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ß-Thalassemia is brought about by defective ß-globin (HBB [hemoglobin subunit ß]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break-based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.
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Therapy via the gene addition of the anti-sickling ßAS3-globin transgene is potentially curative for all ß-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for ßAS3-globin addition and evaluates strategies for an increased ß-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-ßAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior ß-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent ßAS3-globin expression. LVs were initially compared for their ability to achieve high ß-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that ß-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased ß-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with ßAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-ßAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-ßAS3 for the treatment of severe ß-hemoglobinopathies.
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Hemoglobinopatías , Talasemia beta , Humanos , Talasemia beta/genética , Talasemia beta/terapia , Interferencia de ARN , Terapia Genética/métodos , Vectores Genéticos/genética , Hemoglobinopatías/genética , Hemoglobinopatías/terapia , Mutación , Globinas beta/genética , ARN Interferente Pequeño/genéticaRESUMEN
The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.
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Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a ß-globin mutation and to reactivate γ-globin expression as two potential therapies for ß-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
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We have previously demonstrated that both the original γ-globin lentiviral vector (LV) GGHI and the optimized GGHI-mB-3D LV, carrying the novel regulatory elements of the 3D HPFH-1 enhancer and the 3' ß-globin UTR, can significantly increase HbF production in thalassemic CD34+ cells and ameliorate the disease phenotype in vitro. In the present study, we investigated whether the GGHI-mB-3D vector can also exhibit an equally therapeutic effect, following the transduction of sickle cell disease (SCD) CD34+ cells at MOI 100, leading to HbF increase coupled with HbS decrease, and thus, to phenotype improvement in vitro. We show that GGHI-mB-3D LV can lead to high and potentially therapeutic HbF levels, reaching a mean 2-fold increase to a mean value of VCN/cell of 1.0 and a mean transduction efficiency of 55%. Furthermore, this increase was accompanied by a significant 1.6-fold HbS decrease, a beneficial therapeutic feature for SCD. In summary, our data demonstrate the efficacy of the optimized γ-globin lentiviral vector to improve the SCD phenotype in vitro, and highlights its potential use in future clinical SCD trials.
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Anemia de Células Falciformes , Talasemia beta , Humanos , gamma-Globinas/genética , Terapia Genética , Hemoglobina Fetal/genética , Vectores Genéticos/genética , Lentivirus/genética , Talasemia beta/genética , Talasemia beta/terapia , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapiaRESUMEN
Advanced therapy medicinal products (ATMPs) are medicines for human use based on genes, cells or tissue engineering. After clear successes in adults, the nascent technology now sees increasing pediatric application. For many still untreatable disorders with pre- or perinatal onset, timely intervention is simply indispensable; thus, prenatal and pediatric applications of ATMPs hold great promise for curative treatments. Moreover, for most inherited disorders, early ATMP application may substantially improve efficiency, economy and accessibility compared with application in adults. Vindicating this notion, initial data for cell-based ATMPs show better cell yields, success rates and corrections of disease parameters for younger patients, in addition to reduced overall cell and vector requirements, illustrating that early application may resolve key obstacles to the widespread application of ATMPs for inherited disorders. Here, we provide a selective review of the latest ATMP developments for prenatal, perinatal and pediatric use, with special emphasis on its comparison with ATMPs for adults. Taken together, we provide a perspective on the enormous potential and key framework parameters of clinical prenatal and pediatric ATMP application.
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The Global Globin Network (GGN) is a project-wide initiative of the Human Variome/Global Variome Project (HVP) focusing on haemoglobinopathies to build the capacity for genomic diagnosis, clinical services, and research in low- and middle-income countries. At present, there is no framework to evaluate the improvement of care, treatment, and prevention of thalassaemia and other haemoglobinopathies globally, despite thalassaemia being one of the most common monogenic diseases worldwide. Here, we propose a universally applicable system for evaluating and grouping countries based on qualitative indicators according to the quality of care, treatment, and prevention of haemoglobinopathies. We also apply this system to GGN countries as proof of principle. To this end, qualitative indicators were extracted from the IthaMaps database of the ITHANET portal, which allowed four groups of countries (A, B, C, and D) to be defined based on major qualitative indicators, supported by minor qualitative indicators for countries with limited resource settings and by the overall haemoglobinopathy carrier frequency for the target countries of immigration. The proposed rubrics and accumulative scores will help analyse the performance and improvement of care, treatment, and prevention of haemoglobinopathies in the GGN and beyond. Our proposed criteria complement future data collection from GGN countries to help monitor the quality of services for haemoglobinopathies, provide ongoing estimates for services and epidemiology in GGN countries, and note the contribution of the GGN to a local and global reduction of disease burden.
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Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3' untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form.
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Células Eritroides/citología , Edición Génica/métodos , MicroARNs/genética , Proteínas Represoras/genética , Regiones no Traducidas 3' , Sistemas CRISPR-Cas , Línea Celular , Reparación del ADN por Unión de Extremidades , Células Eritroides/metabolismo , Células HEK293 , Humanos , Prueba de Estudio ConceptualRESUMEN
Accurate and consistent interpretation of sequence variants is integral to the delivery of safe and reliable diagnostic genetic services. To standardize the interpretation process, in 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published a joint guideline based on a set of shared standards for the classification of variants in Mendelian diseases. The generality of these standards and their subjective interpretation between laboratories has prompted efforts to reduce discordance of variant classifications, with a focus on the expert specification of the ACMG/AMP guidelines for individual genes or diseases. Herein, we describe our experience as a ClinGen Variant Curation Expert Panel to adapt the ACMG/AMP criteria for the classification of variants in three globin genes (HBB, HBA2, and HBA1) related to recessively inherited hemoglobinopathies, including five evidence categories, as use cases demonstrating the process of specification and the underlying rationale.
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Genoma Humano , Hemoglobinopatías , Humanos , Pruebas Genéticas , Variación Genética , Hemoglobinopatías/diagnóstico , Hemoglobinopatías/genética , Patología Molecular , Estados UnidosRESUMEN
Nuclear envelope (NE) and endoplasmic reticulum (ER) collaborate to control a multitude of nuclear and cytoplasmic actions. In this context, the transmembrane protein TMEM147 localizes to both NE and ER, and through direct and indirect interactions regulates processes as varied as production and transport of multipass membrane proteins, neuronal signaling, nuclear-shape, lamina and chromatin dynamics and cholesterol synthesis. Aiming to delineate the emerging multifunctionality of TMEM147 more comprehensively, we set as objectives, first, to assess potentially more fundamental effects of TMEM147 on the ER and, second, to identify significantly TMEM147-associated cell-wide protein networks and pathways. Quantifying curved and flat ER markers RTN4 and CLIMP63/CKAP4, respectively, we found that TMEM147 silencing causes area and intensity increases for both RTN4 and CLIMP63, and the ER in general, with a profound shift toward flat areas, concurrent with reduction in DNA condensation. Protein network and pathway analyses based on comprehensive compilation of TMEM147 interactors, targets and co-factors then served to manifest novel and established roles for TMEM147. Thus, algorithmically simplified significant pathways reflect TMEM147 function in ribosome binding, oxidoreductase activity, G protein-coupled receptor activity and transmembrane transport, while analysis of protein factors and networks identifies hub proteins and corresponding pathways as potential targets of TMEM147 action and of future functional studies.
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Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Retículo Endoplásmico/ultraestructura , Silenciador del Gen , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Nogo/metabolismo , Mapas de Interacción de Proteínas , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Receptor de Lamina BRESUMEN
Accessibility of hematopoietic stem cells (HSCs) for the manipulation and repopulation of the blood and immune systems has placed them at the forefront of cell and gene therapy development. Recent advances in genome-editing tools, in particular for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and CRISPR/Cas-derived editing systems, have transformed the gene therapy landscape. Their versatility and the ability to edit genomic sequences and facilitate gene disruption, correction or insertion, have broadened the spectrum of potential gene therapy targets and accelerated the development of potential curative therapies for many rare diseases treatable by transplantation or modification of HSCs. Ongoing developments seek to address efficiency and precision of HSC modification, tolerability of treatment and the distribution and affordability of corresponding therapies. Here, we give an overview of recent progress in the field of HSC genome editing as treatment for inherited disorders and summarize the most significant findings from corresponding preclinical and clinical studies. With emphasis on HSC-based therapies, we also discuss technical hurdles that need to be overcome en route to clinical translation of genome editing and indicate advances that may facilitate routine application beyond the most common disorders.
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Sistemas CRISPR-Cas , Edición Génica , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Células Madre Hematopoyéticas , HumanosRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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Células Eritroides/clasificación , Células Eritroides/metabolismo , MicroARNs/genética , Adulto , Factores de Edad , Diferenciación Celular/genética , Línea Celular , Eritropoyesis/genética , Sangre Fetal/citología , Hemoglobina Fetal/genética , Feto/metabolismo , Humanos , ARN Mensajero/genética , Factores de Transcripción , gamma-Globinas/genéticaAsunto(s)
Talasemia beta , Femenino , Humanos , Masculino , Talasemia beta/diagnóstico , Talasemia beta/genéticaRESUMEN
The ß-thalassemias are an increasing challenge to health systems worldwide, caused by absent or reduced ß-globin (HBB) production. Of particular frequency in many Western countries is HBBIVSI-110(G > A) ß-thalassemia (HGVS name: HBB:c.93-21G > A). Its underlying mutation creates an abnormal splice acceptor site in the HBB gene, and while partially retaining normal splicing of HBB, it severely reduces HBB protein expression from the mutant locus and HBB loci in trans. For the assessment of the underlying mechanisms and of therapies targeting ß-thalassemia, accurate quantification of aberrant and normal HBB mRNA is essential, but to date, has only been performed by approximate methods. To address this shortcoming, we have developed an accurate, duplex reverse-transcription quantitative PCR assay for the assessment of the ratio and absolute quantities of normal and aberrant mRNA species as a tool for basic and translational research of HBBIVSI-110(G > A) ß-thalassemia. The method was employed here to determine mRNA ratios and quantities in blood and primary cell culture samples and correlate them with HBB protein levels. Moreover, with its immediate utility for ß-thalassemia and the mutation in hand, the approach can readily be adopted for analysis of alternative splicing or for quantitative assays of any disease-causing mutation that interferes with normal splicing.
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Empalme Alternativo/genética , Mutación/genética , Globinas beta/genética , Talasemia beta/genética , Células Cultivadas , Humanos , ARN Mensajero/genéticaRESUMEN
The structurally and functionally complex endoplasmic reticulum (ER) hosts critical processes including lipid synthesis. Here, we focus on the functional characterization of transmembrane protein TMEM147, and report that it localizes at the ER and nuclear envelope in HeLa cells. Silencing of TMEM147 drastically reduces the level of lamin B receptor (LBR) at the inner nuclear membrane and results in mistargeting of LBR to the ER. LBR possesses a modular structure and corresponding bifunctionality, acting in heterochromatin organization via its N-terminus and in cholesterol biosynthesis via its sterol-reductase C-terminal domain. We show that TMEM147 physically interacts with LBR, and that the C-terminus of LBR is essential for their functional interaction. We find that TMEM147 also physically interacts with the key sterol reductase DHCR7, which is involved in cholesterol biosynthesis. Similar to what was seen for LBR, TMEM147 downregulation results in a sharp decline of DHCR protein levels and co-ordinate transcriptional decreases of LBR and DHCR7 expression. Consistent with this, lipidomic analysis upon TMEM147 silencing identified changes in cellular cholesterol levels, cholesteryl ester levels and profile, and in cellular cholesterol uptake, raising the possibility that TMEM147 is an important new regulator of cholesterol homeostasis in cells.This article has an associated First Person interview with the first author of the paper.
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Membrana Nuclear , Receptores Citoplasmáticos y Nucleares , Colesterol , Células HeLa , Homeostasis , Humanos , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Membrana Nuclear/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptor de Lamina BRESUMEN
The ASAH1 gene encodes acid ceramidase (AC), an enzyme that is implicated in the metabolism of ceramide (Cer). Mutations in the ASAH1 gene cause two different disorders, Farber disease (FD), a rare lysosomal storage disorder, and a rare form of spinal muscular atrophy combined with progressive myoclonic epilepsy (SMA-PME). In the absence of human in vitro neuronal disease models and to gain mechanistic insights into pathological effects of ASAH1 deficiency, we established and characterized a stable ASAH1 knockdown (ASAH1KD) SH-SY5Y cell line. ASAH1KD cells displayed reduced proliferation due to elevated apoptosis and G1/S cell cycle arrest. Distribution of LAMP1-positive lysosomes towards the cell periphery and significantly shortened and less branched neurites upon differentiation, implicate AC for lysosome positioning and neuronal development, respectively. Lipidomic analysis revealed changes in the intracellular levels of distinct sphingolipid species, importantly without Cer accumulation, in line with altered gene transcription within the sphingolipid pathway. Additionally, the transcript levels for Rho GTPases (RhoA, Rac1, and Cdc42), which are key regulators of axonal orientation, neurite branching and lysosome positioning were found to be dysregulated. This study shows the critical role of AC in neurons and suggests how AC depletion leads to defects seen in neuropathology of SMA-PME and FD.
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Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Supervivencia Celular/fisiología , Neuritas/metabolismo , Esfingolípidos/metabolismo , Transcripción Genética , Apoptosis , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Ceramidas/metabolismo , Miopatías Distales/genética , Lipogranulomatosis de Farber/genética , Técnicas de Silenciamiento del Gen , Homeostasis , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Mutación , Mioclonía/congénito , Mioclonía/genética , Neuroblastoma/genética , ARN Mensajero/metabolismo , Transcriptoma , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The common IVSI-110 (G>A) ß-thalassemia mutation is a paradigm for intronic disease-causing mutations and their functional repair by non-homologous end joining-mediated disruption. Such mutation-specific repair by disruption of aberrant regulatory elements (DARE) is highly efficient, but to date, no systematic analysis has been performed to evaluate disease-causing mutations as therapeutic targets. Here, DARE was performed in highly characterized erythroid IVSI-110(G>A) transgenic cells and the disruption events were compared with published observations in primary CD34+ cells. DARE achieved the functional correction of ß-globin expression equally through the removal of causative mutations and through the removal of context sequences, with disruption events and the restriction of indel events close to the cut site closely resembling those seen in primary cells. Correlation of DNA-, RNA-, and protein-level findings then allowed the extrapolation of findings to other mutations by in silico analyses for potential repair based on the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, Cas12a, and transcription activator-like effector nuclease (TALEN) platforms. The high efficiency of DARE and unexpected freedom of target design render the approach potentially suitable for 14 known thalassemia mutations besides IVSI-110(G>A) and put it forward for several prominent mutations causing other inherited diseases. The application of DARE, therefore, has a wide scope for sustainable personalized advanced therapy medicinal product development for thalassemia and beyond.
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In this work, wild-type and heterozygous ß-thalassaemic mice were enriched with 57Fe via gastrointestinal absorption to characterize in greater detail the iron complexes then identifiable via Mössbauer spectroscopy. The 57Fe enrichment method was validated and Mössbauer spectra were obtained at 80 K from blood samples from wild-type and ß-thalassaemic mice at 1, 3, 6, and 9 months of age. As expected, the haemoglobin levels of the thalassaemic mice were lower than from normal mice, indicating anaemia. Furthermore, significant amounts of ferritin-like iron were observed in the thalassaemic mice samples, which decreased with mouse age, reflecting the pattern of reticulocyte count reduction reported in the literature.
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Isótopos de Hierro/metabolismo , Isótopos de Hierro/farmacología , Espectroscopía de Mossbauer , Talasemia beta/sangre , Talasemia beta/metabolismo , Animales , Absorción Intestinal , Ratones , Ratones Endogámicos C57BLRESUMEN
Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental disease-specific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead.