Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.Ministerio de Economía y competitividad and Fondo Europeo de Desarrollo Regional (SAF2015-63868-R (MINECO/FEDER) to N.G., and SAF2016-75988-R (MINECO/FEDER) to M.F.); Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (PGC2018-096132-BI00 (MICINN/FEDER) to N.G.); Universidad Autónoma de Madrid-Banco de Santander Inter-University Cooperation Grant with Latin América (CEAL-AL/2015-12 to N.G.); Red de Investigación de Centros de Enfermedades Tropicales (RICET RD12/0018/0004 to M.F.); and Comunidad de Madrid (S-2010/BMD-2332 to M.F.). CBMSO institutional grants from Fundación Ramón Areces and Banco de Santande

Hematopoietic stem cell gene therapy for the treatment of beta-hemoglobinopathies

β-hemoglobinopathies, including β-thalassemia and sickle cell disease (SCD), are autosomal recessive inherited disorders caused by various mutations in the β-globin gene. The most effective curative therapy involves allogenic hematopoietic stem cell transplantation (HSCT) from an immunologically-matched donor. However, this approach presents some limitations in terms of finding a suitable donor and transplantation related risks, such as a graft-versus-host disease (GvHD). In this thesis, we strongly emphasized that autologous HSCT in combination with gene therapy tools will develop novel treatments for β-hemoglobinopathies. A promising technique for gene editing has emerged in recent years based on the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology. This revolutionary tool enables to perform gene disruption, gene correction, and gene addition in a specific locus of interest with high reliability and efficiency. Through this technology, in our first study, we demonstrated that targeting KLF1 and BCL11A, which control the expression of γ-globin, as well as the promoter region of HBG1 and HBG2 (HBG1/2), resulted in upregulation of γ-globin gene expression and fetal hemoglobin (HbF). Furthermore, after a deep comparison between the three gene editing strategies, we confirmed that BCL11A approach, which is currently in clinical phase, is the safest gene therapy treatment for β-hemoglobinopathies. Nevertheless, HBG1/2 strategy also holds potential for clinical translation. Alternatively, since β-globin gene correction is also a feasible gene therapy approach, in our second study, we attained successfully gene addition in HSPCs by inserting a NheI-tag at the common aberrant splicing mutation point, HBB IVS1-110 by means of Cas9 mRNA and ssODN electroporation. In the last project, we thoroughly compared three lentiviral transgenes encoding for IGF2BP1, shRNA BCL11A, and ꝩ-globin to reactivate HbF production in HSPCs. Also, we assessed whether baboon envelope proteins (BaEV and BaEV-RLess) have a beneficial advantage over the regularly used vesicular-stomatitis-virus-G envelope protein (VSV-G). Our results showed 4 that all treatments using VSV-G envelope proteins resulted in therapeutic levels of HbF. In addition, baboon envelopes, especially BaEV-RLess, achieved decent levels of HbF with less viral particles, which might ameliorate the symptoms of the disease. Finally, even though IGF2BP1 and BCL11A approaches induced higher HbF levels than ꝩ-globin strategy, their role in gene regulation might cause undesired iatrogenic effects. Therefore, we considered ꝩ-globin the best lentiviral gene therapy strategy for the treatment of β-hemoglobinopathies. During this thesis we evidenced that both gene therapy tools, lentiviral gene transfer and genome editing, provide a successful platform for gene treatment of blood disorders. In addition to the current clinical trial approaches, we strongly believe that resurgence of HbF is the most straight forward strategy together with β-globin gene correction. However, due to the low occurrence of gene correction events in HSPCs, further investigation is required