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Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNA F7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNA F7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8 , whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNA F8.1 , ~8-fold and 3-fold; sgRNA F8.2 , ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models. • CRISPR activation (CRISPRa) is a versatile tool to transactivate target promoters. • CRISPRa increased F7 and F8 promoter activity in luciferase-based reporter assays. • CRISPRa promoted expression of endogenous functional factor VII from hepatic cells. • Tailored CRISPRa enhances expression, or rescue disease-causing, promoter mutations. • Transactivation efficiency of CRISPRa resulted to be greater than that of TALE-TF. [ABSTRACT FROM AUTHOR]

Vitamin D (VD) deficiency (VDD) correlates to obesity, with VD a recognized mediator of metabolic diseases. From a previous proteomic study identifying adiponectin as a link between VDD and pediatric obesity, herein we analysed another protein (SSP2301) increased with VDD. A focused 2D-electrophoretic analysis identified 4 corresponding plasma proteins, with one predicted to be fetuin B (FETUB). FETUB was studied due to its emerging role in metabolic diseases and cytogenetic location (3q27.3) with adiponectin. Results were confirmed in obese children, where plasma FETUB was higher with VDD. A direct effect by 1α,25-(OH)2D3 on hepatocellular FETUB synthesis was observed, with a time and dose dependent reduction. Further, we demonstrated the VD-receptor (VDR) is key, with FETUB “released” with VDR silencing. Finally, VD supplementation (6weeks) to juvenile mice fed a standard diet, reduced plasma FETUB. Only at 22weeks did liver FETUB correspond to plasma FETUB, highlighting the contribution of other VD-responsive tissues. Overall, FETUB is a key protein linking VDD to pediatric obesity. With an emerging role in metabolic diseases, we demonstrate that VD/VDR directly regulate FETUB. [ABSTRACT FROM AUTHOR]

Nanomaterials conjugated or complexed with biological moieties such as antibodies, polymers or peptides appear to be suitable not only for drug delivery but also for specific cancer treatment. Here, biocompatible iron oxide magnetic nanoparticles (MNPs) with or without a silica shell coupled with lentiviral vectors (LVs) are proposed as a combined therapeutic approach to specifically target gene expression in a cancer mouse model. Initially, four different MNPs were synthesized and their physical properties were characterized to establish and discriminate their behaviors. MNPs and LVs strictly interacted and transduced cells in vitro as well as in vivo , with no toxicity or inflammatory responses. By injecting LV-MNPs complexes intravenously, green fluorescent protein (GFP) resulted in a sustained long-term expression. Furthermore, by applying a magnetic field on the abdomen of intravenous injected mice, GFP positive cells increased in livers and spleens. In liver, LV-MNPs were able to target both hepatocytes and non-parenchymal cells, while in a mouse model with a grafted tumor, intra-tumor LV-MNPs injection and magnetic plaque application next to the tumor demonstrated the efficient uptake of LV-MNPs complexes with high number of transduced cells and iron accumulation in the tumor site. More important, LV-MNPs with the application of the magnetic plaque spread in all the tumor parenchyma and dissemination through the body was prevented confirming the efficient uptake of LV-MNPs complexes in the tumor. Thus, these LV-MNPs complexes could be used as multifunctional and efficient tools to selectively induce transgene expression in solid tumor for therapeutic purposes. Statement of Significance Our study describes a novel approach of combining magnetic properties of nanomaterials with gene therapy. Magnetic nanoparticles (MNPs) coated with or without a silica shell coupled with lentiviral vectors (LVs) were used as vehicle to target biological active molecules in a mouse cancer model. After in situ injection, the presence of MNP under the magnetic field improve the vector distribution in the tumor mass and after systemic administration, the application of the magnetic field favor targeting of specific organs for LV transduction and specifically can direct LV in specific cells (or avoiding them). Thus, our findings suggest that LV-MNPs complexes could be used as multifunctional and efficient tools to selectively induce transgene expression in solid tumor for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Lentiviral vectors (LVs)-mediated gene transfer is an efficient method for ex vivo and in vivo gene therapy. Actually, LVs have been used in several clinical trials and therapeutic correction was reached in affected patients. However, in order to be effective gene therapy needs to be efficient without detrimental effects for target cells. Successful cell transduction by LVs can be hampered by several factors such as the activation of innate immune sensors during cell transduction and different restriction factors (RFs) inhibiting viral replication inside the cells. Therefore, a better knowledge of host–vector interactions is important for the development of more efficient gene therapy strategies improving the LVs platform by limiting harmful responses. [ABSTRACT FROM AUTHOR]

Background & Aims: Hepatocyte transplantation-induced liver inflammation impairs cell engraftment. We defined whether proinflammatory cytokines and chemokines played roles in regulation of hepatocyte engraftment in the liver. Methods: We performed studies over up to 3 weeks in rat hepatocyte transplantation systems. Expression of 84 cytokine-chemokine genes was studied by quantitative real-time polymerase chain reactions. Expression of selected up-regulated genes was verified by immunohistochemistry. Hepatic recruitment of neutrophils was demonstrated by myeloperoxidase activity assays, and Kupffer cell activation was established by carbon phagocytosis assays. The role of neutrophils and Kupffer cells in regulating expression of cytokine-chemokine genes as well as cell engraftment was determined by cell depletion studies. Results: Within 6 hours after syngeneic cell transplantation, expression of 25 cytokine-chemokine genes increased by 2- to 123-fold, P < .05. These genes were largely associated with activated neutrophils and macrophages, including chemokine ligands, CXCL1, CXCL2, CCL3, CCL4; chemokine receptors, CXCR1 or CXCR2, CCR1, CCR2; and regulatory cytokines tumor necrosis factor α and interleukin-6. Inflammatory cells in the liver immunostained for CCR1, CCR2, CXCR1, and CXCR2, which indicated that up-regulated messenger RNA was appropriately translated. When neutrophils and Kupffer cells were depleted with neutrophil antiserum and gadolinium chloride, respectively, before transplanting cells, cell transplantation-induced cytokine-chemokine responses were attenuated. Virtually all abnormalities subsided in animals treated with neutrophil antiserum plus gadolinium chloride. Moreover, depletion of neutrophils or Kupffer cells improved engraftment of transplanted cells. Conclusions: Cell transplantation-induced liver inflammation involves proinflammatory cytokine-chemokine systems capable of modulation by neutrophils and Kupffer cells. This offers new directions for optimizing cell therapy strategies. [Copyright &y& Elsevier]

This study explored the clinical placement experiences of nursing students during the Covid-19 pandemic. The health emergency caused by Covid-19 required a rapid reorganisation of care settings. This reorganisation entailed revisiting the clinical placements settings and learning programs of Italian nursing faculties. Some Italian universities wanted to seize the health emergency as a learning opportunity enabling the nursing student to acquire additional knowledge and skills. We conducted a descriptive qualitative study employing a phenomenological approach. The study population was second and third-year nursing students. The students did their clinical placement in 5 Northern Italy hospitals, mainly in infectious diseases wards, intensive care and sub-intensive care units, emergency department, short-stay surgical units and internal medicine wards. In these departments, the inpatient wards were entirely converted into Covid-19 units. Ethical approval was obtained from the local ethics committee. Semi-structured, open-ended interviews were conducted in March-April 2021 and analysed following a phenomenological approach. Twenty-one nursing students in their 2nd and 3rd academic year participated. Their average age was 24 years. 81% were female and 19% were male. Three main themes were generated: (i) Learning which surpasses technicalities; (ii) Confronting dignity issues; (iii) Feeling treated as an equal in the workspace. Students had to learn how to lower their fear and self-manage the emotional burden to be a caring presence for the patients who were intensely suffering from the disease and isolation. Attending a clinical practice placement in Covid-19 wards led them to focus on human dignity issues: participants realised how dignity was questioned and how they could become patients' advocates. Students also described that they felt part of the team, with their student role almost fading. This study describes that the most unpredictable public health emergency, such as Covid-19, can provide learning opportunities in the practice environment for nursing students. Students described feeling useful and capitalising on new competencies. Designing educational activities for nursing students concerning pandemic emergencies may be strategic for dealing with similar situations in the future. [ABSTRACT FROM AUTHOR]

Hepatic endothelial cells constitute 25% of all liver cells and exhibit unique properties, including coagulation factor synthesis, participation in host immune responses, paracrine cell signaling, etc. In short-term assays, we recently showed that intraperitoneal transplantation of healthy LSEC in hemophilia A mice restored FVIII activity with phenotypic correction and intraportally transplanted LSEC could be targeted to mouse liver. To determine the potential of genetically-manipulated LSEC, we isolated LSEC by collagenase perfusion of mouse liver followed by Percoll-gradient separation. LSEC were 75% CD31+ and 25% were CD45+, 10% of which were F4/80+. Isolated LSEC were efficiently transduced by lentiviral vectors (LV) and expressed the GFP in culture conditions under an ubiquitous PGK promoter and several endothelial-specific promoters. Further analysis of these endothelial promoters, Tie2, Flk-1, VE-cadherin and ICAM-2, in LSEC and hepatocytes showed that the promoters retained activity in LSEC but were largely inactive in hepatocytes. Moreover, LV-modified primary LSEC survived in the liver of recipients. In parallel studies, to further address whether LSEC will engraft, proliferate and function in the liver of intact animals, we isolated LSEC from transgenic FVB/N Tie-2-GFP mice. Cells were transplanted intraportally into syngeneic FVB/N mice followed by analysis of recipient livers at multiple intervals. Transplanted LSEC expressing the endothelially-restricted Tie2-GFP were observed in the liver of FVB/N mice for the entire 3 month-duration of the studies using cyclophosphamide to inhibit host responses against GFP. EM studies established that transplanted LSEC were incorporated in the sinusoidal endothelial lining of the liver. Immunostaining studies verified that GFP-expressing transplanted LSEC were distinct from Kupffer cells, stained with the F4/80 marker. To further determine whether engraftment and proliferation in transplanted LSEC could be induced by prior endothelial injury in recipients, we used monocrotaline (MCT), a pyrollizidine alkaloid, which damages the vascular endothelium. In MCT-conditioned mice, engraftment of transplanted Tie2-GFP- LSEC significantly improved (e.g., 5-fold after 1 week). Moreover, transplanted LSEC proliferated in mice preconditioned with MCT, such that FACS analysis of Tie-2-GFP LSEC recovered from liver of FVB/N recipients over a 3-month period indicated that 25% of the LSEC compartment was reconstituted. The identity of recovered Tie-2-GFP LSEC was verified by CD31 expression. Also, transplanted Tie-2-GFP LSEC incorporated Dil-Ac-LDL in vivo, which indicated their functional integrity. Conclusions: Primary LSEC were amenable to LV-mediated gene transfer and correctly regulated endothelial promoters. Transplanted LSEC survived and functioned indefinitely in the liver. The ability to reconstitute the liver with transgenic LSEC will offer new ways to investigate biological mechanisms and to pursue cell and gene therapy in hemophilia A and in other suitable disorders.Molecular Therapy (2006) 13, S169–S169; doi: 10.1016/j.ymthe.2006.08.506 [ABSTRACT FROM AUTHOR]

Wiskott–Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients'' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naïve WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients. [Copyright &y& Elsevier]

Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor β (TRβ). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRβ axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. Collectively, these findings suggest that reactivation of the T3/TRβ axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRβ, could be useful tools in HCC therapy. Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC. • The T3/TRβ axis is altered in human HCC. • T3 induces a rapid differentiation program in hepatic preneoplastic lesions. • Repeated cycles of T3 impair HCC progression. • The antitumorigenic effect of T3 is long-lasting and is maintained after withdrawal. • T3 reverts the metabolic profile of HCC to that of a normal hepatocyte. [ABSTRACT FROM AUTHOR]

Breast cancer chemotherapy can cause side effects due to nonspecific drug delivery, low solubility and fast metabolism of drugs used in conventional therapy. Moreover, the therapeutic effect of the drugs is often reduced by the strengthening of chemoresistance, which occurs via a variety of mechanisms. Different strategies have been developed to reduce multidrug resistance (MDR)-associated gene expressions including the use of surfactants and polymers. In this study superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with conjugated linoleic acid (CLA) reduced the number and viability of cells in comparison with both untreated cells or cells treated with SPIONs alone. This cytostatic effect correlated with the increase of peroxisome proliferator-activated receptors γ (PPARγ). The necrotic cell death induced, as a consequence, an inflammatory process, as evidenced by the decrease of the anti-inflammatory PPARα and increase of pro-inflammatory TNFα and IL-1β. PPARs were examined because CLA is one of their natural ligands. The antitumor effect observed was accompanied by a down-regulation of p-glycoprotein (P-gp), which was the first important discovered efflux transporter belonging to MDR, and of ALDH3A1, an enzyme able to metabolize some drugs, reducing their effects. The down-regulation of P-gp correlated with the increase of cytokines. The ALDH3A1 decrease correlated with the increase of PPARγ. Based on these results, PPARs are molecular mediators of anti-cancer effect of SPIONs functionalized with CLA, being changes in these nuclear receptors correlated with induction of cytotoxicity and inflammation, and decreased ability of cancer cells in blocking anti-cancer drug effect. [ABSTRACT FROM AUTHOR]

Steatosis intensifies hepatic ischemia/reperfusion (I/R) injury increasing hepatocyte damage and hepatic inflammation. This study evaluates if this process is associated to a differential response of steatotic hepatocytes (HP) and Kupffer cells (KC) to I/R injury and investigates the molecular mechanisms involved. Control or steatotic (treated with 50 μmol palmitic acid, PA) mouse HP or KC were exposed to hypoxia/reoxygenation (H/R). C57BL/6 mice fed 9 week with control or High Fat diet underwent to partial hepatic IR. PA increased H/R damage of HP and further activated the ASK1-JNK axis stimulated by ER stress during H/R. PA also induced the production of oxidant species (OS), and OS prevention nullified the capacity of PA to increase H/R damage and ASK1/JNK stimulation. ASK1 inhibition prevented JNK activation and entirely protected HP damage. In KC, PA directly activated ER stress, ASK1 and p38 MAPK and increased H/R damage. However, in contrast to HP, ASK1 inhibition further increased H/R damage by preventing p38 MAPK activation. In mice liver, steatosis induced the expression of activated ASK1 in only KC, whereas I/R exposure of steatotic liver activated ASK1 expression also in HP. “In vivo”, ASK1 inhibition prevented ASK1, JNK and p38 MAPK activation and protected I/R damage and expression of inflammatory markers. Conclusions Lipids-induced ASK1 stimulation differentially affects HP and KC by promoting cytotoxic or protective signals. ASK1 increases H/R damage of HP by stimulating JNK and protects KC activating p38MAPK. These data support the potentiality of the therapeutic employment of ASK1 inhibitors that can antagonize the damaging effects of I/R upon fatty liver surgery by the contextual reduction of HP death and of KC-mediated reactions. [ABSTRACT FROM AUTHOR]

An abstract of the article "Cellular Therapy with Transgene Expressing APC Activates CD4 + CD25+ Regulatory T Cells Which Modulate the Immune Response to Gene Therapy Derived Products in Immunocompetent Mice," by Andrea Annoni and colleagues is presented.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells andBcells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHCII peptidome are currently unclear.Todetermine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosinβ4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II selfpeptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent andindependent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. [Copyright &y& Elsevier]

Abstract: The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels. [Copyright &y& Elsevier]

Summary: Autophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVBs). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

An abstract of the article "Lentivirus-Mediated Ex Vivo Gene Therapy in ADA-Deficient SCID Mice," by Alessandra Mortellaro, Matteo Guerrini, Antonella Tabucchi, Filippo Carlucci, Antonia Follenzi, Luigi Naldini, Claudio Bordignon, Maria Grazia Roncarolo and Alessandro Aiuti is presented.