A “Shot in the Arm” for Sickle Cell Disease
Authors: Tim M. Townes
Journal: Molecular Therapy (2021)

In this issue of Molecular Therapy, Li et al.1Li C. Wang H. Georgakopoulou A. Gil S. Yannaki E. Lieber A. In vivo HSC gene therapy using a bi-modular HDAd5/35++ vector cures sickle cell disease in a mouse model.Mol. Ther. 2020; 29 (this issue): 645-657Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar introduce a clever in vivo gene therapy approach that could be more broadly adapted for the correction of a number of hereditary blood disorders. After mobilization of bone marrow HSPCs (hematopoietic stem and progenitor cells), a helper-dependent adenoviral vector that targets HSPCs is introduced intravenously. The vector contains a therapeutic gene, which is engineered to insert itself into the stem cell genome by the action of sleeping beauty transposase, and a cassette encoding a gRNA (guide RNA) and Cas9, which disrupt specific gene regulatory sequences to activate endogenous γ-globin genes in adult erythroid cells (see Figure 1). The authors convincingly demonstrate that this combination of independent activities cures a humanized mouse model of severe sickle cell disease (SCD). SCD is characterized by hemoglobin sickle (HbS) tetramers (α2βS2) that polymerize at low oxygen concentrations. These polymers convert normally pliable red blood cells into fragile, sickle-shaped structures that occlude small vessels and that lyse and release a host of products causing extensive tissue and organ damage. Several gene addition and gene editing approaches have recently demonstrated significant amelioration of the disease in phase I/II clinical trials.2Townes T.M. Cavassana M. Gene Therapy for Sickle Cell Disease.in: Gladwin M. Kato G. Novelli E. Sickle Cell Disease. McGraw-Hill, 2021Google Scholar, 3Frangoul H. Altshuler D. Cappellini M.D. Chen Y.S. Domm J. Eustace B.K. Foell J. de la Fuente J. Grupp S. Handgretinger R. et al.CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.N. Engl. J. Med. 2020; (Published online December 5, 2020)https://doi.org/10.1056/NEJMoa2031054Crossref PubMed Scopus (175) Google Scholar, 4Esrick E.B. Lehmann L.E. Biffi A. Achebe M. Brendel C. Ciuculescu M.F. Daley H. MacKinnon B. Morris E. Federico A. et al.Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease.N. Engl. J. Med. 2020; (Published online December 5, 2020)https://doi.org/10.1056/NEJMoa2029392Crossref PubMed Scopus (55) Google Scholar These are outstanding achievements, yet all involve ex vivo approaches. HSPCs must be isolated from the patient, modified in culture, and infused back into the patient. These approaches require the isolation and manipulation of large numbers of cells and the ablation of remaining endogenous HSPCs before the modified cells can be reinfused. As an alternate approach, Li et. al.1Li C. Wang H. Georgakopoulou A. Gil S. Yannaki E. Lieber A. In vivo HSC gene therapy using a bi-modular HDAd5/35++ vector cures sickle cell disease in a mouse model.Mol. Ther. 2020; 29 (this issue): 645-657Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar developed an in vivo gene therapy that involves mobilization of HSPCs from the bone marrow into the peripheral blood stream followed by intravenous injection of a helper-dependent adenovirus (HDAd) vector designated HDAd5/35++. The HDAd5/35++ virus binds to CD46 on the surface of human HSPCs but does not bind to mouse HSPCs. Therefore, the authors bred CD46 transgenic mice with a humanized mouse model of severe SCD to obtain a model to test their in vivo gene therapy approach. After mobilization of HSPCs with granulocyte colony-stimulating factor (GCSF) and AMD3100, an immunostimulant also known as Plerixaflor, HDAd5/35++ containing two transgene cassettes was administered by intravenous injection. Cassette 1 contains a human γ-globin gene driven by a β-globin gene promoter and a powerful locus control region (LCR); cassette 2 encodes a gRNA and Cas9 nuclease that target the BCL11A repressor binding site in the “endogenous” human γ-globin gene promoter of knockin mice. Neither cassette alone provides sufficient γ-globin gene expression to correct the sickle phenotype, but the combination of cassettes results in fetal hemoglobin (HbF, α2γ2) levels of 30%, which cures the disease. This result is consistent with the phenotypes of individuals who are homozygous for the sickle gene (SS) but also express high levels of HbF (hereditary persistence of HbF [HPFH]) and are therefore asymptomatic. The dramatic correction of the sickle phenotype is an exciting result; however, there are some caveats. The intravenous injection of high-titer adenoviral vectors has been problematic in clinical trials in the past because of innate immune responses and because many individuals in the human population have antibodies at least to human adenovirus serotypes.5Somanathan S. Calcedo R. Wilson J.M. Adenovirus-antibody complexes contributed to lethal systemic inflammation in a gene therapy trial.Mol. Ther. 2020; 28: 784-793Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Also, transposase-mediated integration of the transgene into the genome is random. The in vivo exposure of large numbers of endothelial cells and many other cell types to random transgene integration exacerbates the problems associated with non-specific genetic alterations. Finally, wild-type Cas9 and many of the variants of this protein have extremely high enzymatic activities that are optimal for protecting bacteria from phage infection but may not provide optimal safety for editing the human genome and may also be targeted by immune responses. Nevertheless, Li et al.1Li C. Wang H. Georgakopoulou A. Gil S. Yannaki E. Lieber A. In vivo HSC gene therapy using a bi-modular HDAd5/35++ vector cures sickle cell disease in a mouse model.Mol. Ther. 2020; 29 (this issue): 645-657Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar are cognizant of these issues and suggest improvements that could mitigate them. The devastating nature of SCD requires the development of multiple therapeutic approaches to provide the best chance of a cure. Fortunately, many scientists worldwide are working diligently on SCD, and these efforts provide hope to millions of patients and their families. In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse ModelLi et al.Molecular TherapySeptember 5, 2020In BriefIn vivo hematopoietic stem cell transduction with HDAd5/35++ vectors containing modules for SB100x-mediated γ-globin gene addition and CRISPR-Cas9-triggered reactivation of γ-globin conferred γ-globin levels that were curative in a humanized mouse model of sickle cell disease. Full-Text PDF