Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner.

Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software's suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.

Generation of an iPSC line (SCTCi014-A) and isogenic control line (SCTCi014-A-1) from an age-related macular degeneration patient carrying the variant c.355G>A in the CFI gene.

Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a male AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). The line can be utilized to study the effects of this variant in disease-specific cell types.

Generation of an iPSC line (SCTCi015-A) and isogenic control line (SCTCi015-A-1) from an age-related macular degeneration patient carrying the variant c.355G>A in the CFI gene.

Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a female AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). This line can be utilized to study the effects of this variant in disease-specific cell types.

Identification of a Complex Allele in IMPG2 as a Cause of Adult-Onset Vitelliform Macular Dystrophy.

Purpose: Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders with approximately 270 genes involved. IMPG2 is associated with adult-onset vitelliform macular dystrophy. Here, we investigated two unrelated patients with vitelliform macular dystrophy to identify the underlying genetic cause. Methods: Whole-exome sequencing identified a putative causal complex allele consisting of c.3023-15T>A and c.3023G>A (p.(Gly1008Asp)) in IMPG2 in both individuals. To assess its effect, in vitro splice assays in HEK293T and further characterization in patient-derived photoreceptor precursor cells (PPCs) were conducted. Results: The results of the midigene splice assays in HEK293T showed that the complex allele causes a variety of splicing defects ranging from a small deletion to (multiple-)exon skipping. This finding was further validated using patient-derived PPCs that showed a significant increase of out-of-frame transcripts lacking one or multiple exons compared to control-derived PPCs. Overall, control PPCs consistently showed low levels of aberrantly spliced IMPG2 transcripts that were highly elevated in patient-derived PPCs. These differences were even more obvious upon inhibition of nonsense-mediated decay with cycloheximide. Conclusions: We report a heterozygous complex allele in IMPG2 causative for adult-onset vitelliform macular dystrophy in two unrelated individuals with mild visual loss and bilateral vitelliform lesions. The predicted causal missense mutation c.3023G>A, located in the consensus splice acceptor site, enhances the splicing effect of the upstream variant c.3023-15T>A, leading to the generation of aberrant transcripts that decrease the full-length IMPG2 levels. These results suggest a haploinsufficiency mechanism of action and highlight the complementarity of using different models to functionally assesses splicing defects.

Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides.

Over the last decades, animal models have become increasingly important in therapeutic drug development and assessment. The use of these models, mainly mice and rats, allow evaluating drugs in the real-organism environment and context. However, several molecular therapeutic approaches are sequence-dependent, and therefore, the humanization of such models is required to assess the efficacy. The generation of genetically modified humanized mouse models is often an expensive and laborious process that may not always recapitulate the human molecular and/or physiological phenotype. In this chapter, we summarize basic aspects to consider before designing and generating humanized models, especially when they are aimed to test antisense-based therapies.

Generation of a patient-derived induced pluripotent cell line (SCTCi016-A) carrying a homozygous variant in RPE65.

Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease / El miR-320c regula la expresión del gen SERPINA1 y se induce en pacientes con enfermedad pulmonar

Introduction: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3′UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. Methods: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. Results: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. Conclusion: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases. (AU)

Introducción: La deficiencia de alfa-1 antitripsina (DAAT) es una condición genética que produce enfermedad pulmonar y hepática con una gran variabilidad clínica. Los microARN se han identificado como modificadores de la gravedad de algunas enfermedades y su desregulación podría desempeñar un papel en la heterogeneidad de esta enfermedad. Los miembros de la familia miR-320 regulan múltiples procesos, incluyendo la inflamación, y tienen lugares de unión en la región 3’UTR del gen SERPINA1. En este estudio exploramos la implicación del miR-320c, un miembro de esta familia, en la DAAT. Métodos: Primero se realizaron estudios in vitro para demostrar la regulación del gen SERPINA1 por parte del miR-320. Además, se analizó la expresión de miR-320c en la sangre de 98 individuos con diferentes niveles de AAT mediante PCR cuantitativa y se correlacionó con los parámetros clínicos. Por último, se utilizaron células HL60 para analizar la inducción de miR-320c en condiciones inflamatorias. Resultados: La sobreexpresión del miR-320 en células HepG2 inhibía la expresión del gen SERPINA1. El análisis de expresión de miR-320c en los pacientes reveló una expresión significativamente aumentada en los casos con enfermedad pulmonar. Por otro lado, las células HL60 tratadas con LPS como factor proinflamatorio mostraron un aumento de expresión de miR-320c, lo que sugiere que este miARN responde a procesos inflamatorios. Conclusión: Nuestros resultados demuestran que el miR-320c inhibe la expresión de SERPINA1 en células hepáticas y que sus niveles en sangre están asociados con la presencia de enfermedad pulmonar en pacientes con diferentes niveles de AAT. Esto sugiere que el miR-320c desempeña un papel en la regulación de los niveles de AAT y podría ser un biomarcador de inflamación en enfermedades pulmonares. (AU)

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

miR-320c Regulates SERPINA1 Expression and Is Induced in Patients With Pulmonary Disease.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease. METHODS: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions. RESULTS: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. CONCLUSION: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases.

Exploiting the passenger ACO1-deficiency arising from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

Molecular Therapies for Inherited Retinal Diseases-Current Standing, Opportunities and Challenges.

Inherited retinal diseases (IRDs) are both genetically and clinically highly heterogeneous and have long been considered incurable. Following the successful development of a gene augmentation therapy for biallelic RPE65-associated IRD, this view has changed. As a result, many different therapeutic approaches are currently being developed, in particular a large variety of molecular therapies. These are depending on the severity of the retinal degeneration, knowledge of the pathophysiological mechanism underlying each subtype of IRD, and the therapeutic target molecule. DNA therapies include approaches such as gene augmentation therapy, genome editing and optogenetics. For some genetic subtypes of IRD, RNA therapies and compound therapies have also shown considerable therapeutic potential. In this review, we summarize the current state-of-the-art of various therapeutic approaches, including the pros and cons of each strategy, and outline the future challenges that lie ahead in the combat against IRDs.

Downregulation of specific FBXW7 isoforms with differential effects in T-cell lymphoblastic lymphoma.

FBXW7 is a driver gene in T-cell lymphoblastic neoplasia acting through proteasome degradation of key proto-oncogenes. FBXW7 encodes three isoforms, α, ß and γ, which differ only in the N-terminus. In this work, massive sequencing revealed significant downregulation of FBXW7 in a panel of primary T-cell lymphoblastic lymphomas characterised by the absence of mutations in its sequence. We observed that decreased expression mainly affected the FBXW7ß isoform and to a lesser extent FBXW7α and may be attributed to the combined effect of epigenetic changes, alteration of upstream factors and upregulation of miRNAs. Transient transfections with miRNA mimics in selected cell lines resulted in a significant decrease of total FBXW7 expression and its different isoforms separately, with the consequent increment of critical substrates and the stimulation of cell proliferation. Transient inhibition of endogenous miRNAs in a T-cell lymphoblastic-derived cell line (SUP-T1) was capable of reversing these proliferative effects. Finally, we show how FBXW7 isoforms display different roles within the cell. Simultaneous downregulation of the α and γ isoforms modulates the amount of CCNE1, whilst the ß-isoform alone was found to have a prominent role in modulating the amount of c-MYC. Our data also revealed that downregulation of all isoforms is a sine qua non condition to induce a proliferative pattern in our cell model system. Taking these data into account, potential new treatments to reverse downregulation of all or a specific FBXW7 isoform may be an effective strategy to counteract the proliferative capacity of these tumour cells.