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CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1 −/− mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites. Substrate reduction therapies (SRT) are a promising therapeutic approach for monogenic inherited metabolic diseases. Here the authors evaluate the therapeutic potential of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I and demonstrate its safety and efficacy.

Multiple myeloma (MM) is an incurable disease, whose clinical heterogeneity makes its management challenging, highlighting the need for biological features to guide improved therapies. Deregulation of specific long non-coding RNAs (lncRNAs) has been shown in MM, nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we identified 40,511 novel lncRNAs in MM samples. lncRNAs accounted for 82% of the MM transcriptome and were more heterogeneously expressed than coding genes. A total of 10,351 overexpressed and 9,535 downregulated lncRNAs were identified in MM patients when compared with normal bone-marrow plasma cells. Transcriptional dynamics study of lncRNAs in the context of normal B-cell maturation revealed 989 lncRNAs with exclusive expression in MM, among which 89 showed de novo epigenomic activation. Knockdown studies on one of these lncRNAs, SMILO (specific myeloma intergenic long non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. We also showed that the expression of lncRNAs, together with clinical and genetic risk alterations, stratified MM patients into several progression-free survival and overall survival groups. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in older individuals. Here we analyze the transcriptome of human HSCs purified from young and older healthy adults, as well as MDS patients, identifying transcriptional alterations following different patterns of expression. While aging-associated lesions seem to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detect the upregulation of the transcription factor DNA Damage Inducible Transcript 3 ( DDIT3 ). Overexpression of DDIT3 in human healthy HSCs induces an MDS-like transcriptional state, and dyserythropoiesis, an effect associated with a failure in the activation of transcriptional programs required for normal erythroid differentiation. Moreover, DDIT3 knockdown in CD34 + cells from MDS patients with anemia is able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythroid differentiation characterizing MDS patients. Myelodysplastic syndromes (MDS) are age-related pathologies in which alterations of hematopoietic stem cells lead to abnormal formation of blood cells. Here, the authors study the lesions that these cells undergo in aging and disease, characterizing a factor whose alteration in MDS leads to abnormal blood cell production.

BackgroundMost patients with advanced non-small-cell lung cancer (NSCLC) fail to derive significant benefit from programmed cell death protein-1 (PD-1) axis blockade, and new biomarkers of response are needed. In this study, we aimed to discover and validate spatially resolved protein markers associated with sensitivity to PD-1 axis inhibition in NSCLC.MethodsWe initially assessed a discovery cohort of 56 patients with NSCLC treated with PD-1 axis inhibitors at Yale Cancer Center. Using the GeoMx Digital Spatial Profiling (DSP) system, 71 proteins were measured in spatial context on each spot in a tissue microarray. We used the AQUA method of quantitative immunofluorescence (QIF) to orthogonally validate candidate biomarkers. For external independent validation, we assessed whole tissue sections derived from 128 patients with NSCLC treated with single-agent PD-1 axis inhibitors at the 12 de Octubre Hospital (Madrid) using DSP. We further analyzed two immunotherapy untreated cohorts to address prognostic significance (n=252 from Yale Cancer Center; n=124 from University Clinic of Navarra) using QIF and DSP, respectively.ResultsUsing continuous log-scaled data, we identified CD44 expression in the tumor compartment (pan-cytokeratin (CK)+) as a novel predictor of prolonged progression-free survival (PFS) (multivariate HR=0.68, p=0.043) in the discovery set. We validated by QIF that tumor CD44 levels assessed as continuous QIF scores were associated with longer PFS (multivariate HR=0.31, p=0.022) and overall survival (multivariate HR=0.29, p=0.038). Using DSP in an independent immunotherapy treated cohort, we validated that CD44 levels in the tumor compartment, but not in the immune compartment (panCK–/CD45+), were associated with clinical benefit (OR=1.22, p=0.018) and extended PFS under PD-1 axis inhibition using the highest tertile cutpoint (multivariate HR=0.62, p=0.03). The effect of tumor cell CD44 in predicting PFS remained significant after correcting for programmed death-ligand 1 (PD-L1) Tumor Proportion Score (TPS) in both cohorts. High tumor cell CD44 was not prognostic in the absence of immunotherapy. Using DSP data, intratumoral regions with elevated tumor cell CD44 expression showed prominent (fold change>1.5, adjusted p<0.05) upregulation of PD-L1, TIM-3, ICOS, and CD40 in two independent cohorts.ConclusionsThis work highlights CD44 as a novel indicative biomarker of sensitivity to PD-1 axis blockade that might help to improve immunotherapy strategies for NSCLC.

Background Co‐morbid Alzheimer’s disease (AD) pathology is a major risk factor for cognitive impairment (CI) in PD, but whether and how AD co‐pathology affects the clinical phenotype of PD‐CI is incompletely understood. Recently validated plasma biomarkers for AD pathology, such as ptau217, hold great promise to revolutionize the diagnosis of neurodegenerative diseases. Here, we used plasma ptau217 to detect AD co‐pathology in a well‐characterized cohort of PD patients with CI and examine its associations with APOE4 genotype, cognitive profile, and cerebral hypometabolism on FDG‐PET. Method Eighty‐eight PD patients were stratified into PD‐CI (N=50; 24 PD‐MCI, 26 PDD) and PD with normal cognition (PD‐CN; N=38) using neuropsychological testing with the PD‐Cognitive Rating Scale. All patients had a blood draw and an FDG‐PET scan at study inclusion. Plasma ptau217 levels were measured using the ALZpath ptau217 Simoa immunoassay, and patients were classified as ptau217(+) and ptau217(‐) using an established threshold (0.4 pg/mL). APOE4 alleles were genotyped and coded as a binary variable. FDG‐PET data was processed using SPM12 and brain‐wide hypometabolism patterns (vs PD‐CN) were assessed across 52 atlas‐defined brain regions. In addition, we explicitly tested whether PD‐CI‐ptau217(+) had specifically more pronounced hypometabolism in an a‐priori region‐of‐interest (ROI) composed of temporo‐parietal areas typically affected in AD. Result Fourteen PD‐CI (28%) and 5 PD‐CN (13%) were classified as ptau217(+). PD‐CN‐ptau217(+) were excluded from further analyses. Compared to PD‐CI‐ptau217(‐), PD‐CI‐ptau217(+) had a higher prevalence of APOE4 carriers (50% vs 16%, p=0.04) and more impaired memory scores (p=0.03), although global cognition (MoCA) was not significantly different (p=0.10) (Table 1). When compared to PD‐CN, both PD‐CI‐ptau217(‐) and PD‐CI‐ptau217(+) showed significant hypometabolism in posterior‐occipital, temporal, and frontal areas (p<0.05, FDR‐corrected), but hypometabolism in PD‐CI‐ptau217(+) was considerably more extensive, particularly in temporo‐parietal areas (Fig‐1). ROI‐based analysis confirmed significantly more pronounced hypometabolism of AD‐related regions in PD‐CI‐ptau217(+) compared to PD‐CI‐ptau217(‐) (p=0.01), whereas occipital hypometabolism, typical for PD‐CI, did not differ (p=0.83). Conclusion AD co‐pathology results in a more memory‐predominant cognitive profile and AD‐like neurodegeneration phenotype in PD‐CI. Novel plasma biomarkers may significantly facilitate clinical detection of AD co‐pathology, which may have important implications for personalized diagnosis, prognosis, and treatment of PD patients.