Terapia Génica enfermedades monogénicas

Autores: Zabaleta, N.; Unzu Ezquerro, Carmen María; Weber, N. D.; et al.

Título: Gene therapy for liver diseases - progress and challenges

Revista: NATURE REVIEWS GASTROENTEROLOGY AND HEPATOLOGY

ISSN: 1759-5045 Vol.20 N° 5 2023 págs. 288 - 305

Resumen

Gene therapy is poised to revolutionize modern medicine, with seemingly unlimited potential for treating and curing genetic disorders. For otherwise incurable indications, including most inherited metabolic liver disorders, gene therapy provides a realistic therapeutic option. In this Review, we discuss gene supplementation and gene editing involving the use of recombinant adeno-associated virus (rAAV) vectors for the treatment of inherited liver diseases, including updates on several ongoing clinical trials that are producing promising results. Clinical testing has been essential in highlighting many key translational challenges associated with this transformative therapy. In particular, the interaction of a patient's immune system with the vector raises issues of safety and the duration of treatment efficacy. Furthermore, several serious adverse events after the administration of high doses of rAAVs suggest greater involvement of innate immune responses and pre-existing hepatic conditions than initially anticipated. Finally, permanent modification of the host genome associated with rAAV genome integration and gene editing raises concerns about the risk of oncogenicity that require careful evaluation. We summarize the main progress, challenges and pathways forward for gene therapy for liver diseases. In this Review, the authors discuss gene therapy involving the use of recombinant adeno-associated virus vectors for the treatment of inherited liver diseases, including ongoing clinical trials that are producing promising results.
Autores: Silva Pilipich, Noelia Romina (Autor de correspondencia); Covo-Vergara, Á.; Smerdou Picazo, Cristian (Autor de correspondencia)

Título: Local delivery of immunomodulatory antibodies for gastrointestinal tumors

Revista: CANCERS

ISSN: 2072-6694 Vol.15 N° 8 2023 págs. 2352

Resumen

Simple Summary Many types of gastrointestinal tumors, such as gastric, colorectal, and pancreatic cancer, do not respond well to immunotherapies based on the use of antibodies against immune checkpoints, which are injected systemically into patients and generate frequent adverse effects. This review focuses on alternative ways to deliver immunostimulatory antibodies based on gene therapy vectors able to produce them locally at the tumor site. In particular, the use of modified viruses as vectors can induce local inflammation, which contributes to generating stronger antitumor responses. Many preclinical studies show that gastrointestinal tumors could respond better to immunotherapy by using these novel delivery approaches. Cancer therapy has experienced a breakthrough with the use of immune checkpoint inhibitors (ICIs) based on monoclonal antibodies (mAbs), which are able to unleash immune responses against tumors refractory to other therapies. Despite the great advancement that ICIs represent, most patients with gastrointestinal tumors have not benefited from this therapy. In addition, ICIs often induce adverse effects that are related to their systemic use. Local administration of ICIs in tumors could concentrate their effect in the malignant tissue and provide a higher safety profile. A new and attractive approach for local delivery of ICIs is the use of gene therapy vectors to express these blocking antibodies in tumor cells. Several vectors have been evaluated in preclinical models of gastrointestinal tumors to express ICIs against PD-1, PD-L1, and CTLA-4, among other immune checkpoints, with promising results. Vectors used in these settings include oncolytic viruses, self-replicating RNA vectors, and non-replicative viral and non-viral vectors. The use of viral vectors, especially when they have replication capacity, provides an additional adjuvant effect that has been shown to enhance antitumor responses. This review covers the most recent studies involving the use of gene therapy vectors to deliver ICIs to gastrointestinal tumors.
Autores: Fernández Montero, Alejandro; Zuaznabar Martinez, Jon; Pina Sanchez, Manuel; et al.

Título: Photodynamic nasal SARS-CoV-2 decolonization shortens infectivity and influences specific T-Cell responses

Revista: FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY

ISSN: 2235-2988 Vol.13 2023 págs. 1110467

Resumen

Background: The main objective was to evaluate the efficacy of intranasal photodynamic therapy (PDT) in SARS-CoV-2 mildly symptomatic carriers on decreasing the infectivity period. SARS-CoV-2-specific immune-stimulating effects and safety were also analysed. Methods: We performed a randomized, placebo-controlled, clinical trial in a tertiary hospital (NCT05184205). Patients with a positive SARS-CoV-2 PCR in the last 48 hours were recruited and aleatorily assigned to PDT or placebo. Patients with pneumonia were excluded. Participants and investigators were masked to group assignment. The primary outcome was the reduction in in vitro infectivity of nasopharyngeal samples at days 3 and 7. Additional outcomes included safety assessment and quantification of humoral and T-cell immune-responses. Findings: Patients were recruited between December 2021 and February 2022. Most were previously healthy adults vaccinated against COVID-19 and most carried Omicron variant. 38 patients were assigned to placebo and 37 to PDT. Intranasal PDT reduced infectivity at day 3 post-treatment when compared to placebo with a ß-coefficient of -812.2 (CI95%= -478660 ¿ -1.3, p<0.05) infectivity arbitrary units. The probability of becoming PCR negative (ct>34) at day 7 was higher on the PDT-group, with an OR of 0.15 (CI95%=0.04-0.58). There was a decay in anti-Spike titre and specific SARS-CoV-2 T cell immunity in the placebo group 10 and 20 weeks after infection, but not in the PDT-group. No serious adverse events were reported. Interpretation: Intranasal-PDT is safe in pauci-symptomatic COVID-19 patients, it reduces SARS-CoV-2 infectivity and decelerates the decline SARS-CoV-2 specific immune-responses.
Autores: Vinueza Gavilanes, Rodrigo; Bravo González, Jorge; Basurco Gogorcena, Leyre; et al.

Título: Stabilization of 14-3-3 protein-protein interactions with fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models

Revista: NEUROBIOLOGY OF DISEASE

ISSN: 0969-9961 Vol.183 2023 págs. 106166

Resumen

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.
Autores: Elurbide Tardio, Jasmin; Carte Abad, Beatriz; Guedes, J.; et al.

Título: NatB catalytic subunit depletion disrupts DNA replication initiation leading to senescence in MEFs

Revista: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

ISSN: 1661-6596 Vol.24 N° 10 2023 págs. 8724

Resumen

Alpha-aminoterminal acetyltransferase B (NatB) is a critical enzyme responsible for acetylating the aminoterminal end of proteins, thereby modifying approximately 21% of the proteome. This post-translational modification impacts protein folding, structure, stability, and interactions between proteins which, in turn, play a crucial role in modulating several biological functions. NatB has been widely studied for its role in cytoskeleton function and cell cycle regulation in different organisms, from yeast to human tumor cells. In this study, we aimed to understand the biological importance of this modification by inactivating the catalytic subunit of the NatB enzymatic complex, Naa20, in non-transformed mammal cells. Our findings demonstrate that depletion of NAA20 results in decreased cell cycle progression and DNA replication initiation, ultimately leading to the senescence program. Furthermore, we have identified NatB substrates that play a role in cell cycle progression, and their stability is compromised when NatB is inactivated. These results underscore the significance of N-terminal acetylation by NatB in regulating cell cycle progression and DNA replication.
Autores: Pastor-Fernández, A.; Bertos, A. R.; Sierra-Ramírez, A.; et al.

Título: Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice

Revista: AGING CELL

ISSN: 1474-9718 Vol.22 N° 3 2023 págs. e13771

Resumen

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.
Autores: Murillo Sauca, Oihana; Gazquez López, Cristina; Moreno Luqui, Daniel; et al.

Título: Therapeutic efficacy of VTX-801 in advanced stage of Wilson's disease in mice

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.31 N° 4 Supl. 1 2023 págs. 325
Autores: Silva Pilipich, Noelia Romina; Blanco, E.; Lozano Moreda, Teresa; et al.

Título: A self-amplifying RNA vector expressing nanobodies against immune checkpoints induces potent antitumor responses

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.31 N° 4 Supl. 1 2023 págs. 724 - 725
Autores: Murillo Sauca, Oihana; Moreno Luqui, Daniel; Gazquez López, Cristina; et al.

Título: Therapeutic efficacy of the combination of long-term penicillamine treatment with a single administration of VTX-801 in Wilson's disease mice

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.31 N° 4 Supl. 1 2023 págs. 725
Autores: Zabaleta, N.; Torella, Laura; Weber, N. D.; et al.

Título: mRNA and gene editing: late breaking therapies in liver diseases

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.76 N° 3 2022 págs. 869 - 887

Resumen

The efficient delivery of RNA molecules to restore the expression of a missing or inadequately functioning protein in a target cell and the intentional specific modification of the host genome using engineered nucleases represent therapeutic concepts that are revolutionizing modern medicine. The initiation of several clinical trials using these approaches to treat metabolic liver disorders as well as the recently reported remarkable results obtained by patients with transthyretin amyloidosis highlight the advances in this field and show the potential of these therapies to treat these diseases safely and efficaciously. These advances have been possible due, firstly, to significant improvements made in RNA chemistry that increase its stability and prevent activation of the innate immune response and, secondly, to the development of very efficient liver-targeted RNA delivery systems. In parallel, the breakout of CRISPR/CRISPR-associated 9-based technology in the gene editing field has marked a turning point in in vivo modification of the cellular genome with therapeutic purposes, which can be based on gene supplementation, correction, or silencing. In the coming years we are likely to witness the therapeutic potential of these two strategies both separately and in combination. In this review we summarize the preclinical data obtained in animal models treated with mRNA as a therapeutic agent and discuss the different gene editing strategies applied to the treatment of liver diseases, highlighting both their therapeutic efficacy as well as safety concerns.
Autores: Martinez Garcia, Javier; Molina Delgado, Angie; González Aseguinolaza, Gloria; et al.

Título: Gene therapy for acquired and genetic cholestasis

Revista: BIOMEDICINES

ISSN: 2227-9059 Vol.10 N° 6 2022 págs. 1238

Resumen

Cholestatic diseases can be caused by the dysfunction of transporters involved in hepatobiliary circulation. Although pharmacological treatments constitute the current standard of care for these diseases, none are curative, with liver transplantation being the only long-term solution for severe cholestasis, albeit with many disadvantages. Liver-directed gene therapy has shown promising results in clinical trials for genetic diseases, and it could constitute a potential new therapeutic approach for cholestatic diseases. Many preclinical gene therapy studies have shown positive results in animal models of both acquired and genetic cholestasis. The delivery of genes that reduce apoptosis or fibrosis or improve bile flow has shown therapeutic effects in rodents in which cholestasis was induced by drugs or bile duct ligation. Most studies targeting inherited cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), have focused on supplementing a correct version of a mutated gene to the liver using viral or non-viral vectors in order to achieve expression of the therapeutic protein. These strategies have generated promising results in treating PFIC3 in mouse models of the disease. However, important challenges remain in translating this therapy to the clinic, as well as in developing gene therapy strategies for other types of acquired and genetic cholestasis.
Autores: Weber, N. D. (Autor de correspondencia); Martinez Garcia, Javier; González Aseguinolaza, Gloria

Título: Comment on "Synthetic human ABCB4 mRNA therapy rescues severe liver disease phenotype in a BALB/c.Abcb4 -/- mouse model of PFIC3"

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.76 N° 3 2022 págs. 749 - 751
Autores: Bazo Ochoa, Andrea; Lantero, A.; Mauleón Mayora, Itsaso; et al.

Título: Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice

Revista: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

ISSN: 1422-0067 Vol.23 N° 23 2022 págs. 14940

Resumen

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.
Autores: Mitxelena Iribarren, Oihane (Autor de correspondencia); Mondragón, B.; Pérez Lorenzo, Eva; et al.

Título: Evaluation of the degradation of materials by exposure to germicide UV-C light through colorimetry, tensile strength and surface microstructure analyses

Revista: MATERIALS TODAY COMMUNICATIONS

ISSN: 2352-4928 Vol.31 2022 págs. 103690

Resumen

Due to the COVID19 pandemic, solutions to automate disinfection using UV-C combined with mobile robots are beginning to be explored. It has been proved that the use of these systems highly reduces the risk of contagion. However, its use in real applications is not being as rapid as it needs to be. One of the main market input barriers is the fear of degrading facilities. For this reason, it is crucial to perform a detailed study on the degradation effect of UV-C light on inert materials. This experimental study proves that, considering exposition times equivalent to several work years in hospital rooms, only the appearance of the material is affected, but not their mechanical functionalities. This relevant result could contribute to accelerate the deployment of these beneficial disinfection technologies. For that purpose, a colorimetry test, tensile strength test, and analysis of the surface microstructure were carried out. The results showed that polymers tend to turn yellow, while fabrics lose intensity depending on the color. Red is hardly affected by UV-C, but blue and green are. Thus, this study contributes to the identification of the best materials and colors to be used in rooms subjected to disinfection processes. In addition, it is shown how the surface microstructure of the materials is altered in most of the materials, but not the tensile strength of the fabrics.
Autores: Murillo Sauca, Oihana; Collantes Martínez, María; Gazquez López, Cristina; et al.

Título: High value of 64Cu as a tool to evaluate the restoration of physiological copper excretion after gene therapy in Wilson's disease

Revista: MOLECULAR THERAPY. METHODS & CLINICAL DEVELOPMENT

ISSN: 2329-0501 Vol.9 N° 26 2022 págs. 98 - 106

Resumen

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.
Autores: Ros-Ganan, I.; Hommel, Mirja; Trigueros-Motos, L.; et al.

Título: Optimising the IgG-degrading enzyme treatment regimen for enhanced adeno-associated virus transduction in the presence of neutralising antibodies

Revista: CLINICAL & TRANSLATIONAL IMMUNOLOGY

ISSN: 2050-0068 Vol.11 N° 2 2022 págs. e1375

Resumen

Objective. Pre-existing neutralising antibodies (NAbs) to adeno-associated viruses (AAVs) remain an impediment for systemically administered AAV-mediated gene therapy treatment in many patients, and various strategies are under investigation to overcome this limitation. Here, IgG-degrading enzymes (Ides) derived from bacteria of the genus Streptococcus were tested for their ability to cleave human IgG and allow AAV-mediated transduction in individuals with pre-existing NAbs. Methods. Cleavage activity of three different Ides was evaluated in vitro in serum from different species. Passively immunised mice or non-human primates (NHP) with naturally occurring anti-AAV NAbs were used to define the optimal IdeS dose and administration window for AAVAnc80 and AAV8 vectors in mice and AAV3B in NHPs. Results. The selected candidate, IdeS, was found to be highly efficient at cleaving human IgG, less efficient against NHP IgG and inefficient against mouse IgG. In vivo, we observed differences in how IdeS affected liver transduction in the presence of NAbs depending on the AAV serotype. For AAVAnc80 and AAV3B, the best transduction levels were achieved when the vector was administered after IgG digestion products were cleared from circulation. However, for AAV8 we only observed a modest and transient inhibition of transduction by IdeS cleavage products. Conclusion. Preconditioning with IdeS represents a unique treatment opportunity for patients primarily excluded from participation in gene therapy clinical trials because of elevated circulating anti-AAV NAb levels. However, careful determination of the optimal IdeS dose and timing for the administration of each AAV serotype is essential for optimal transduction.
Autores: Martinez Garcia, Javier; Molina González, Manuela; Odriozola Moncayola, Leticia; et al.

Título: A minimal bile salt excretory pump promoter allows bile acid-driven physiological regulation of transgene expression from a gene therapy vector

Revista: CELL & BIOSCIENCE

ISSN: 2045-3701 Vol.12 N° 1 2022 págs. 79

Resumen

Background Bile acid (BA) homeostasis is mainly regulated by bile salt excretory pump (BSEP), a hepatocyte transporter that transfers BAs to the bile. BSEP expression is regulated by BA levels through activation of farnesoid X receptor transcription factor, which binds to the inverted repeat (IR-1) element in the BSEP promoter. Gene therapy of cholestatic diseases could benefit from using vectors carrying endogenous promoters physiologically regulated by BAs, however their large size limits this approach, especially when using adeno-associated viral vector (AAV) vectors. Results We evaluated the functionality and BA-mediated regulation of minimal versions of human and mouse BSEP promoters containing IR-1 using AAV vectors expressing luciferase. Unexpectedly, a minimal mouse BSEP promoter (imPr) showed higher BA-mediated expression and inducibility than a minimal human promoter (ihPr) or than full-length BSEP promoters in human hepatic cells. In addition, in mice receiving an AAV8 vector carrying imPr promoter-driven luciferase expression was efficiently regulated by administration of a BA-enriched diet. Interestingly, this vector also expressed significantly higher luciferase levels in Abcb4(-/-) mice, which have high levels of BAs, compared to wild type mice, or to mice receiving a vector containing the luciferase gene downstream of the constitutive alpha-1 antitrypsin promoter. In contrast, the AAV vector containing ihPr showed very low luciferase expression with no inducibility. Finally, we optimized imPr by adding three IR-1 repeats at its 5 ' end. This new promoter provided higher levels of luciferase than imPr both in vitro and in vivo. Conclusions The imPr could represent a useful tool for gene therapy approaches in which physiological BA regulation is desired.
Autores: Olague Micheltorena, María Cristina; Mitxelena Iribarren, Oihane; Sierra-García, J.E.; et al.

Título: Rapid SARS-CoV-2 disinfection on distant surfaces with UV-C: The inactivation is affected by the type of material

Revista: JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY

ISSN: 2666-4690 Vol.11 2022 págs. 100138 - *

Resumen

SARS-CoV-2 is responsible for the COVID-19 pandemic, which has caused almost 570 million infections and over six million deaths worldwide. To help curb its spread, solutions using ultraviolet light (UV) for quick virus inactivation inside buildings without human intervention could be very useful to reduce chances of contagion. The UV dose must be sufficient to inactivate the virus considering the different materials in the room, but it should not be too high, not to degrade the environment. In the present study, we have analyzed the ability of a 254nm wavelength UV-C lamp to inactivate dried samples of SARS-CoV-2 exposed at a distance of two meters, simulating a full-scale scenario. Our results showed that virus inactivation was extremely efficient in most tested materials, which included plastic, metal, wood, and textile, with a UV-C exposure of only 42s (equivalent to 10mJ/cm2). However, porous materials like medium density fibreboard, were hard to decontaminate, indicating that they should be avoided in hospital rooms and public places.
Autores: Pacin-Ruiz, B.; Cortese, M. F. (Autor de correspondencia); Tabernero, D. (Autor de correspondencia); et al.

Título: Inspecting the ribozyme region of hepatitis delta virus genotype 1: conservation and variability

Revista: VIRUSES-BASEL

ISSN: 1999-4915 Vol.14 N° 2 2022 págs. 215

Resumen

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688-771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715-745. No difference in QS was observed over time. The most variable region was between nt 739-769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.
Autores: Kronborg Hansen, A. K.; Dubik, M.; Marczynska, J.; et al.

Título: Targeting signaling pathway downstream of RIG-I/MAVS in the CNS stimulates production of endogenous type I IFN and suppresses EAE

Revista: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

ISSN: 1422-0067 Vol.23 N° 19 2022 págs. 11292

Resumen

Type I interferons (IFN), including IFN beta, play a protective role in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Type I IFNs are induced by the stimulation of innate signaling, including via cytoplasmic RIG-I-like receptors. In the present study, we investigated the potential effect of a chimeric protein containing the key domain of RIG-I signaling in the production of CNS endogenous IFN beta and asked whether this would exert a therapeutic effect against EAE. We intrathecally administered an adeno-associated virus vector (AAV) encoding a fusion protein comprising RIG-I 2CARD domains (C) and the first 200 amino acids of mitochondrial antiviral-signaling protein (MAVS) (M) (AAV-CM). In vivo imaging in IFN beta/luciferase reporter mice revealed that a single intrathecal injection of AAV-CM resulted in dose-dependent and sustained IFN beta expression within the CNS. IFN beta expression was significantly increased for 7 days. Immunofluorescent staining in IFN beta-YFP reporter mice revealed extraparenchymal CD45+ cells, choroid plexus, and astrocytes as sources of IFN beta. Moreover, intrathecal administration of AAV-CM at the onset of EAE induced the suppression of EAE, which was IFN-I-dependent. These findings suggest that accessing the signaling pathway downstream of RIG-I represents a promising therapeutic strategy for inflammatory CNS diseases, such as MS.
Autores: Silva Pilipich, Noelia Romina; Lasarte-Cía, A.; Lozano Moreda, Teresa; et al.

Título: Intratumoral electroporation of a self-amplifying RNA expressing IL-12 induces antitumor effects in mouse models of cancer

Revista: MOLECULAR THERAPY - NUCLEIC ACIDS

ISSN: 2162-2531 Vol.29 2022 págs. 387 - 399

Resumen

Alphavirus vectors based on self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce innate immune responses, making them an interesting tool for antitumor therapy. These vectors are usually delivered as viral particles, but it is also possible to administer them as RNA. We evaluated this possibility by invivo electroporation of Semliki Forest virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. Optimization of saRNA electroporation conditions in tumors was performed using an SFV vector coding for luciferase. Then we evaluated the therapeutic potential of this approach using an SFV saRNA coding for interleukin-12 (SFV-IL-12), a proinflammatory cytokine with potent antitumor effects. Delivery of SFV-IL-12 saRNA by electroporation led to improvement in tumor control and higher survival compared with mice treated with electroporation or with SFV-IL-12 saRNA alone. The antitumor efficacy of SFV-IL-12 saRNA electroporation increased by combination with systemic PD-1 blockade. This therapy, which was also validated in a hepatocellular carcinoma tumor model, suggests that local delivery of saRNA by electroporation could be an attractive strategy for cancer immunotherapy. This approach could have easy translation to the clinical practice, especially for percutaneously accessible tumors.
Autores: Camps Ramón, Gracián; Maestro Galilea, Sheila; Usai, C.; et al.

Título: RIPK1 plays a survival role in the liver of mice with active HDV replication

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.77 N° Suppl. 1 2022 págs. S248 - S248
Autores: Milagros, S.; Uribarri, A.; Castro, C.; et al.

Título: Characterization of AAV9 infectivity when it presents CY5.5 linked to the capsid at positions 470 and 471

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.33 N° 23-24 2022 págs. A48 - A48
Autores: Klermund, J.; Loiacono, L.; Torella, Laura; et al.

Título: An in vivo CAST-Seq workflow identifies and quantifies off-target activity as well as chromosomal translocations in organs edited in vivo with CRISPR-Cas nucleases or nickases

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.33 N° 23-24 2022 págs. A6 - A7
Autores: Saande, Cassondra Jeanette; Bazo Ochoa, Andrea; Fernández-Huarte, L.; et al.

Título: Magnetic targeting of iron oxide nanoparticles to the kidneys

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.33 N° 23-24 2022 págs. A179 - A179
Autores: Torella, Laura; Tamayo Uria, Ibon; Vales, A.; et al.

Título: Liver gene-editing based on nickase Cas9 for the treatment of primary hyperoxaluria type I (PH1) is more efficient when using an all-in-one delivery system

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.30 N° 4 2022 págs. 211 - 211
Autores: Molina Delgado, Angie; Odriozola Moncayola, Leticia; Trigueros-Motos, L.; et al.

Título: CpG content reduction in codon-optimized AAV transgene sequences does not reduce hTLR9 activation in an in vitro model

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.30 N° 4 2022 págs. 524 - 525
Autores: Weber, N. D.; Odriozola Moncayola, Leticia; Gazquez López, Cristina; et al.

Título: Increased in vitro transgene expression driven by rAAV expression cassette modifications is not predictive of the in vivo outcome

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.33 N° 23-24 2022 págs. A54 - A55
Autores: González-de Zarate, A.; Covo-Vergara, A.; Igea-Sucunza, A.; et al.

Título: Development of a self-amplifying RNA vector expressing a high affinity PD-1 domain for cancer therapy

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.33 N° 23-24 2022 págs. A185 - A186
Autores: Tamarit, B.; González Aseguinolaza, Gloria; Brossel, A.; et al.

Título: Safety and biodistribution of VTX-801, an AAV3B gene therapy vector, in healthy cynomolgus monkeys

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.30 N° 4 2022 págs. 592 - 592
Autores: Pacin-Ruiz, B.; Camps Ramón, Gracián; Cortese, M. F.; et al.

Título: HDV-mediated inhibition of HBV in superinfection mouse model: the role of type I Interferon

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.77 N° S1 2022 págs. S257 - S258
Autores: Motos, L. T.; Ros-Ganan, I.; Hommel, Mirja; et al.

Título: Optimizing the IdeS treatment regimen for enhanced adeno-associated virus transduction in the presence of neutralizing antibodies

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.30 N° 4 (Supplement 1) 2022 págs. 528 - 528

Resumen

Objective: Pre-existing neutralising antibodies (NAbs) to adeno-associated viruses (AAVs) remain an impediment for systemically administered AAV-mediated gene therapy treatment in many patients, and various strategies are under investigation to overcome this limitation. Here, IgG-degrading enzymes (Ides) derived from bacteria of the genus Streptococcus were tested for their ability to cleave human IgG and allow AAV-mediated transduction in individuals with pre-existing NAbs. Methods: Cleavage activity of three different Ides was evaluated in vitro in serum from different species. Passively immunised mice or non-human primates (NHP) with naturally occurring anti-AAV NAbs were used to define the optimal IdeS dose and administration window for AAVAnc80 and AAV8 vectors in mice and AAV3B in NHPs. Results: The selected candidate, IdeS, was found to be highly efficient at cleaving human IgG, less efficient against NHP IgG and inefficient against mouse IgG. In vivo, we observed differences in how IdeS affected liver transduction in the presence of NAbs depending on the AAV serotype. For AAVAnc80 and AAV3B, the best transduction levels were achieved when the vector was administered after IgG digestion products were cleared from circulation. However, for AAV8 we only observed a modest and transient inhibition of transduction by IdeS cleavage products. Conclusion: Preconditioning with IdeS represents a unique treatment opportunity for patients primarily excluded from participation in gene therapy clinical trials because of elevated circulating anti-AAV NAb levels. However, careful determination of the optimal IdeS dose and timing for the administration of each AAV serotype is essential for optimal transduction.
Autores: Cartón García, Fernando; Saande, Cassondra Jeanette; Meraviglia-Crivelli, D.; et al.

Título: Oligonucleotide-based therapies for renal diseases

Revista: BIOMEDICINES

ISSN: 2227-9059 Vol.9 N° 3 2021 págs. 303

Resumen

The global burden of chronic kidney disease (CKD) is increasing every year and represents a great cost for public healthcare systems, as the majority of these diseases are progressive. Therefore, there is an urgent need to develop new therapies. Oligonucleotide-based drugs are emerging as novel and promising alternatives to traditional drugs. Their expansion corresponds with new knowledge regarding the molecular basis underlying CKD, and they are already showing encouraging preclinical results, with two candidates being evaluated in clinical trials. However, despite recent technological advances, efficient kidney delivery remains challenging, and the presence of off-targets and side-effects precludes development and translation to the clinic. In this review, we provide an overview of the various oligotherapeutic strategies used preclinically, emphasizing the most recent findings in the field, together with the different strategies employed to achieve proper kidney delivery. The use of different nanotechnological platforms, including nanocarriers, nanoparticles, viral vectors or aptamers, and their potential for the development of more specific and effective treatments is also outlined.
Autores: Maestro Galilea, Sheila; Weber, N. D.; Zabaleta, N.; et al.

Título: Novel vectors and approaches for gene therapy in liver diseases

Revista: JHEP REPORTS

ISSN: 2589-5559 Vol.3 N° 4 2021 págs. 100300

Resumen

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.
Autores: Silva Pilipich, Noelia Romina; Smerdou Picazo, Cristian (Autor de correspondencia); Vanrell, L. (Autor de correspondencia)

Título: A small virus to deliver small antibodies: new targeted therapies based on AAV delivery of nanobodies

Revista: MICROORGANISMS

ISSN: 2076-2607 Vol.9 N° 9 2021 págs. 1956

Resumen

Nanobodies are camelid-derived single-domain antibodies that present some advantages versus conventional antibodies, such as a smaller size, and higher tissue penetrability, stability, and hydrophilicity. Although nanobodies can be delivered as proteins, in vivo expression from adeno-associated viral (AAV) vectors represents an attractive strategy. This is due to the fact that AAV vectors, that can provide long-term expression of recombinant genes, have shown an excellent safety profile, and can accommodate genes for one or several nanobodies. In fact, several studies showed that AAV vectors can provide sustained nanobody expression both locally or systemically in preclinical models of human diseases. Some of the pathologies addressed with this technology include cancer, neurological, cardiovascular, infectious, and genetic diseases. Depending on the indication, AAV-delivered nanobodies can be expressed extracellularly or inside cells. Intracellular nanobodies or "intrabodies" carry out their function by interacting with cell proteins involved in disease and have also been designed to help elucidate cellular mechanisms by interfering with normal cell processes. Finally, nanobodies can also be used to retarget AAV vectors, when tethered to viral capsid proteins. This review covers applications in which AAV vectors have been used to deliver nanobodies, with a focus on their therapeutic use.
Autores: González Aseguinolaza, Gloria (Autor de correspondencia); Izeta, A.; Martín Molina, F.

Título: Spanish society of gene and cell therapy

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 23-24 2021 págs. 1425 - 1426
Autores: Pérez Iturralde, Andrea; Carte Abad, Beatriz; Aldabe Arregui, Rafael (Autor de correspondencia)

Título: Consequences of mammalian target of rapamycin inhibition on adeno-associated virus hepatic transduction efficacy

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 19 - 20 2021 págs. 1242 - 1250

Resumen

The efficiency of recombinant adeno-associated virus (AAV) vectors transducing host cells is very low, limiting their therapeutic potential in patients. There are several cellular pathways interacting and interfering with the journey of the AAV from the cell surface to the nucleus, opening the possibility to enhance AAV transduction by modifying these interactions. In this study, we explored the results of AAV hepatic transduction when different mammalian target of rapamycin (mTOR) inhibitors, rapamycin, MLN0128, RapaLink-1, were used in preconditioned juvenile and adult mice. We confirmed rapamycin as an AAV hepatic transduction enhancer in juvenile and adult mice; however, RapaLink-1, a stronger mTOR inhibitor and a clear hepatic autophagy inducer, had no positive effect. Moreover, MLN0128 reduced AAV hepatic transduction. Therefore, our results show a complex interaction between the mTOR pathway and AAV-mediated hepatic transduction and indicate that mTOR inhibition is not a straightforward strategy for improving AAV transduction. More studies are necessary to elucidate the molecular mechanisms involved in the positive and negative effects of mTOR inhibitors on AAV transduction efficiency.</p>
Autores: Casales Zoco, Erkuden; Martisová, Eva; Villanueva, H.; et al.

Título: Idiotype vaccines produced with a non-cytopathic alphavirus self-amplifying RNA vector induce antitumor responses in a murine model of B-cell lymphoma

Revista: SCIENTIFIC REPORTS

ISSN: 2045-2322 Vol.11 N° 1 2021 págs. 21427

Resumen

A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 mu g of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.
Autores: Rodríguez García, Estefanía; Zabaleta Lasarte, Nerea; Gil Fariña, Irene; et al.

Título: AdrA as a potential immunomodulatory candidate for STING-mediated antiviral therapy that required both type I IFN and TNF-alpha production

Revista: JOURNAL OF IMMUNOLOGY

ISSN: 0022-1767 Vol.206 N° 2 2021 págs. 376 - 385

Resumen

Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrAwas addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrAwt-treatedmice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-alpha R (IFNAR)(-/-) animals were additionally treated with anti- TNF-alpha (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR(-/-) and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-alpha-treated IFNAR(-/-) animals. No effect of AdrA wt was seen in STING- deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-alpha are both required and act synergistically.
Autores: Kaiser, R. A.; Weber, N. D.; Trigueros-Motos, L.; et al.

Título: Use of an adeno-associated virus serotype Anc80 to provide durable cure of phenylketonuria in a mouse model

Revista: JOURNAL OF INHERITED METABOLIC DISEASE

ISSN: 0141-8955 Vol.44 N° 6 2021 págs. 1369 - 1381

Resumen

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pah(enu2) mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pah(enu2) mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 x 10(12) VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration.
Autores: Nistal-Villán, E. (Autor de correspondencia); Argemí Ballbé, José María; de Jaime Soguero, A.; et al.

Título: Linking the expression of therapeutic genes to unfolded protein response: a new option for anti-hepatitis B virus gene therapy

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 7-8 2021 págs. 341 - 348

Resumen

Tight control of transgene expression is key to ensure the efficacy of a wide range of gene therapy interventions, in which the magnitude and duration of gene expression have to be adjusted to therapeutic needs, thereby limiting secondary effects. The development of upgraded strategies to link transgene expression to pathological stress episodes is an unmet need in gene therapy. Here, we propose an expression strategy that associates transgene expression to an intracellular stress coping mechanism, the unfolded protein response. Specifically, we harnessed the cis elements required to sustain the noncanonical splicing of X-box binding protein 1 (XBP1) messenger RNA (mRNA) in response to the dysfunction of the endoplasmic reticulum (ER), a situation commonly known as ER stress, to drive the expression of heterologous genes. Since ER stress features a wide variety of pathological conditions, including viral infections, cancer, or metabolic disorders, this new expression module stimulates the synthesis of therapeutic genes as a response to cellular damage, and ensures their expression only when necessary. Validation of this inducible expression system was performed in vitro and in vivo, and its potential to limit/inhibit viral infections has been shown in proof-of principle experiments.
Autores: Weber, N. D.; Odriozola Moncayola, Leticia; Bouquet, C.; et al.

Título: AAV-based gene therapy for PFIC3 (VTX-803) shows further promise with durable efficacy in PFIC3 mice at different disease stages

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.29 N° 4 2021 págs. 245 - 245
Autores: Bazo Ochoa, Andrea; Lantero, A.; Mauleón Mayora, Itsaso; et al.

Título: rAAV-mediated gene therapy in combination with short-term nitrogen-scavenger treatment corrects biochemical and behavioral abnormalities and increases lifespan in infant citrullinemia type 1 (CTLN-1) mice

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.29 N° 4 Supl. 1 2021 págs. 254 - 255
Autores: Bazo Ochoa, Andrea; Lantero, A.; Neri, L.; et al.

Título: rAAV-mediated gene therapy in combination with short-term nitrogen-scavenger treatment corrects biochemical and behavioral abnormalities and increases lifespan in infant Citrullinemia Type 1 (CTLN-1) mice

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 19-20 2021 págs. A105 - A105
Autores: Weber, N. D.; Salas Gómez, David; Odriozola Moncayola, Leticia; et al.

Título: Coadministration of AAV expressing MDR3 (VTX-803) and ImmTOR allows for vector re-administration to treat progressive familial intrahepatic cholestasis type 3 (PFIC3) in juvenile Abcb4(-/-) mice

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.29 N° 4 2021 págs. 14 - 15
Autores: Martinez Garcia, Javier; Molina González, Manuela; Odriozola Moncayola, Leticia; et al.

Título: A minimal BSEP promoter allows bile acid-driven physiological regulation of transgene expression from an AAV vector

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 19-20 2021 págs. A105
Autores: Palmeiro, E. B.; Ibáñez, M.; Hernández Sáez, Carlos; et al.

Título: The STAT3 phosphorylation status discriminates tumor associated populations in myeloid cell tumor-infiltrating model

Revista: ANNALS OF ONCOLOGY

ISSN: 0923-7534 Vol.32 N° Supl. 5 2021 págs. S368 - S368
Autores: Lumbreras Roche, Sara; Ricobaraza Abarquero, Ana; Baila-Rueda, L.; et al.

Título: Gene supplementation of CYP27A1 in the liver restores bile acid metabolism in a mouse model of Cerebrotendinous Xanthomatosis

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 19-20 2021 págs. A105 - A106
Autores: Torella, Laura; Tamayo Uria, Ibon; Vales Aranguren, África; et al.

Título: In vivo deletion of glycolate oxidase (GO) using double-nicking CRISPR/Cas9 for an efficient and safer treatment of Primary Hyperoxaluria type I (PH1)

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.32 N° 19-20 2021 págs. A16
Autores: Ballesteros Briones, M. C. ; Silva Pilipich, Noelia Romina; Herrador Cañete, Guillermo; et al.

Título: A new generation of vaccines based on alphavirus self-amplifying RNA

Revista: CURRENT OPINION IN VIROLOGY

ISSN: 1879-6257 Vol.44 2020 págs. 145 - 153

Resumen

DNA or mRNA vaccines have potential advantages over conventional vaccines since they are easier to manufacture and have higher safety profiles. In particular, self-amplifying RNA (saRNA) derived from alphavirus expression vectors has shown to be very efficient to induce humoral and cellular responses against many antigens in preclinical models, being superior to non-replicating mRNA and DNA. This is mainly due to the fact that saRNA can provide very high expression levels and simultaneously induces strong innate responses, potentiating immunity. saRNA can be administered as viral particles or DNA, but direct delivery as RNA represents a safer and more simple approach. Although saRNA can be delivered as naked RNA, in vivo transfection can be enhanced by electroporation or by complexing it with cationic lipids or polymers. Alphavirus saRNA could have broad application to vaccinate against human pathogens, including emerging ones like SARS-CoV-2, for which clinical trials have been recently initiated.
Autores: González Aseguinolaza, Gloria (Autor de correspondencia)

Título: Givosiran - Running RNA Interference to Fight Porphyria Attacks

Revista: NEW ENGLAND JOURNAL OF MEDICINE

ISSN: 0028-4793 Vol.382 N° 24 2020 págs. 2366 - 2367

Resumen

The porphyrias are a group of rare and ultra-rare devastating disorders of heme biosynthesis. The majority of these disorders are inherited, and patients present with disabling symptoms that have a profound effect on their quality of life. The subtypes of acute hepatic porphyria - including acute intermittent porphyria (the most common subtype), hereditary coproporphyria, variegate porphyria, and delta-aminolevulinic acid (ALA) dehydratase-deficiency porphyria - are characterized by the occurrence of severe neurovisceral attacks. The genetic deficit that causes acute hepatic porphyria reduces heme availability under conditions of increased heme demands, which leads to activation of the heme-synthesis pathway and up-regulation of . . .
Autores: Zalba Oteiza, Sara; Contreras Sandoval, Ana Margarita; Martisová, Eva; et al.

Título: Quantification of Pharmacokinetic Profiles of PD-1/PD-L1 Antibodies by Validated ELISAs

Revista: PHARMACEUTICS

ISSN: 1999-4923 Vol.12 N° 6 2020 págs. 595

Resumen

Immunotherapy has changed the paradigm of cancer treatments. In this way, several combinatorial strategies based on monoclonal antibodies (mAb) such as anti (a)-PD-1 or anti (a)-PD-L1 are often reported to yield promising clinical benefits. However, the pharmacokinetic (PK) behavior of these mAbs is a critical issue that requires selective analytical techniques. Indeed, few publications report data on a-PD1/a-PD-L1 exposure and its relationship with therapeutic or toxic effects. In this regard, preclinical assays allow the time profiles of antibody plasma concentrations to be characterized rapidly and easily, which may help to increase PK knowledge. In this study, we have developed and validated two in-house ELISAs to quantify a-PD-1 and a-PD-L1 in plasma collected from tumor-bearing mice. The linear range for the a-PD-1 assay was 2.5-125 ng/mL and 0.11-3.125 ng/mL for the a-PD-L1 assay, whereas the intra-and inter-day precision was lower than 20% for both analytes. The PK characterization revealed a significant decrease in drug exposure after administration of multiple doses. Plasma half-life for a-PD-1 was slightly shorter (22.3 h) than for a-PD-L1 (46.7 h). To our knowledge, this is the first reported preclinical ELISA for these immune checkpoint inhibitors, which is sufficiently robust to be used in different preclinical models. These methods can help to understand the PK behavior of these antibodies under different scenarios and the relationship with response, thus guiding the choice of optimal doses in clinical settings.
Autores: Vinueza Gavilanes, Rodrigo; Íñigo Marco, Ignacio; Larrea, L. ; et al.

Título: N-terminal acetylation mutants affect alpha-synuclein stability, protein levels and neuronal toxicity

Revista: NEUROBIOLOGY OF DISEASE

ISSN: 0969-9961 Vol.137 2020 págs. 104781

Resumen

Alpha-synuclein (aSyn) protein levels are sufficient to drive Parkinson's disease (PD) and other synucleinopathies. Despite the biomedical/therapeutic potential of aSyn protein regulation, little is known about mechanisms that limit/control aSyn levels. Here, we investigate the role of a post-translational modification, N-terminal acetylation, in aSyn neurotoxicity. N-terminal acetylation occurs in all aSyn molecules and has been proposed to determine its lipid binding and aggregation capacities; however, its effect in aSyn stability/neurotoxicity has not been evaluated. We generated N-terminal mutants that alter or block physiological aSyn N-terminal acetylation in wild-type or pathological mutant E46K aSyn versions and confirmed N-terminal acetylation status by mass spectrometry. By optical pulse-labeling in living primary neurons we documented a reduced half-life and accumulation of aSyn N-terminal mutants. To analyze the effect of N-terminal acetylation mutants in neuronal toxicity we took advantage of a neuronal model where aSyn toxicity was scored by longitudinal survival analysis. Salient features of aSyn neurotoxicity were previously investigated with this approach. aSyn-dependent neuronal death was recapitulated either by higher aSyn protein levels in the case of WT aSyn, or by the combined effect of protein levels and enhanced neurotoxicity conveyed by the E46K mutation. aSyn N-terminal mutations decreased E46K aSyn-dependent neuronal death both by reducing protein levels and, importantly, by reducing the intrinsic E46K aSyn toxicity, being the D2P mutant the least toxic. Together, our results illustrate that the N-terminus determines, most likely through its acetylation, aSyn protein levels and toxicity, identifying this modification as a potential therapeutic target.
Autores: Anasagasti, A. ; Lara-Lopez, A.; Milla-Navarro, S. ; et al.

Título: Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa

Revista: PHARMACEUTICS

ISSN: 1999-4923 Vol.12 N° 10 2020 págs. 913

Resumen

Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.
Autores: Silva-Pilipich, N.; Martisová, Eva; Ballesteros-Briones, M. C.; et al.

Título: Long-term systemic expression of a novel PD-1 blocking nanobody from an AAV vector provides antitumor activity without toxicity

Revista: BIOMEDICINES

ISSN: 2227-9059 Vol.8 N° 12 2020 págs. 562

Resumen

Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However, it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects. We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector downstream of four different promoters (CMV, CAG, EF1 alpha, and SFFV) and its expression was analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels in vitro reaching 2-20 micrograms/mL with all promoters, except SFFV, which showed lower levels. Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8 vectors. Nb11 serum levels increased steadily along time, reaching 1-3 microgram/mL two months post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that continuous expression of immunomodulatory nanobodies from long-term expression vectors could have antitumor effects with low toxicity.
Autores: Simoes, I. T.; Aranda, F. (Autor de correspondencia); Casado-Llombart, S. ; et al.

Título: Multifaceted effects of soluble human CD6 in experimental cancer models

Revista: JOURNAL FOR IMMUNOTHERAPY OF CANCER

ISSN: 2051-1426 Vol.8 N° 1 2020 págs. e000172

Resumen

Background CD6 is a lymphocyte surface co-receptor physically associated with the T-cell receptor (TCR)/CD3 complex at the center of the immunological synapse. There, CD6 assists in cell-to-cell contact stabilization and modulation of activation/differentiation events through interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), its main reported ligand. While accumulating evidence is attracting new interest on targeting CD6 for therapeutic purposes in autoimmune disorders, little is known on its potential in cancer. In an attempt to elucidate the in vivo relevance of blocking CD6-mediated interactions in health and disease, we explored the consequences of expressing high circulating levels of a soluble form CD6 (sCD6) as a decoy receptor. Methods High sCD6 serum levels were achieved by using transgenic C57BL/6 mice expressing human sCD6 under the control of lymphoid-specific transcriptional elements (shCD6LckE mu Tg) or wild type either transduced with hepatotropic adeno-associated virus coding for mouse sCD6 or undergoing repeated infusions of recombinant human sCD6 protein. Characterization of sCD6-induced changes was performed by ex vivo flow cytometry and functional analyses of mouse lymphoid organ cells. The in vivo relevance of those changes was explored by challenging mice with subcutaneous or metastatic tumors induced by syngeneic cancer cells of different lineage origins. Results Through a combination of in vitro and in vivo studies, we show that circulating sCD6 expression induces defective regulatory T cell (Treg) generation and function, decreased CD166/ALCAM-mediated tumor cell proliferation/migration and impaired galectin-induced T-cell apoptosis, supporting the fact that sCD6 modulates antitumor lymphocyte effector function and tumorigenesis. Accordingly, sCD6 expression in vivo resulted in delayed subcutaneous tumor growth and/or reduced metastasis on challenge of mice with syngeneic cancer cells. Conclusions Evidence is provided for the disruption of CD6 receptor-ligand interactions as a feasible immunomodulatory approach in cancer.
Autores: Lasa Ventura, Marta; Neri, L.; Carte Abad, Beatriz; et al.

Título: Maturation of NAA20 Aminoterminal End Is Essential to Assemble NatB N-Terminal Acetyltransferase Complex

Revista: JOURNAL OF MOLECULAR BIOLOGY

ISSN: 0022-2836 Vol.432 N° 22 2020 págs. 5889 - 5901

Resumen

Protein lifespan is regulated by co-translational modification by several enzymes, including methionine aminopeptidases and N-alpha-aminoterminal acetyltransferases. The NatB enzymatic complex is an N-terminal acetyltransferase constituted by two subunits, NAA20 and NAA25, whose interaction is necessary to avoid NAA20 catalytic subunit degradation. We found that deletion of the first five amino acids of hNAA20 or fusion of a peptide to its amino terminal end abolishes its interaction with hNAA25. Substitution of the second residue of hNAA20 with amino acids with small, uncharged side-chains allows NatB enzymatic complex formation. However, replacement by residues with large or charged side-chains interferes with its hNAA25 interaction, limiting functional NatB complex formation. Comparison of NAA20 eukaryotic sequences showed that the residue following the initial methionine, an amino acid with a small uncharged side-chain, has been evolutionarily conserved. We have confirmed the relevance of second amino acid characteristics of NAA20 in NatB enzymatic complex formation in Drosophila melanogaster. Moreover, we have evidenced the significance of NAA20 second residue in Saccharomyces cerevisiae using different NAA20 versions to reconstitute NatB formation in a yNAA20-KO yeast strain. The requirement in humans and in fruit flies of an amino acid with a small uncharged side-chain following the initial methionine of NAA20 suggests that methionine aminopeptidase action may be necessary for the NAA20 and NAA25 interaction. We showed that inhibition of MetAP2 expression blocked hNatB enzymatic complex formation by retaining the initial methionine of NAA20. Therefore, NatB-mediated protein N-terminal acetylation is dependent on methionine aminopeptidase, providing a regulatory mechanism for protein N-terminal maturation. (C) 2020 Elsevier Ltd. All rights reserved.
Autores: Mevel, M. (Autor de correspondencia); Bouzelha, M. ; Leray, A.; et al.

Título: Chemical modification of the adeno-associated virus capsid to improve gene delivery

Revista: CHEMICAL SCIENCE

ISSN: 2041-6520 Vol.11 N° 4 2020 págs. 1122 - 1131

Resumen

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling.
Autores: López-Manzaneda, S.; Ojeda-Pérez, I.; Zabaleta Lasarte, Nerea; et al.

Título: In vitro and in vivo genetic disease modeling via NHEJ-Precise deletions using CRISPR-Cas9

Revista: MOLECULAR THERAPY. METHODS & CLINICAL DEVELOPMENT

ISSN: 2329-0501 Vol.19 2020 págs. 426 - 437

Resumen

The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30-60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.
Autores: Lantero, A.; Mauleón Mayora, Itsaso; Neri, L.; et al.

Título: rAAV Gene Therapy for the Treatment of Citrullinemia Type I (CTLN-1) in Immature Mice

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.28 N° 4 2020 págs. 121 - 121
Autores: Ros, I.; Salas Gómez, David; Rodríguez Parra, Estefanía Maite; et al.

Título: Sensitivity of different AAV serotypes to pre-existing human nabs, in vivo impact

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.28 N° 4 2020 págs. 181 - 182
Autores: Prieto Valtueña, Jesús María; González Aseguinolaza, Gloria (Autor de correspondencia)

Título: Acute Intermittent Porphyria: Novel Etiologic and Pathogenic Therapies Based on RNA Transfer to the Liver

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.70 N° 3 2019 págs. 1061 - 1063
Autores: Gulias, P.; Guerra-Varela, J. ; González Aparicio, Manuela; et al.

Título: Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Revista: GENES

ISSN: 2073-4425 Vol.10 N° 12 2019

Resumen

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how El/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.
Autores: Salas Gómez, David; Kwikkers, K. L.; Zabaleta Lasarte, Nerea; et al.

Título: Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates

Revista: BLOOD ADVANCES

ISSN: 2473-9529 Vol.3 N° 17 2019 págs. 2632 - 2641

Resumen

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.
Autores: Godoy, C. ; Tabernero, D. (Autor de correspondencia); Sopena, S.; et al.

Título: Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Revista: WORLD JOURNAL OF GASTROENTEROLOGY

ISSN: 1007-9327 Vol.25 N° 13 2019 págs. 1566 - 1579

Resumen

BACKGROUND Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses. AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients. METHODS Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity. RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype. CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.
Autores: Weber, N. D. (Autor de correspondencia); Odriozola, L. ; Martinez Garcia, Javier; et al.

Título: Gene therapy for progressive familial intrahepatic cholestasis type 3 in a clinically relevant mouse model

Revista: NATURE COMMUNICATIONS

ISSN: 2041-1723 Vol.10 N° 1 2019 págs. 5694

Resumen

Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare monogenic disease caused by mutations in the ABCB4 gene, resulting in a reduction in biliary phosphatidylcholine. Reduced biliary phosphatidylcholine cannot counteract the detergent effects of bile salts, leading to cholestasis, cholangitis, cirrhosis and ultimately liver failure. Here, we report results from treating two- or five-week-old Abcb4(-/-) mice with an AAV vector expressing human ABCB4, resulting in significant decreases of PFIC3 disease biomarkers. All male mice achieved a sustained therapeutic effect up through 12 weeks, but the effect was achieved in only 50% of females. However, two-week-old females receiving a second inoculation three weeks later maintained the therapeutic effect. Upon sacrifice, markers of PFIC3 disease such as, hepatosplenomegaly, biliary phosphatidylcholine and liver histology were significantly improved. Thus, AAV-mediated gene therapy successfully prevented PFIC3 symptoms in a clinically relevant mouse model, representing a step forward in improving potential therapy options for PFIC3 patients.
Autores: Murillo Sauca, Oihana; Moreno Luqui, Daniel; Gazquez López, Cristina; et al.

Título: Liver expression of a miniATP7B gene results in long-term restoration of copper homeostasis in a Wilson disease model in mice

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.70 N° 1 2019 págs. 108 - 126

Resumen

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8¿ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal¿binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (delta57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper.
Autores: Lopez-Pastor, A. R. ; Gomez-Hernandez, A.; Diaz-Castroverde, S.; et al.

Título: Liver-specific insulin receptor isoform A expression enhances hepatic glucose uptake and ameliorates liver steatosis in a mouse model of diet-induced obesity

Revista: DISEASE MODELS AND MECHANISMS

ISSN: 1754-8403 Vol.12 N° 2 2019 págs. dmm036186

Resumen

Among the main complications associated with obesity are insulin resistance and altered glucose and lipid metabolism within the liver. It has previously been described that insulin receptor isoform A (IRA) favors glucose uptake and glycogen storage in hepatocytes compared with isoform B (IRB), improving glucose homeostasis in mice lacking liver insulin receptor. Thus, we hypothesized that IRA could also improve glucose and lipid metabolism in a mouse model of high-fat-diet-induced obesity. We addressed the role of insulin receptor isoforms in glucose and lipid metabolism in vivo. We expressed IRA or IRB specifically in the liver by using adeno-associated viruses (AAVs) in a mouse model of diet-induced insulin resistance and obesity. IRA, but not IRB, expression induced increased glucose uptake in the liver and muscle, improving insulin tolerance. Regarding lipid metabolism, we found that AAV-mediated IRA expression also ameliorated hepatic steatosis by decreasing the expression of Fasn, Pgc1a, Acaca and Dgat2 and increasing Scd-1 expression. Taken together, our results further unravel the role of insulin receptor isoforms in hepatic glucose and lipid metabolism in an insulin-resistant scenario. Our data strongly suggest that IRA is more efficient than IRB at favoring hepatic glucose uptake, improving insulin tolerance and ameliorating hepatic steatosis. Therefore, we conclude that a gene therapy approach for hepatic IRA expression could be a safe and promising tool for the regulation of hepatic glucose consumption and lipid metabolism, two key processes in the development of non-alcoholic fatty liver disease associated with obesity.
Autores: Weber, N. D.; Odriozola, L.; Martinez Garcia, Javier; et al.

Título: AAV Expressing MDR3 (VTX-803) Mediates the Correction of Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3) in a Clinically Relevant Mouse Model

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.27 N° 4 2019 págs. 203 - 203
Autores: Ballesteros-Briones, M. C.; Martisová, Eva; Casales Zoco, Erkuden; et al.

Título: Short-term local expression of a PD-L1 blocking antibody from a self-replicating RNA vector induces potent antitumor responses

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.27 N° 4 2019 págs. 268 - 268
Autores: Maestro Galilea, Sheila; Camps, G.; Usai, C.; et al.

Título: Elucidating the role of hepatitis delta components during viral cycle and pathogenesis

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.70 2019 págs. 640A - 640A
Autores: Weber, N. D.; Odriozola, L.; Martinez Garcia, Javier; et al.

Título: VTX-803 AAV-mediated correction of progressive familiar intrahepatic cholestasis type 3 in a clinically relevant mouse model

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.70 N° 1 2019 págs. E590
Autores: Ganan, I. R.; Salas, D.; Rodriguez, E.; et al.

Título: Sensitivity of different AAV serotypes to pre-existing NAbs

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.30 N° 11 2019 págs. A115 - A115
Autores: Murillo Sauca, Oihana; Moreno Luqui, Daniel; Gazquez López, Cristina; et al.

Título: Therapeutic efficacy of VTX-801 Wilson's disease mice in mice with at an advanced stage the disease

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.30 N° 11 2019 págs. A151 - A152
Autores: Torella, Laura; Raimondi, Ivan; Vales Aranguren, África; et al.

Título: Paired-nickase S.aureus Cas9 system is an efficient and potentially safer in vivo treatment for Primary Hyperoxaluria Type 1

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.30 N° 11 2019 págs. A2 - A2
Autores: Zabaleta Lasarte, Nerea; Torella, L.; Vales Aranguren, África; et al.

Título: In Vivo glycolate Oxidase Disruption by AAV-Mediated Delivery of Paired S. Aureus Cas9 Nickase to Treat Primary Hiperoxaluria

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.27 N° 4 2019 págs. 368 - 368
Autores: Ramon, G. C.; Maestro Galilea, Sheila; Usai, C. ; et al.

Título: Elucidating the mechanisms of hdv-induced liver damage in an aav-hbv/hdv coinfection mouse model

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.70 2019 págs. 632A - 632A
Autores: Rodríguez Parra, Estefanía Maite; Zabaleta Lasarte, Nerea; Gil-Farina, I.; et al.

Título: AdrA as a potential immunomodulatory candidate for STING-mediated anti-viral therapy

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.30 N° 11 2019 págs. A219 - A220
Autores: Silva Pilipich, Noelia Romina; Martisová, Eva; Ballesteros-Briones, M. C.; et al.

Título: Long-term systemic expression of a new PD-1 blocking antibody from an AAV vector provides antitumor activity without toxicity

Revista: HUMAN GENE THERAPY

ISSN: 1043-0342 Vol.30 N° 11 2019 págs. A57 - A57
Autores: Moreno Luqui, Daniel; Murillo Sauca, Oihana; Gazquez López, Cristina; et al.

Título: Visualization of the therapeutic efficacy of a gene correction approach in Wilson's disease by laser-ablation inductively coupled mass spectrometry

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.68 N° 5 2018 págs. 1088 - 1090
Autores: Suarez-Amaran, L.; Usai, C.; Di Scala, M.; et al.

Título: A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-beta induction (vol 67, pg 669, 2017)

Revista: JOURNAL OF HEPATOLOGY

ISSN: 1600-0641 Vol.69 N° 1 2018 págs. 262 - 264
Autores: Torralba, D.; Baixauli, F. ; Villarroya-Beltri, C.; et al.

Título: Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

Revista: NATURE COMMUNICATIONS

ISSN: 2041-1723 Vol.9 2018

Resumen

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate antipathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.
Autores: Rodríguez-García, E. ; Olague Micheltorena, María Cristina; Ríus-Rocabert, S. ; et al.

Título: TMEM173 alternative spliced isoforms modulate viral replication through STING pathway

Revista: IMMUNOHORIZONS

ISSN: 2573-7732 Vol.2 N° 11 2018 págs. 363 - 376

Resumen

The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-ß induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-ß, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-ß by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron¿exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology.
Autores: González Aseguinolaza, Gloria; Murillo Sauca, Oihana; Moreno Luqui, Daniel; et al.

Título: Nonclinical Safety Evaluation of VTX-801, an AAV Vector for Treatment of Wilson's Disease

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.26 N° 5 2018 págs. 388 - 388
Autores: Murillo Sauca, Oihana; Moreno Luqui, Daniel; Gazquez López, Cristina; et al.

Título: Gene therapy optimization for Wilson's disease

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.68 N° Supl. 1 2018 págs. S83 - S83
Autores: Neri, L. ; Dominguez, V.; Elurbide Tardio, Jasmin; et al.

Título: NAA25 and Tropomyosin regulate liver metabolic zonation, hepatocyte polarity and ploidy

Revista: JOURNAL OF HEPATOLOGY (ONLINE)

ISSN: 0168-8278 Vol.68 N° Supl. 1 2018 págs. S54 - S54
Autores: Vinueza Gavilanes, Rodrigo; Adin Marcos, Íñigo; Larrea Urcola, Luis María; et al.

Título: N-terminal acetylation stabilizes alpha-synuclein protein and modulates its toxicity

Revista: FEBS OPEN BIO

ISSN: 2211-5463 Vol.8 N° Supl. 1 2018 págs. 412 - 412
Autores: Maestro, S.; Cortese, M. F.; Gonzalez, C. ; et al.

Título: Antiviral Activity of a New siRNA Targeting a Hyper-Conserved Region in Hepatitis B X Gene Transcripts: Preliminary Data.

Revista: HEPATOLOGY

ISSN: 0270-9139 Vol.68 N° Supl. 1 2018 págs. 239A - 240A
Autores: González Aseguinolaza, Gloria; Murillo Sauca, Oihana; Moreno Luqui, Daniel; et al.

Título: Therapeutic Efficacy of VTX-801, an Optimized AAV Vector for the Treatment of Wilson's Disease

Revista: MOLECULAR THERAPY

ISSN: 1525-0016 Vol.26 N° 5 2018 págs. 249 - 250

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Título: Generación de vectores de terapia génica quiméricos para tratar la poliquistosis renal autosómica dominante tipo II

Código de expediente: 0011-1408-2020-000028

Investigador principal: SERGIO MILAGROS SOLCHAGA.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: FIMA 2020 -GN DOCTORANDOS INDUSTRIALES 2020- 2021

Fecha de inicio: 29-09-2020

Fecha fin: 28-09-2023

Importe concedido: 78.897,12€

Otros fondos: -
Título: Optimización de vectores de terapia génica para el tratamiento del síndrome de Dravet

Código de expediente: 0011-1307-2020-000049

Investigador principal: RUBEN HERNANDEZ ALCOCEBA.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: FIMA 2020 -GN DOCTORANDOS INDUSTRIALES 2020- 2021

Fecha de inicio: 24-09-2020

Fecha fin: 30-09-2021

Importe concedido: 14.000,00€

Otros fondos: -
Título: Gene therapy of colorectal cancer using a self-replicating RNA vector expressing inhibitors of tumor cell adhesion in combination with immunostimulatory antibodies

Código de expediente: 0011-0537-2019-000006

Investigador principal: GUILLERMO HERRADOR CAÑETE.

Financiador: GOBIERNO DE NAVARRA / DPTO. EDUCACIÓN CULTURA Y TURISMO

Convocatoria: FIMA GNE 2019 BECAS PREDOCTORALES FIMA GNE 2019 BECAS PREDOCTORALES

Fecha de inicio: 01-06-2020

Fecha fin: 31-10-2022

Importe concedido: 68.743,32€

Otros fondos: -
Título: Creación de una plataforma para el desarrollo de vectores de terápia génica con tropismo renal.(Drones Génicos)

Código de expediente: 0011-1411-2019-000074

Investigador principal: RAFAEL ALDABE ARREGUI.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: FIMA 2019 GN PROYECTOS ESTRATEGICOS DE I+D 2019-2021

Fecha de inicio: 01-04-2019

Fecha fin: 30-11-2021

Importe concedido: 631.682,12€

Otros fondos: -
Título: Análisis de la interación del virus de la hepatitis delta (HDV) con el hospedador y con el virus de la hepatitis B (HBV) (INTERDELTA

Código de expediente: RTI2018-101936-B-I00

Investigador principal: GLORIA GONZALEZ ASEGUINOLAZA.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2018 - PROYECTOS DE I+D RETOS INVESTIGACION

Fecha de inicio: 01-01-2019

Fecha fin: 30-09-2022

Importe concedido: 338.800,00€

Otros fondos: Fondos FEDER
Título: RNA autorreplicativo armado con nanobodies inmunoestimuladores para terapia del cáncer colorectal

Código de expediente: 64/2018

Investigador principal: CRISTIAN SMERDOU PICAZO.

Financiador: GOBIERNO DE NAVARRA. DEPARTAMENTO DE SALUD

Convocatoria: 2018 PROYECTOS DE I+D EN SALUD

Fecha de inicio: 31-12-2018

Fecha fin: 30-12-2021

Importe concedido: 78.775,00€

Otros fondos: Fondos FEDER
Título: Terapia génica con glucocerebrosidasa para el tratamiento de la enfermedad de Parkinson.

Código de expediente: 0011-1383-2019-000006

Investigador principal: JOSE LUIS LANCIEGO PEREZ.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: 2019 - GN INDUSTRIA PROYECTOS CENTROS TECNOLOGICOS Y ORGANISMOS DE INVESTIGACION

Fecha de inicio: 01-12-2018

Fecha fin: 30-11-2019

Importe concedido: 132.648,00€

Otros fondos: -
Título: Desarrollo de una plataforma de terapia génica para enfermedades genéticas renales

Código de expediente: RTC-2017-6600-1

Investigador principal: RAFAEL ALDABE ARREGUI.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2017 - PROYECTOS RETOS-COLABORACION

Fecha de inicio: 01-04-2018

Fecha fin: 30-04-2022

Importe concedido: 150.913,59€

Otros fondos: Fondos FEDER
Título: Selección y optimización de nuevas moléculas antitumorales. Validación de la acetilación aminoterminal de proteínas como diana terapéutica antitumoral.

Código de expediente: 0011-1383-2018-000011

Investigador principal: RAFAEL ALDABE ARREGUI.

Financiador: GOBIERNO DE NAVARRA

Convocatoria: 2018 - GN INDUSTRIA PROYECTOS CENTROS TECNOLOGICOS Y ORGANISMOS DE INVESTIGACION

Fecha de inicio: 01-02-2018

Fecha fin: 30-11-2018

Importe concedido: 175.851,50€

Otros fondos: -
Título: CREACION DE UNA PLATAFORMA EUROPEA INNOVADORA DE TERAPIA GENICA PARA ENFERMEDADES HEREDITARIAS POCO FRECUENTES.

Código de expediente: EUIN2017-89279

Investigador principal: GLORIA GONZALEZ ASEGUINOLAZA.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2017 MINECO Europa Investigación

Fecha de inicio: 01-10-2017

Fecha fin: 30-09-2019

Importe concedido: 20.700,00€

Otros fondos: Fondos FEDER
Título: PREPARACION DE LA PROPUESTA ITN-NMATTERS

Código de expediente: EUIN2017-89194

Investigador principal: RAFAEL ALDABE ARREGUI.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2017 MINECO Europa Investigación

Fecha de inicio: 01-07-2017

Fecha fin: 31-12-2018

Importe concedido: 18.130,00€

Otros fondos: Fondos FEDER
Título: Desarrollo y caracterización de un modelo de infeccíón crónica por el virus de la Hepatitis Delta en ratón y desarrollo de nuevos tratamientos

Código de expediente: SAF2015-70028-R

Investigador principal: GLORIA GONZALEZ ASEGUINOLAZA.

Financiador: MINISTERIO DE CIENCIA E INNOVACIÓN

Convocatoria: 2015 - PROYECTOS DE I+D RETOS

Fecha de inicio: 01-01-2016

Fecha fin: 31-12-2018

Importe concedido: 210.240,00€

Otros fondos: Fondos FEDER

Título: Curing Dravet Syndrome by Gene Therapy

Código de expediente: AC17/00029

Investigador principal: ROCIO SANCHEZ-CARPINTERO ABAD

Financiador: INSTITUTO DE SALUD CARLOS III

Convocatoria: E-RARE

Fecha de inicio: 01-01-2018

Fecha fin: 31-05-2021

Importe concedido: 59.290,00€

Otros fondos: Fondos FEDER

Título: Desarrollo de nuevas estrategias de inmunoterapia para el tratamiento del cáncer colorectal usando vectores basados en RNA autorreplicativo

Investigador principal: CRISTIAN SMERDOU PICAZO

Financiador: INSTITUTO DE SALUD CARLOS III

Convocatoria: 2017 - PROYECTOS DE I+D EN SALUD

Fecha de inicio: 01-01-2018

Fecha fin: 31-12-2021

Importe concedido: 141.570,00€
Título: Terapia Génica para el Síndrome de Dravet

Investigador principal: MIGUEL VALENCIA USTARROZ

Financiador: FUNDACION INOCENTE INOCENTE

Convocatoria: FUNDACION INOCENTE INOCENTE 2017

Fecha de inicio: 19-10-2017

Fecha fin: 31-01-2019

Importe concedido: 29.755,00€

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