THERAPEUTIC GENE EDITING FOR HEMOGLOBINOPATHIES Gene therapy for Hemoglobinopathies.
Main Article Content
Keywords
Gene editing; gene therapy; Hemoglobinopathies; Thalassemia; Sickle Cell Anemia.
Abstract
In the last ten years, a consistent number of clinical studies have evaluated different gene approaches for the treatment of patients with sickle cell disease (SCD) and transfusion-dependent b-thalassemia (TDT). Initial studies of gene therapy for hemoglobinopathies involved the use of lentiviral vectors to add functional copies of the gene encoding b-globin in defective CD34 cells; more recently, gene editing techniques have been used involving either CRISPR-Cas9, transcription activation-like effector protein nuclease, zinc finger nuclease, and base editing to either induce fetal hemoglobin production at therapeutic levels or to genetically repair the underlying molecular defect causing the disease.
Here, we review recent gene editing studies that have started the development of a new era in the treatment of hemoglobinopathies and, in general, monoallelic hereditary diseases.
Keywords: Gene editing; gene therapy; Hemoglobinopathies; Thalassemia; Sickle Cell Anemia.
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References
1. Masuda T, Wang X, Maeda M, Canver MC, Sher F, Funnell A, Fisher C, Suclu M, Martyn G, Norton LJ. Transcription factors LRF and BCL11A independently repress expression of fetal hemoglobin. Science 2016; 351: 285-289.
2. Lidonnici MR, Scaramuzza S, Ferrari G. Gene therapy for hemoglobinopathies. Gene Therapy 2023; 34: 793-807.
3. Locatelli F, Thompson AA, Kwiatowski JL, Porter JB, Thraser AJ, Hongeng S, Sauer MG, Thuret I, Lai A, Thraser AJ, et al. Betibeglogene autotemcel gene therapy for non-0 /0 genotype -thalassemia. N Engl J Med 2022; 386: 415-427.
4. Liu N, Hargreaves VV, Zhu Q, Kurland JV, Hong J, Kim W, Sher F, Marcias-Travino C, Rogers JM, Kurita R, et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 2018; 173: 430-442.
5. Huang P, Pesiak CA, Ren R, Khandros E, Qin K, Keller CA, Giardine B, Bell HW, Lan X, Sharma M, et al. HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription. Nat Genet 2022; 54: 1417-1426.
6. Huang P, Pesiak SA, Shehu V, Keller CA, Giardine B, Shi J, Hardison RC, Blobel GA, Khandros E. Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching. Blood 2024; 143: 1980-1991.
7. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin C, Shao Z, Canver MC, Smith EC, Pinello L, et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 2013; 342: 2453-257.
8. Smith EC, Luc S, Croney DM, Woodworth MB, Greig LC, Fujiwara Y, Nguyen M, Sher F, Macklis JD, Bauer DE, et al. Strict in vivo specificity of the Bcl11a erythroid enhancer. Blood 2016; 128: 2338-2342.
9. Psatha N, Reik A; Phelps S, Zhou Y, Dalas D, Yannaki E, Levasseur DN, Urnov FD, Holmes MC, Papayannopoulou T. Disruption of the BCL11A erythroid enhancer reactivates fetal hemoglobin in erythroid cells of patients with -thalassemia major. Mol Therapy Methods & Clin Dev 2018; 10: 313-320.
10. Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 2015; 527: 192-197.
11. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger R, et al. CRISPR-Casp gene editing for sickle cell disease and -thalassemia. N Engl J Med 2022; 384: 252-260.
12. Locatelli F, Lang P, Wall D, Meisel R, Carbacioglu S, Li AM, de la Fuente J, Shah AJ, Carpenter B, Kwiatkowski JL, et al. Examglogene autotemcel for transfusion-dependent -thalassemia. N Engl J Med 2024; 390: 1663-1676.
13. Fu B, Liao J, Chen S, Li W, Wang Q, Hu J, Yang F, Hsiao S, Jiang Y, Wang L, et al. CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric °/° transfusion-dependent -thalassemia. Nat Med 2022; 28: 1573-1580.
14. Zheng B, Liu R, Zhang X, Fu B, Xu Y, Shi J, Feng X, Wang L, Wang C, Liang R, et al. Efficacy and safety of Brl-101, CRISPR-Cas9-mediated gene editing of the BCL11A enhancer in transfusion-dependent -thalassemia. Blood 2023; 142 (suppl.1): 4995.
15. Frangoul H, Locatelli F, Bhatia M, Mapara M, Molinari L, Wall D, Liem RI, Telfer P, Shah AJ, Cavazzana M, et al. Exagamglogene autotemcel for severe sickle cell disease. N Engl J Med 2024; 390: 1649-1662.
16. Ye L, Wang J, Tan Y, Beyer AI, Xie F, Muench MO, Kan YW. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: an approach for treating sickle cell disease and -thalassemia. Proc Natl Acad Sci USA 2016; 113: 10661-10665.
17. Traxler EA, Yao Y, Wang YD, Woodard KJ, Kurita R, Nakamura Y, Hughes JR, Hardison RC, Blobel GA, Li C, et al. A genome-editing strategy to treat -hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 2016; 22: 987-990.
18. Métais JY, Doerfler PA, Mayuranathan T, Bauer DE, Fowler SC, Hsieh MM, Katta V, Kerwala S, Lazzarotto CR, Luk K, et al. Genome editing of HBG1 and HBG2 to induce fetal hemoglobin. Blood Adv 2019; 3: 3379-3392.
19. Sharma A, Boelens JJ, Cancio M, Hanking JS, Bhad P, Azizy M, Lewandowski A, Zhao X, Chitnis S, Peddinti R, et al. CRISPR-Cas9 editing of the HBBG1 and HBG2 promoters to treat sickle cell disease. N Engl J Med 2023; 389: 820-832.
20. Paul B, Montoya G. CRISPR-Cas12a: functional overview and applications. Biochem J 2020; 43: 8-17.
21. De Dreuzy E, Haeth J, Zuris JA, Sousa P, Viswanathan R, Scott S, Da Silva J, Ta T, Copehart S, Wang T, et al. EDIT-301: an experimental autologous cell therapy comprising Cas12a-RNP modified mPB-CD34+ cells for the potential treatment of SCD. Blood 2019; 134(suppl.1): 4636.
22. Hanna R, Frangoul H, McKinney C, Pineiro L, Mapara M, Chang KH, Jaskolka M, Kim K, Farrington DL, Wally M, et al. AsCas12a gene editing of HBG1/2 promoters with EDIT-301 results in rapid and sustained normalization of hemoglobin and increased fetal hemoglobin in patients with severe sickle cell disease and transfusion-dependent beta-thalassemia. Blood 2023; 142(suppl.1): 4996.
23. Esrick EB, Lehmann LE, Biffi A, Achebe M, Brendel C, Ciuculescu MF, 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 2021; 384: 205-215.
24. Spencer-Chapman M, Cull AH, Ciuculescu MF, Esrick EB, Mitchell E, Jung H, O’Neill L, Roberts K, Fabre MA, et al. Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease. Nat Med 2023; 29: 3175-3183.
25. Lee BC, Gin A, Wu C, Singh K, Grice M, Mortlock R, Abraham D, Fan X, Zhou Y, AIJanahi A, et al. Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSCPCs in rhesus macaques. Cell Stem Cell 2024; 31: 455-466.
26. Smith AR, Schiller GJ, Vercellotti GM, Kwiatowski JL, Krishnamurti L, Esrick EB, Williams DA, Miller WP, Woolfson A; Walters MC. Preliminary results of a phase 1 / 2 clinical study of zinc finger nuclease-mediated editing of BCL11A in autologous hematopoietic stem cells for transfusion-dependent beta thalassemia. Blood 2019; 134(suppl.1): 3544.
27. Alavi A, Krishnamurti L, Abedi M, Galeon I, Reiner D, Smith SE, Wang L, Ramezi A, Rendo P, Walters MC. Preliminary safety and efficacy results from Precizn-1: an ongoing phase 1 / 2 study on zinc finger nuclease-modified autologous CD34+ HSPCs for sickle cell disease (SCD). Blood 2021; 138(suppl.1): Blood 2021; 138(suppl.1): 2930.
28. Alavi A, Abedi M, Parikh S, Boismenu R, Chen M, Hsu BL, Cockroft BM, Galeon I, Rendo P, Walters MC. Inetrin safety and efficacy results from a phase 1 / 2 study of zinc finger nuclease-modified autologous hematopoietic stem cells for sickle cell disease (PRECIZN-1). Blood 2022; 140(suppl.1): 4907-4909.
29. Newby GA, Yen JS, Woodard KJ, Mayuranathan T, Lazzarotto CR, Li Y, Sheppard-Tillman H, Porter SN, Yao Y, Mayberry K, et al. Base editing of hematopoietic stem cells rescues sickle cell disease in mice. Nature 2021, 595: 295-302.
30. Mayuranathan T, Newby GA, Feng R, Yao Y, Mayberry KD, Lazzarotto CR, Li Y, Levine RM, Nimmagadda N, Dempsey E, et al. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet 2023; 55: 1210-1220.
31. Chu SH, Ortega M, Feliciano P, Winton V, Xu C, Haupt D, McDonald T, Martinez S, Liquori A, Marshall J, et al. Conversion of HbS to Nb G-Makassar by adenine base editing is compatible with normal hemoglobin function. Blood 2021; 138(suppl.1): 951-952.
32. Hardouin G, Anatoniou P, Martinucci P, Felix T, Manceau S, Joseph L, Masson C, Scaramuzza S, Ferrari G, Cavazzana M, et al. Adenine base editor-mediated correction of the common and severe IVS1-110 (G>A) -thalassemia mutation. Blood 2023; 141: 1169-1177.
33. Naiiseh B, Papasavva PL, Papaioannu NY, Tomazou M, Koniali L, Felekis X, Constantinou C, Sitarou M, Christou S, Kleanthous M, et al. Context base editing for splice correction of IVSI-110 -thalassemia. Molecular Therapy Nucleic Acids 2024; 35: 1-12.
34. Han W, Qiu HY, Sun S, Fu ZC, Wang GQ, Qian X, Wang L, Zhai X, Wei J, Wang Y, et al. Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs. Cell Stem Cell 2023; 30: 1624-1639.
35. Anzalone AW, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby JA, Raguram A, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019; 576: 149-157.
36. Everette KA, Newby GA, Levine RM, Meyberry K, Jang Y, Jang Y, Mayuranathan T, Nimmagadda N, Dempsey E, Li Y, et al. Ex vivo prime editing of patient hematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice. Nat Biomed Eng 2023; 7: 616-628.
37. Li C, Georgakopoulou A, Newby GA, Chen PJ, Everette KA, Paschoudi K, Viachaki E, Gil S, Anderson AK, Koob T, et al. In vivo HSC prime editing rescues sickle cell disease in a mouse model. Blood 2023; 141: 2085-2089.
2. Lidonnici MR, Scaramuzza S, Ferrari G. Gene therapy for hemoglobinopathies. Gene Therapy 2023; 34: 793-807.
3. Locatelli F, Thompson AA, Kwiatowski JL, Porter JB, Thraser AJ, Hongeng S, Sauer MG, Thuret I, Lai A, Thraser AJ, et al. Betibeglogene autotemcel gene therapy for non-0 /0 genotype -thalassemia. N Engl J Med 2022; 386: 415-427.
4. Liu N, Hargreaves VV, Zhu Q, Kurland JV, Hong J, Kim W, Sher F, Marcias-Travino C, Rogers JM, Kurita R, et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 2018; 173: 430-442.
5. Huang P, Pesiak CA, Ren R, Khandros E, Qin K, Keller CA, Giardine B, Bell HW, Lan X, Sharma M, et al. HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription. Nat Genet 2022; 54: 1417-1426.
6. Huang P, Pesiak SA, Shehu V, Keller CA, Giardine B, Shi J, Hardison RC, Blobel GA, Khandros E. Let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching. Blood 2024; 143: 1980-1991.
7. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin C, Shao Z, Canver MC, Smith EC, Pinello L, et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 2013; 342: 2453-257.
8. Smith EC, Luc S, Croney DM, Woodworth MB, Greig LC, Fujiwara Y, Nguyen M, Sher F, Macklis JD, Bauer DE, et al. Strict in vivo specificity of the Bcl11a erythroid enhancer. Blood 2016; 128: 2338-2342.
9. Psatha N, Reik A; Phelps S, Zhou Y, Dalas D, Yannaki E, Levasseur DN, Urnov FD, Holmes MC, Papayannopoulou T. Disruption of the BCL11A erythroid enhancer reactivates fetal hemoglobin in erythroid cells of patients with -thalassemia major. Mol Therapy Methods & Clin Dev 2018; 10: 313-320.
10. Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 2015; 527: 192-197.
11. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK, Foell J, de la Fuente J, Grupp S, Handgretinger R, et al. CRISPR-Casp gene editing for sickle cell disease and -thalassemia. N Engl J Med 2022; 384: 252-260.
12. Locatelli F, Lang P, Wall D, Meisel R, Carbacioglu S, Li AM, de la Fuente J, Shah AJ, Carpenter B, Kwiatkowski JL, et al. Examglogene autotemcel for transfusion-dependent -thalassemia. N Engl J Med 2024; 390: 1663-1676.
13. Fu B, Liao J, Chen S, Li W, Wang Q, Hu J, Yang F, Hsiao S, Jiang Y, Wang L, et al. CRISPR-Cas9-mediated gene editing of the BCL11A enhancer for pediatric °/° transfusion-dependent -thalassemia. Nat Med 2022; 28: 1573-1580.
14. Zheng B, Liu R, Zhang X, Fu B, Xu Y, Shi J, Feng X, Wang L, Wang C, Liang R, et al. Efficacy and safety of Brl-101, CRISPR-Cas9-mediated gene editing of the BCL11A enhancer in transfusion-dependent -thalassemia. Blood 2023; 142 (suppl.1): 4995.
15. Frangoul H, Locatelli F, Bhatia M, Mapara M, Molinari L, Wall D, Liem RI, Telfer P, Shah AJ, Cavazzana M, et al. Exagamglogene autotemcel for severe sickle cell disease. N Engl J Med 2024; 390: 1649-1662.
16. Ye L, Wang J, Tan Y, Beyer AI, Xie F, Muench MO, Kan YW. Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: an approach for treating sickle cell disease and -thalassemia. Proc Natl Acad Sci USA 2016; 113: 10661-10665.
17. Traxler EA, Yao Y, Wang YD, Woodard KJ, Kurita R, Nakamura Y, Hughes JR, Hardison RC, Blobel GA, Li C, et al. A genome-editing strategy to treat -hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 2016; 22: 987-990.
18. Métais JY, Doerfler PA, Mayuranathan T, Bauer DE, Fowler SC, Hsieh MM, Katta V, Kerwala S, Lazzarotto CR, Luk K, et al. Genome editing of HBG1 and HBG2 to induce fetal hemoglobin. Blood Adv 2019; 3: 3379-3392.
19. Sharma A, Boelens JJ, Cancio M, Hanking JS, Bhad P, Azizy M, Lewandowski A, Zhao X, Chitnis S, Peddinti R, et al. CRISPR-Cas9 editing of the HBBG1 and HBG2 promoters to treat sickle cell disease. N Engl J Med 2023; 389: 820-832.
20. Paul B, Montoya G. CRISPR-Cas12a: functional overview and applications. Biochem J 2020; 43: 8-17.
21. De Dreuzy E, Haeth J, Zuris JA, Sousa P, Viswanathan R, Scott S, Da Silva J, Ta T, Copehart S, Wang T, et al. EDIT-301: an experimental autologous cell therapy comprising Cas12a-RNP modified mPB-CD34+ cells for the potential treatment of SCD. Blood 2019; 134(suppl.1): 4636.
22. Hanna R, Frangoul H, McKinney C, Pineiro L, Mapara M, Chang KH, Jaskolka M, Kim K, Farrington DL, Wally M, et al. AsCas12a gene editing of HBG1/2 promoters with EDIT-301 results in rapid and sustained normalization of hemoglobin and increased fetal hemoglobin in patients with severe sickle cell disease and transfusion-dependent beta-thalassemia. Blood 2023; 142(suppl.1): 4996.
23. Esrick EB, Lehmann LE, Biffi A, Achebe M, Brendel C, Ciuculescu MF, 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 2021; 384: 205-215.
24. Spencer-Chapman M, Cull AH, Ciuculescu MF, Esrick EB, Mitchell E, Jung H, O’Neill L, Roberts K, Fabre MA, et al. Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease. Nat Med 2023; 29: 3175-3183.
25. Lee BC, Gin A, Wu C, Singh K, Grice M, Mortlock R, Abraham D, Fan X, Zhou Y, AIJanahi A, et al. Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSCPCs in rhesus macaques. Cell Stem Cell 2024; 31: 455-466.
26. Smith AR, Schiller GJ, Vercellotti GM, Kwiatowski JL, Krishnamurti L, Esrick EB, Williams DA, Miller WP, Woolfson A; Walters MC. Preliminary results of a phase 1 / 2 clinical study of zinc finger nuclease-mediated editing of BCL11A in autologous hematopoietic stem cells for transfusion-dependent beta thalassemia. Blood 2019; 134(suppl.1): 3544.
27. Alavi A, Krishnamurti L, Abedi M, Galeon I, Reiner D, Smith SE, Wang L, Ramezi A, Rendo P, Walters MC. Preliminary safety and efficacy results from Precizn-1: an ongoing phase 1 / 2 study on zinc finger nuclease-modified autologous CD34+ HSPCs for sickle cell disease (SCD). Blood 2021; 138(suppl.1): Blood 2021; 138(suppl.1): 2930.
28. Alavi A, Abedi M, Parikh S, Boismenu R, Chen M, Hsu BL, Cockroft BM, Galeon I, Rendo P, Walters MC. Inetrin safety and efficacy results from a phase 1 / 2 study of zinc finger nuclease-modified autologous hematopoietic stem cells for sickle cell disease (PRECIZN-1). Blood 2022; 140(suppl.1): 4907-4909.
29. Newby GA, Yen JS, Woodard KJ, Mayuranathan T, Lazzarotto CR, Li Y, Sheppard-Tillman H, Porter SN, Yao Y, Mayberry K, et al. Base editing of hematopoietic stem cells rescues sickle cell disease in mice. Nature 2021, 595: 295-302.
30. Mayuranathan T, Newby GA, Feng R, Yao Y, Mayberry KD, Lazzarotto CR, Li Y, Levine RM, Nimmagadda N, Dempsey E, et al. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet 2023; 55: 1210-1220.
31. Chu SH, Ortega M, Feliciano P, Winton V, Xu C, Haupt D, McDonald T, Martinez S, Liquori A, Marshall J, et al. Conversion of HbS to Nb G-Makassar by adenine base editing is compatible with normal hemoglobin function. Blood 2021; 138(suppl.1): 951-952.
32. Hardouin G, Anatoniou P, Martinucci P, Felix T, Manceau S, Joseph L, Masson C, Scaramuzza S, Ferrari G, Cavazzana M, et al. Adenine base editor-mediated correction of the common and severe IVS1-110 (G>A) -thalassemia mutation. Blood 2023; 141: 1169-1177.
33. Naiiseh B, Papasavva PL, Papaioannu NY, Tomazou M, Koniali L, Felekis X, Constantinou C, Sitarou M, Christou S, Kleanthous M, et al. Context base editing for splice correction of IVSI-110 -thalassemia. Molecular Therapy Nucleic Acids 2024; 35: 1-12.
34. Han W, Qiu HY, Sun S, Fu ZC, Wang GQ, Qian X, Wang L, Zhai X, Wei J, Wang Y, et al. Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs. Cell Stem Cell 2023; 30: 1624-1639.
35. Anzalone AW, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby JA, Raguram A, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019; 576: 149-157.
36. Everette KA, Newby GA, Levine RM, Meyberry K, Jang Y, Jang Y, Mayuranathan T, Nimmagadda N, Dempsey E, Li Y, et al. Ex vivo prime editing of patient hematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice. Nat Biomed Eng 2023; 7: 616-628.
37. Li C, Georgakopoulou A, Newby GA, Chen PJ, Everette KA, Paschoudi K, Viachaki E, Gil S, Anderson AK, Koob T, et al. In vivo HSC prime editing rescues sickle cell disease in a mouse model. Blood 2023; 141: 2085-2089.
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Aug 31, 2024
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Testa, U., Leone, G. and Cappellini, M. D. (2024) “THERAPEUTIC GENE EDITING FOR HEMOGLOBINOPATHIES: Gene therapy for Hemoglobinopathies”., Mediterranean Journal of Hematology and Infectious Diseases, 16(1), p. e2024068. doi: 10.4084/MJHID.2024.068.
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