Context base editing for splice correction of IVSI-110 ß-thalassemia.

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.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of ß-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

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.

High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.

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.

Effect of HBB genotype on survival in a cohort of transfusion-dependent thalassemia patients in Cyprus.

Initiation of regular transfusion in transfusion-dependent thalassemia (TDT) is based on the assessment of clinical phenotype. Pathogenic HBB variants causing ß-thalassemia are important determinants of phenotype and could be used to aid decision making. We investigated the association of HBB genotype with survival in a cohort study in the four thalassemia centres in Cyprus. HBB genotype was classified as severe (ß0/ß0 or ß+/ß0), moderate (ß+/ß+), or mild (ß0/ß++ or ß+/ß++). Risk factors for mortality were evaluated using multivariate Cox proportional-hazards regression. 537 subjects were followed for a total of 20,963 person years. 80.4% (95% CI 76.4-84.7) of individuals survived to 50 years of age with increasing rates of liver, infection and malignancy-related deaths observed during recent follow-up. We evaluated non-modifiable risk factors and found worse outcomes associated with male sex (Hazard ratio 1.9, 95% CI 1.1-3.0, p=0.01) and milder genotype (Hazard ratio 1.6, 95% CI 1.1-2.3, p=0.02). The effect of genotype was confirmed in a second model, which included treatment effects. Patients with a milder genotype initiated transfusion significantly later and had reduced blood requirements compared to those with moderate or severe genotypes, although pre-transfusion hemoglobin levels did not differ between genotypes. Our results suggest that early treatment decisions to delay transfusion and different long-term treatment strategies in milder genotypes have led to adverse long-term effects of under-treated thalassemia and worse survival. We propose that HBB genotype determination and use of this information to aid in decision making can improve long-term outcomes of thalassaemia patients.

A Concise Review on the Frequency, Major Risk Factors and Surveillance of Hepatocellular Carcinoma (HCC) in ß-Thalassemias: Past, Present and Future Perspectives and the ICET-A Experience.

Due to the recent alarming increase in the incidence of hepatocellular carcinoma (HCC) in thalassemias, the present report reviews briefly the frequency, the major risk factors, and the surveillance of HCC in ß-thalassemias. Over the past 33 years, 153 cases of HCC were reported in patients with thalassemia, mainly in Italy and Greece. Among HCV-infected patients, additional factors promoting the development of HCC included: advanced age, male sex, chronic hepatitis B (CHB) co-infection, and iron overload. For early diagnosis of HCC, sequential ultrasound screening is recommended especially for thalassemia patients with chronic hepatitis C (CHC), which coincides with (one or more) additional risk factors for HCC. Here we report also the preliminary data from thalassemic patients, above the age of 30 years, followed in 13 ICET-A centers. The total number of enrolled patients was 1,327 (males: 624 and 703 females). The prevalence of HCC in thalassemia major patients [characterized by transfusion-dependency (TDT)] and thalassemia intermedia [characterized by nontransfusion dependency (NTDT)] was 1.66 % and 1.96 %, respectively. The lowest age at diagnosis of HCC was 36 years for TDT and 47 years for NTDT patients. We hope that this review can be used to develop more refined and prospective analyses of HCC magnitude and risk in patients with thalassemia and to define specific international guidelines to support clinicians for early diagnosis and treatment of HCC in thalassemic patients.

An ICET-A survey on occult and emerging endocrine complications in patients with ß-thalassemia major: Conclusions and recommendations.

In adult thalassemia major (TM) patients, a number of occult and emerging endocrine complications, such as: central hypothyroidism (CH), thyroid cancer, latent hypocortisolism, and growth hormone deficiency (GHD) have emerged and been reported. As the early detection of these complications is essential for appropriate treatment and follow-up, the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescent Medicine (ICET-A) promoted a survey on these complications in adult TM patients, among physicians (pediatricians, hematologists and endocrinologists) caring for TM patients in different countries. The data reported by 15 countries are presented.The commonest endocrine complications registered in 3.114 TM adults are CH and GHD (4.6 % and 3.0 %, respectively), followed by latent hypocortisolism (1.2%). In 13 patients (0.41%) a cytological papillary or follicular thyroid carcinoma was diagnosed in 11 and 2 patients, respectively, and a lobectomy or thyroidectomy was carried out. Of 202 TM patients below the age of 18 years, the  reported endocrine complications were: GHD in 4.5%, latent hypocortisolism in 4.4% and central hypothyrodisim in 0.5%. Transition phase was an area of interest for many clinicians, especially as patients with complex chronic health conditions are responding to new treatments extending their lifespan beyond imagination.. In conclusion, our survey provides a better understanding of  physicians' current clinical practices and beliefs in the detection, prevention and treatment of some endocrine complications prevailing in adult TM patients. Regular surveillance, early diagnosis, treatment and follow-up in a multi-disciplinary specialized setting are recommended.

First study on iron complexes in blood and organ samples from thalassaemic and normal laboratory mice using Mössbauer spectroscopy.

Measurements of iron complexes and iron stores in the body are crucial for evaluation and management of chelation therapy targeted against iron accumulation or overload in blood and organs. In this work, blood and tissue samples from one normal and one thalassaemic laboratory mouse were studied using 57Fe Mössbauer spectroscopy at 78 K for the first time. In contrast to human patients, these laboratory mice did not receive any medical treatment, thus the iron components present in the samples are not altered from their natural state. The Mössbauer spectra of blood, liver and spleen samples of the thalassaemic mouse were found to differ in shape and iron content compared with corresponding spectra of the normal mouse. These results demonstrate a basis for further exploitation of the thalassaemic mouse model to study thalassaemia and its treatment in more detail using Mössbauer spectroscopy.