell infusion. CART-meso cells were engineered by lentiviral transduction with a construct composed of the anti-mesothelin single-chain variable fragment derived from the mouse monoclonal antibody SS1 fused to intracellular signaling domains of 4-1BB and CD3zeta. Patients received 1–3 × 107 or 1–3 × 108 CART-meso cells/m2 with or without 1.5 g/m2 cyclophosphamide. Lentiviral-transduced CART-meso cells were well tolerated; one dose-limiting toxicity (grade 4, sepsis) occurred at 1–3 × 107/m2 CART-meso without cyclophosphamide. The best overall response was stable disease (11/15 patients). CART-meso cells expanded in the blood and reached peak levels by days 6–14 but persisted transiently. Cyclophosphamide pre-treatment enhanced CART-meso expansion but did not improve persistence beyond 28 days. CART-meso DNA was detected in 7/10 tumor biopsies. Human anti-chimeric antibodies (HACA) were detected in the blood of 8/14 patients. CART-meso cells were well tolerated and expanded in the blood of all patients but showed limited clinical activity. Studies evaluating a fully human anti-mesothelin CAR are ongoing.

Recent evidence suggests that microRNAs (miRNAs) can be released to the extracellular microenvironment and mediate cell-cell communication through exosomes. The aim of this study was to identify exosomal miR-301a (exo-miR-301a) involved in glioblastoma (GBM) radioresistance and reveal the possible mechanisms. The exo-miR-301a specifically secreted by hypoxic GBM cells could transfer to corresponding normoxia-cultured cells and promote radiation resistance. Hypoxic exo-miR-301a directly targeted TCEAL7 genes, which were identified as a tumor suppressor in GBM malignancy and actively repressed its’ expression in normoxic glioma cells. Our studies indicated that TCEAL7 negatively regulated the Wnt/β-catenin pathway by blocking β-catenin translocation from cytoplasm to nucleus. Interestingly, we clarified that the Wnt/β-catenin signaling was activated by miR-301a and TCEAL7 mediated the important procession. The exo-miR-301a was involved in the resistance to radiotherapy, and the effects would be reversed by miR-301a inhibition or TCEAL7 overexpression to regulate the Wnt/β-catenin axis. Here we show that exo-miR-301a, which is characteristically expressed and secreted by hypoxic glioma cells, is a potent regulator of Wnt/β-catenin and then depresses radiation sensitivity through targeting anti-oncogene TCEAL7. The newly identified exo-miR-301a/TCEAL7-signaling axis could present a novel target for cellular resistance to cancer therapeutic radiation in GBM patients.

Many patients with systemic lupus erythematosus (SLE) have lupus nephritis, one of the severe complications of SLE. We previously reported that CD8+CD103+ T regulatory cells induced ex vivo with transforming growth factor β (TGF-β) (iTregs) inhibited immune cells responses to ameliorate excessive autoimmune inflammation. However, the molecular mechanism(s) underlying the role of these CD8+ iTregs is still unclear. Here we identified that CD39, which is highly expressed on CD8+ iTregs, crucially contributes to the immunosuppressive role of the CD8+CD103+ iTregs. We showed that adoptive transfer of CD8+CD103+ iTregs significantly relieves the chronic graft-versus-host disease with lupus nephritis and CD39 inhibitor mostly abolished the functional activities of these CD8+ iTregs in vitro and in vivo. CD39+ cells sorted from CD8+CD103+ iTregs were more effective in treating lupus nephritis than CD39− partner cells in vivo. Furthermore, human CD8+ iTregs displayed increased CD103 and CD39 expressions, and CD39 was involved in the suppressive function of human CD8+ iTregs. Thus, our data implicated a crucial role of CD39 in CD8+CD103+ iTregs in treating lupus nephritis, and CD39 could be a new phenotypic biomarker for the identification of highly qualified CD8+ Tregs. This subpopulation may have therapeutic potential in patients with SLE nephritis and other autoimmune diseases.

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c+ cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases.

Multiple clinical trials employing recombinant adeno-associated viral (rAAV) vectors have been initiated for neuromuscular disorders, including Duchenne and limb-girdle muscular dystrophies, spinal muscular atrophy, and recently X-linked myotubular myopathy (XLMTM). Our previous work on a canine model of XLMTM showed that a single rAAV8-cMTM1 systemic infusion corrected structural abnormalities within the muscle and restored contractile function, with affected dogs surviving more than four years post injection. This remarkable therapeutic efficacy presents a unique opportunity to identify the downstream molecular drivers of XLMTM pathology, and to what extent the whole muscle transcriptome is restored to normal after gene transfer. Herein, RNA-sequencing was used to examine the transcriptomes of the Biceps femoris and Vastus lateralis in a previously-described canine cohort that showed dose-dependent clinical improvements after rAAV8-cMTM1 gene transfer. Our analysis confirmed several dysregulated genes previously observed in XLMTM mice, but also identified transcripts linked to XLMTM pathology. We demonstrated XLMTM transcriptome remodeling and dose-dependent normalization of gene expression after gene transfer and created metrics to pinpoint potential biomarkers of disease progression and correction.

Background NL003 is a plasmid engineered to simultaneously express two isoforms of hepatocyte growth factor. This phase II study was performed to assess the clinical safety and efficacy of intramuscular injection of NL003 in critical limb ischemia (CLI) patients for 6 months. Methods and results Two hundred patients (Rutherford scale 4-5) were randomly assigned: placebo (n=50) and low-dose (n=50), middle-dose (n=50) or high-dose NL003 (n=50). Drug was administered in the affected limb of 197 patients on days 0, 14, and 28. No significant differences in the incidence of adverse events (AEs) or serious AEs were found among the groups. At 6 months, pain severity was significantly reduced in all NL003 groups but not in the placebo group (p<0.05). The proportion of patients with complete ulcer healing in the high-dose group was significantly higher than that of the placebo group (p=0.0095). There were no statistically significant differences in transcutaneous oxygen pressure(TcPO2), ankle-brachial index(ABI) or toe-brachial index(TBI) value among the four groups throughout the study period. Conclusions These results provide the first effective evidence of significant improvements in total healing of ulcers in treated legs, complete pain relief without analgesics, and safety for NL003 in patients with Rutherford stage 4 to 5.

Extracellular vesicles (EVs) are membrane vesicles released virtually by all cell types. Several studies have shown that stem cell derived EVs may mimic both in vitro and in vivo the biological effects of the cells. We recently demonstrated that non-alcoholic steatohepatitis (NASH) is inhibited by treatment with stem cell-like population derived from human adult liver, defined as HLSCs. The aim of the present study was to evaluate whether EVs released by HLSCs influence the progression of NASH, induced by a diet deprived of methionine and choline, in immunocompromised mice. EV treatment was initiated after 2 weeks of diet with a biweekly administration of three different doses. Bio-distribution evaluated by optical imaging showed a preferential accumulation in normal and in particular in fibrotic liver. EV treatment significantly improved liver function and reduced signs of liver fibrosis and inflammation, at both morphological and molecular levels. In particular, we observed that out of 29 fibrosis-associated genes up-regulated in NASH liver, 28 were significantly downregulated by EV treatment. In conclusion, HLSC-derived EVs display anti-fibrotic and anti-inflammatory effects in a model of chronic liver disease leading to an improvement of liver function.

miR24-2 is associated with human tumorigenesis, however, its molecular mechanisms are poorly understood. Herein, our findings demonstrate that miR24-2 promotes the proliferation ability in vitro and the tumorigenic ability in vivo in human liver cancer stem cells. Mechanically, the miR24-2 targets for 3'-UTR (2627-2648) of protein arginine methyltransferase 7 inhibits the translational ability of prmt7 gene. Moreover, miR24-2 inhibits the di-/tri-methylation of histone H4 arginine 3 by reducing PRMT7, and then promotes the expression of Nanog via long noncoding RNA HULC. Notably, miR24-2 inhibits histone deacetylase HDAC3 through miR675 which promotes the acetylation of histone H4 at lysine 16. Subsequently, miR24-2 enhances the interaction between LC3 and ATG4 dependent on PI3K and triggers cellular autophagy. Strikingly, miR24-2 inhibits the degradation of pyruvate kinase M1 via autophagosome-P62 in human liver cancer stem cells. Furthermore, miR-24-2 enhances the activity of Src by promoting the binding of PKM1 to the Src promoter regions in human liver cancer stem cells. In particular, our results also indicate that src gene determines the oncogenic fuctions of miR24-2. These results provided a valuable theoretical basis for the discovery of liver cancer therapeutic targets and diagnosis markers based on miR24-2.

Chimeric antigen receptor (CAR) T cell therapy remains relatively ineffective against solid tumors due to inadequate infiltration and in vivo expansion of CAR-T cells. Unlike hematological malignancies, solid tumors have vascular barriers that hinder CAR-T cells from reaching the tumor site. Here, we demonstrated that combretastatin A-4 phosphate (CA4P), a vascular disrupting agent (VDA), can significantly improve the infiltration ability of CAR-T cells in solid tumors as evidenced by elevated levels of IFN-γ. Moreover, combined treatment with CA4P and CAR-T cells greatly increased the therapeutic efficiency of the CAR-T cells in subcutaneous ovarian cancer mouse xenograft models and patient-derived xenograft (PDX) models of colon and ovarian carcinoma. Our findings highlight CA4P as an effective antitumor agent candidate for combination with CAR-T cells in clinical applications to treat solid tumors.