Shedding light on the immune response to AAV gene therapy

AAV vectors are one of the most promising tools in retinal gene therapy. However, accumulating evidence points to the relevance of AAV's immunogenicity in the clinical setting. In light of this, it is critical to have a better knowledge of the mechanisms underlying immunological responses to AAV vectors. In this PhD project we focused on the innate immune responses to AAV vector- and AAV production system-dependent factors that modulate vector immunogenicity in different in vitro models and its impact on AAV-mediated retinal gene therapy. In order to do that, a wide range of basic-research techniques, including biochemistry, molecular biology, microscopy, cell culture and organotypic culturing methodologies as well as preclinical-research analyses and interpretation of SD-OCT and FAF were performed. The main results achieved in this dissertation may be summarised as follows: 1. Experimental AAV8 and AAV2 vectors induced lot-specific innate immune responses in human pDCs which were neither specific to the capsid/vg ratio nor the production platform nor the manufacturer. Also, innate immune responses in pDCs were dependent on TLR9 signalling pathway, which could be reduced by pre-treatment with DNase. Furthermore, DNase treatment increased the transduction rate across all AAV8 vector lots in HEK293T cells. This suggests that both HEK293- and Sf9-cell derived preparations of experimental AAV vectors may contain a variety of extra-viral DNA impurities. Some of them cause lot-specific innate immune responses in human pDCs and impair transduction in HEK293T cells. 2. cgAAV lots without any known extra-viral DNA impurities but carrying different amounts of HCP were not able to elicit any innate immune response in neither pDCs nor in PMA-differentiated THP-1 cells. This may suggest that either the amount of HCP content found in our cgAAV vectors lots may not be high enough to induce immune responses in our in vitro models, or that HCP impurities are less immunogenic than extra-viral DNA impurities. 3. PRR ligands induced innate immune responses in ROs at early timepoints suggesting the presence of at least TLR2, 4 and 9. On the other hand, immunogenic AAV vectors that were sensed by pDCs were not able to elicit innate immune responses in retinal models. However, AAV vectors were able to transduce ROs. 4. SD-OCT analysis of NHPs after clinical grade AAV-mediated subretinal gene therapy revealed that, although the surgical procedure by itself induced the appearance of HRF in the ONL, the number of HRF increased over time in the high dose AAV-treated eyes. In addition, immune cell infiltration was detected in retinal sections from AAV treated eyes. Interestingly, the cgAAV lot used in this preclinical study contained lower HCP levels than the cgAAV lots used in THP-1 and pDCs in which no immune response was detected. This implies that an immune response to AAV and not to extra-viral impurities, appears to be the most plausible explanation for the group of animals injected with the high dose, and the HRF observed on SD-OCT could represent activated microglia activation into outer retinal layers. Finally, we showed that extra-viral DNA contaminants can affect the immunogenicity and potency of AAV vector lots. This has significant implications for the study of innate immune mechanisms involved in AAV vector recognition (basic research), the safety and efficacy of AAV-mediated gene therapy in animal models or human patients (translational and clinical research), and it also provides new information on how to adapt the AAV production process to generate safer and more effective gene therapy vectors (vector production methods)

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy

Modelo animal; Medicina regenerativa; RetinaModel animal; Medicina regenerativa; RetinaAnimal model; Regenerative medicine; RetinaBackground: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.This work was supported by Spanish Ministry of Science and Innovation (MICINN, RTI2018-095377-B-100), Instituto de Salud Carlos III ISCIII/FEDER (PRB2 PT13/0001/0041; TerCel RD16/0011/0024 and Oftared-RETICS RD16/0008), ERA-NET EuroNanoMed III/ISCIII (AC19/00080), the Catalan Government, AGAUR (2017-SGR-899), CERCA Programme/Generalitat de Catalunya, and by Fundació Carmen i Mª José Godó (Fundació CMJ Godó 2017)

Bone metabolism and clinical study of 44 patients with bisphosphonate-related osteonecrosis of the jaws

Osteonecrosis of the jaws is a clinical entity described and linked to treatment with bisphosphonates in 2003. Its real incidence is unknown and it could increase due to the large number of patients treated with these drugs, and its cumulative effect on the bone. State of the art knowledge regarding its etiopathogeny, clinical course and suitable treatments is limited. Objectives: To study the clinical characteristics of 44 patients with bisphosphonate-related osteonecrosis of the jaws and the state of their bone mineral metabolism: bone remodeling state, prevalence of fractures, bone mineral density study, and assessment of the different treatment strategies. Design of the Study: Observational. Information was gathered prospectively through interviews, clinical examinations, additional tests and review of medical records. Results: We studied 16 men and 28 women with a mean age of 64.7 years. Breast cancer was the most frequent underlying disease. Zoledronate was used in 82% of the cases and in the non-oncology group of patients; alendronate was the most frequently used bisphosphonate. The mean duration of the zoledronate and alendronate treatments was 25 months and 88 months respectively. The lower jaw was the most frequent location, and previous exodontias- among the triggering factors known-were the most closely linked to its onset. We found considerable osteoblastic activity in patients suffering from neoplasia, with artifacts present in their bone densitometry and a high percentage of vertebral fractures. Conclusions: According to our results, osteonecrosis of the jaws affects elderly patients. We found a direct relationship between the duration of exposure and the accumulated dose. Other relevant factors are: Poor oral and dental health, corticoids, diabetes and teeth extractions. In essence, it is a clinical diagnosis. Prevention is the best strategy to handle this clinical entity

Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins

Background Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. Methods In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. Results We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Conclusions Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.This project has been financed through a) The ISCIII (Miguel Servet-I, 2015), co-financed by the European Regional Development Fund (ERDF), No CP15/00071. b) The European Union’s Horizon 2020 research and innovation program, under grant agreement No 634479. c) Regional Ministry of Economy, Innovation and Science of the Junta de Andalucía, No P09-CTS-04967.info:eu-repo/semantics/publishedVersio

Obtención y caracterización de células de pluripotencia inducida a partir de monocitos humanos

La obtención de células de pluripotencia inducida (iPS) a partir de células somáticas ha proporcionado al campo de la medicina regenerativa una nueva herramienta para la terapia celular, evitando los problemas éticos relacionados con las células madre embrionarias (ESC) (Ronen et al. 2012). Las iPS, al igual que las ESC, son capaces de diferenciarse hacia distintos tipos celulares por lo que pueden derivarse a modelos celulares de enfermedades, apropiados tanto para la investigación básica como para el ensayo de nuevas terapias. La fuente habitual de iPS son fibroblastos obtenidos de una biopsia de piel. En nuestro caso, el objetivo es poner a punto un sistema de generación de modelos celulares a partir de sangre periférica, que es el tejido adulto más accesible, por lo que la obtención de muestras es mucho menos invasivo, y además permite el acceso a numerosas muestras almacenadas en los bancos de sangre (Phillips et al. 2012). Concretamente, en nuestro proyecto pretendemos obtener epitelio pigmentario de la retina (RPE) a partir de monocitos de pacientes con degeneración macular (AMD) portadores de un genotipo de susceptibilidad a la enfermedad. La metodología seguida ha consistido en el cultivo de monocitos sanguíneos y su transformación a iPS mediante la expresión de factores de reprogramación introducidos en la célula mediante el virus Sendai (SeV). El SeV es un virus no integrativo y se emplea como portador de los factores de transcripción claves para activar la pluripotencia, es decir, permite la expresión de transgenes sin el riesgo de modificación del genoma del hospedador (Fusaki et al. 2009). Además, la eficiencia de generación de iPS con el SeV es significativamente mayor que con otros métodos como la reprogramación por mARN o mediante vectores episomales.Los clones de iPS obtenidos, antes de su diferenciación hacia RPE, han de ser caracterizados mediante la comprobación de la ausencia del SeV (por PCR), la expresión de marcadores de pluripotencia, mediante inmunocitoquímica y PCR (OCT4, SOX2, SSEA-4, NANOG) y ensayos de fosfatasa alcalina. Los resultados obtenidos nos permiten comprobar que la reprogramación ha sido satisfactoria.Hasta el momento, hemos sido capaces de reprogramar células sanguíneas hacia iPS y mantenerlas. El siguiente paso será empezar a optimizar los protocolos de diferenciación hacia RPE. Tras caracterizarlo procederemos a realizar todo el proceso de reprogramación y diferenciación con muestras de pacientes con AMD

Molecular and microbiological report of a hospital outbreak of NDM-1-carrying Enterobacteriaceae in Mexico

Abstract Objectives To characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico. Methods From September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates. Results Fifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates. The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments. Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10. The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenemsusceptible isolates (p =<0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p =<0.05). Conclusions Four Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy

Background: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). Methods: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. Results: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. Conclusions: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA. Keywords: age-related macular degeneration (AMD); geographic atrophy; pig; animal model; stem cells; iPSC; RPE; retina; regenerative medicine; advanced cell therap

Risk factors and outcome associated with the acquisition of linezolid-resistant Enterococcus faecalis

Objectives: Linezolid is a synthetic oxazolidinone antibiotic frequently used to treat vancomycin-resistant enterococcal infections. Vancomycin-susceptible Enterococcus faecalis can develop resistance to linezolid in environments with excessive linezolid use. The aim of this study was to define risk factors and outcome associated with the acquisition of linezolid-resistant E. faecalis (LREfs). Methods: A retrospective case–control study was designed including patients hospitalised from January 2014 to October 2017 at Hospital Civil de Guadalajara ‘Fray Antonio Alcalde’ in Guadalajara, Mexico. A total of 50 patients culture-positive for LREfs and 100 control patients hospitalised in the same room and time as the cases were included. Clinical and demographic data were collected and analysed. Results: Risk factors for the presence of LREfs included prior linezolid use [odds ratio (OR) = 6.74], prior clindamycin use (OR = 6.72) and previous surgery (OR = 5.79). The mortality rate was 18% for LREfs cases versus 9% for controls. Conclusion: LREfs has emerged and spread in our hospital, an environment in which linezolid use is considerable. Risk factors for LREfs are prior antibiotic use, including linezolid, and previous surgery

Risk factors and molecular mechanisms associated with trimethoprim–sulfamethoxazole resistance in Stenotrophomonas maltophilia in Mexico

Abstract Purpose. Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen causing an increasing number of nosocomial infections. Our aim was to evaluate the risk factors and mechanisms associated with trimethoprim– sulfamethoxazole (SXT) resistance in S. maltophilia infections in Mexico. Methodology. Clinical isolates and patients’ demographic and clinical data were collected from February 2007 to August 2015 in two tertiary-care hospitals in Mexico. Antimicrobial susceptibility and analysis of sul and SmeABC and SmeDEF efflux pump overexpression were performed in all isolates. Results/Key findings. In the 9-year period, 196 patients infected with S. maltophilia were identified. Most patients were male, and the mean age was 46.2years. The mean Charlson score was 1.42, and the most frequent comorbidities were arterial hypertension (26.7%), type 2 diabetes (21.2%) and cerebral infarction (11.6%). High drug resistance to meropenem (93.4%), gentamicin (55.1%), ceftazidime (52.3%), cefotaxime (51.5%), amikacin (42.3%) and cefepime (32.1%), and lower resistance to ciprofloxacin (26.0%), SXT (25.0%), chloramphenicol (14.3%) and levofloxacin (2.6%) were detected. SXT resistance was not associated with the sul genes. SmeABC overexpression was associated with gentamicin (P=0.001) and levofloxacin resistance (P=0.041), whereas SmeDEF overexpression was associated with ceftazidime resistance (P=0.003). Prolonged hospitalization (�15days) was an independent risk factor for SXT-resistant S. maltophilia infections (OR=3.05; 95%CI=1.12– 8.86; P=0.029). Conclusion. Given the high SXT resistance rate, SXT is not an effective first-line therapy for our patients; instead, levofloxacin could be used as an appropriate therapeutic option against S. maltophilia infections