Advertisement

Under the hood: The molecular biology driving gene therapy for the treatment of sickle cell disease

  • Evan Waldron
    Affiliations
    Department of Pathology and Cell Biology, Columbia University Irving Medical Center, 622W. 168th Street, New York, NY 10032, United States
    Search for articles by this author
  • Yvette C. Tanhehco
    Correspondence
    Corresponding author at: Department of Pathology and Cell Biology, Division of Transfusion Medicine, Columbia University Irving Medical Center, 622W 168th Street Harkness Pavilion, 4-418A, New York, NY 10032, United States.
    Affiliations
    Department of Pathology and Cell Biology, Columbia University Irving Medical Center, 622W. 168th Street, New York, NY 10032, United States
    Search for articles by this author

      Highlights

      • Lentiviral vectors are used for gene therapy for sickle cell disease.
      • Modified β-globin, βT87Q, prevents HbS polymerization.
      • CRISPR ribonucleoprotein is used to modify patient genomes to increase expression of hemoglobin F.
      • BCL11a expression can be suppressed with RNAi to increase expression of hemolgobin F.

      Abstract

      Gene therapy will soon become the dominant modality for treating of sickle cell disease (SCD). Currently, three technologies are the most promising: expression of transgenic globin genes via a lentiviral vector, controlled mutation of the β-globin control cluster by transgenic CRISPR-based ribonucleoprotein, and suppression of BCL11a mRNA by shRNA. In this review, we discuss the mechanism of each technology and how they correct the SCD pathology at the molecular level. We conclude by discussing potential directions future therapy may take.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Transfusion and Apheresis Science
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Tanhehco Y.C.
        Gene therapy for hemoglobinopathies.
        Transfus Apher Sci. 2021; 60103061
        • Kavanagh P.L.
        • Fasipe T.A.
        • Wun T.
        Sickle cell disease: a review.
        JAMA. 2022; 328: 57-68
        • Piel F.B.
        • Steinberg M.H.
        • Rees D.C.
        Sickle cell disease.
        N Engl J Med. 2017; 376: 1561-1573
        • Rees D.C.
        • Williams T.N.
        • Gladwin M.T.
        Sickle-cell disease.
        Lancet. 2010; 376: 2018-2031
      1. Weatherall D., Akinyanju O., Fucharoen S., Olivieri N., Musgrove P. Inherited Disorders of Hemoglobin. In: nd, Jamison D.T., Breman J.G., Measham A.R., Alleyne G., Claeson M., et al., editors. Disease Control Priorities in Developing Countries. Washington (DC) 2006.

        • Bihoreau M.T.
        • Baudin V.
        • Marden M.
        • Lacaze N.
        • Bohn B.
        • Kister J.
        • et al.
        Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.
        Protein Sci. 1992; 1: 145-150
        • Eaton W.A.
        Hemoglobin S polymerization and sickle cell disease: a retrospective on the occasion of the 70th anniversary of Pauling's Science paper.
        Am J Hematol. 2020; 95: 205-211
        • Cretegny I.
        • Edelstein S.J.
        Double strand packing in hemoglobin S fibers.
        J Mol Biol. 1993; 230: 733-738
        • Carragher B.
        • Bluemke D.A.
        • Gabriel B.
        • Potel M.J.
        • Josephs R.
        Structural analysis of polymers of sickle cell hemoglobin. I. Sickle hemoglobin fibers.
        J Mol Biol. 1988; 199: 315-331
        • Wishner B.C.
        • Ward K.B.
        • Lattman E.E.
        • Love W.E.
        Crystal structure of sickle-cell deoxyhemoglobin at 5 A resolution.
        J Mol Biol. 1975; 98: 179-194
        • Dykes G.
        • Crepeau R.H.
        • Edelstein S.J.
        Three-dimensional reconstruction of the fibres of sickle cell haemoglobin.
        Nature. 1978; 272: 506-510
        • Dykes G.W.
        • Crepeau R.H.
        • Edelstein S.J.
        Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S.
        J Mol Biol. 1979; 130: 451-472
        • Pauling L.
        • Itano H.A.
        • et al.
        Sickle cell anemia a molecular disease.
        Science. 1949; 110: 543-548
        • Schlenz A.M.
        • Phillips S.M.
        • Mueller M.
        • Melvin C.L.
        • Adams R.J.
        • Kanter J.
        Barriers and facilitators to chronic red cell transfusion therapy in pediatric sickle cell anemia.
        J Pedia Hematol Oncol Nurs. 2022; 39: 209-220
        • Porter J.B.
        • Shah F.T.
        Iron overload in thalassemia and related conditions: therapeutic goals and assessment of response to chelation therapies.
        Hematol Oncol Clin North Am. 2010; 24: 1109-1130
        • Yazdanbakhsh K.
        • Ware R.E.
        • Noizat-Pirenne F.
        Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management.
        Blood. 2012; 120: 528-537
        • Nickel R.S.
        • Hendrickson J.E.
        • Fasano R.M.
        • Meyer E.K.
        • Winkler A.M.
        • Yee M.M.
        • et al.
        Impact of red blood cell alloimmunization on sickle cell disease mortality: a case series.
        Transfusion. 2016; 56: 107-114
        • Canver M.C.
        • Orkin S.H.
        Customizing the genome as therapy for the beta-hemoglobinopathies.
        Blood. 2016; 127: 2536-2545
        • Kutlar A.
        • Kanter J.
        • Liles D.K.
        • Alvarez O.A.
        • Cancado R.D.
        • Friedrisch J.R.
        • et al.
        Effect of crizanlizumab on pain crises in subgroups of patients with sickle cell disease: a SUSTAIN study analysis.
        Am J Hematol. 2019; 94: 55-61
        • Ataga K.I.
        • Kutlar A.
        • Kanter J.
        • Liles D.
        • Cancado R.
        • Friedrisch J.
        • et al.
        Crizanlizumab for the prevention of pain crises in sickle cell disease.
        N Engl J Med. 2017; 376: 429-439
        • Niihara Y.
        • Miller S.T.
        • Kanter J.
        • Lanzkron S.
        • Smith W.R.
        • Hsu L.L.
        • et al.
        A phase 3 trial of l-glutamine in sickle cell disease.
        N Engl J Med. 2018; 379: 226-235
        • Vichinsky E.
        • Hoppe C.C.
        • Ataga K.I.
        • Ware R.E.
        • Nduba V.
        • El-Beshlawy A.
        • et al.
        A phase 3 randomized trial of voxelotor in sickle cell disease.
        N Engl J Med. 2019; 381: 509-519
        • Walters M.C.
        • De Castro L.M.
        • Sullivan K.M.
        • Krishnamurti L.
        • Kamani N.
        • Bredeson C.
        • et al.
        Indications and results of HLA-identical sibling hematopoietic cell transplantation for sickle cell disease.
        Biol Blood Marrow Transpl. 2016; 22: 207-211
        • Orkin S.H.
        • Bauer D.E.
        Emerging genetic therapy for sickle cell disease.
        Annu Rev Med. 2019; 70: 257-271
        • Milone M.C.
        • O'Doherty U.
        Clinical use of lentiviral vectors.
        Leukemia. 2018; 32: 1529-1541
        • Hughes S.H.
        Reverse transcription of retroviruses and LTR retrotransposons.
        Microbiol Spectr. 2015; (3:MDNA3-0027-2014)
        • Miller M.D.
        • Wang B.
        • Bushman F.D.
        Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro.
        J Virol. 1995; 69: 3938-3944
        • Demeulemeester J.
        • De Rijck J.
        • Gijsbers R.
        • Debyser Z.
        Retroviral integration: site matters: mechanisms and consequences of retroviral integration site selection.
        Bioessays. 2015; 37: 1202-1214
        • Hacein-Bey-Abina S.
        • von Kalle C.
        • Schmidt M.
        • Le Deist F.
        • Wulffraat N.
        • McIntyre E.
        • et al.
        A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.
        N Engl J Med. 2003; 348: 255-256
        • Dull T.
        • Zufferey R.
        • Kelly M.
        • Mandel R.J.
        • Nguyen M.
        • Trono D.
        • et al.
        A third-generation lentivirus vector with a conditional packaging system.
        J Virol. 1998; 72: 8463-8471
        • Laetsch T.W.
        • Maude S.L.
        • Milone M.C.
        • Davis K.L.
        • Krueger J.
        • Cardenas A.M.
        • et al.
        False-positive results with select HIV-1 NAT methods following lentivirus-based tisagenlecleucel therapy.
        Blood. 2018; 131: 2596-2598
        • Kanter J.
        • Walters M.C.
        • Krishnamurti L.
        • Mapara M.Y.
        • Kwiatkowski J.L.
        • Rifkin-Zenenberg S.
        • et al.
        Biologic and clinical efficacy of lentiglobin for sickle cell disease.
        N Engl J Med. 2022; 386: 617-628
        • Pawliuk R.
        • Westerman K.A.
        • Fabry M.E.
        • Payen E.
        • Tighe R.
        • Bouhassira E.E.
        • et al.
        Correction of sickle cell disease in transgenic mouse models by gene therapy.
        Science. 2001; 294: 2368-2371
        • Cavazzana-Calvo M.
        • Payen E.
        • Negre O.
        • Wang G.
        • Hehir K.
        • Fusil F.
        • et al.
        Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia.
        Nature. 2010; 467: 318-322
        • Sankaran V.G.
        • Orkin S.H.
        The switch from fetal to adult hemoglobin.
        Cold Spring Harb Perspect Med. 2013; 3a011643
        • Sankaran V.G.
        • Menne T.F.
        • Xu J.
        • Akie T.E.
        • Lettre G.
        • Van Handel B.
        • et al.
        Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
        Science. 2008; 322: 1839-1842
        • Barrangou R.
        • Fremaux C.
        • Deveau H.
        • Richards M.
        • Boyaval P.
        • Moineau S.
        • et al.
        CRISPR provides acquired resistance against viruses in prokaryotes.
        Science. 2007; 315: 1709-1712
        • Lander E.S.
        The heroes of CRISPR.
        Cell. 2016; 164: 18-28
        • Jiang F.
        • Doudna J.A.
        CRISPR-Cas9 structures and mechanisms.
        Annu Rev Biophys. 2017; 46: 505-529
        • Makarova K.S.
        • Wolf Y.I.
        • Alkhnbashi O.S.
        • Costa F.
        • Shah S.A.
        • Saunders S.J.
        • et al.
        An updated evolutionary classification of CRISPR-Cas systems.
        Nat Rev Microbiol. 2015; 13: 722-736
        • Wang J.Y.
        • Pausch P.
        • Doudna J.A.
        Structural biology of CRISPR-Cas immunity and genome editing enzymes.
        Nat Rev Microbiol. 2022;
        • Doudna J.A.
        • Charpentier E.
        Genome editing. The new frontier of genome engineering with CRISPR-Cas9.
        Science. 2014; 3461258096
        • Jinek M.
        • Chylinski K.
        • Fonfara I.
        • Hauer M.
        • Doudna J.A.
        • Charpentier E.
        A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
        Science. 2012; 337: 816-821
        • Dong D.
        • Ren K.
        • Qiu X.
        • Zheng J.
        • Guo M.
        • Guan X.
        • et al.
        The crystal structure of Cpf1 in complex with CRISPR RNA.
        Nature. 2016; 532: 522-526
        • Yamano T.
        • Nishimasu H.
        • Zetsche B.
        • Hirano H.
        • Slaymaker I.M.
        • Li Y.
        • et al.
        Crystal structure of Cpf1 in complex with guide RNA and target DNA.
        Cell. 2016; 165: 949-962
        • Gasiunas G.
        • Barrangou R.
        • Horvath P.
        • Siksnys V.
        Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.
        Proc Natl Acad Sci USA. 2012; 109: E2579-E2586
        • Jiang F.
        • Doudna J.A.
        The structural biology of CRISPR-Cas systems.
        Curr Opin Struct Biol. 2015; 30: 100-111
        • Jiang F.
        • Zhou K.
        • Ma L.
        • Gressel S.
        • Doudna J.A.
        Structural biology. A Cas9-guide RNA complex preorganized for target DNA recognition.
        Science. 2015; 348: 1477-1481
        • Nishimasu H.
        • Ran F.A.
        • Hsu P.D.
        • Konermann S.
        • Shehata S.I.
        • Dohmae N.
        • et al.
        Crystal structure of Cas9 in complex with guide RNA and target DNA.
        Cell. 2014; 156: 935-949
        • Cong L.
        • Ran F.A.
        • Cox D.
        • Lin S.
        • Barretto R.
        • Habib N.
        • et al.
        Multiplex genome engineering using CRISPR/Cas systems.
        Science. 2013; 339: 819-823
        • Mali P.
        • Yang L.
        • Esvelt K.M.
        • Aach J.
        • Guell M.
        • DiCarlo J.E.
        • et al.
        RNA-guided human genome engineering via Cas9.
        Science. 2013; 339: 823-826
        • LaFountaine J.S.
        • Fathe K.
        • Smyth H.D.
        Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9.
        Int J Pharm. 2015; 494: 180-194
        • Roth D.B.
        • Wilson J.H.
        Relative rates of homologous and nonhomologous recombination in transfected DNA.
        Proc Natl Acad Sci USA. 1985; 82: 3355-3359
        • Deriano L.
        • Roth D.B.
        Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage.
        Annu Rev Genet. 2013; 47: 433-455
        • Frangoul H.
        • Altshuler D.
        • Cappellini M.D.
        • Chen Y.S.
        • Domm J.
        • Eustace B.K.
        • et al.
        CRISPR-Cas9 gene editing for sickle cell disease and beta-thalassemia.
        N Engl J Med. 2021; 384: 252-260
        • Metais J.Y.
        • Doerfler P.A.
        • Mayuranathan T.
        • Bauer D.E.
        • Fowler S.C.
        • Hsieh M.M.
        • et al.
        Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.
        Blood Adv. 2019; 3: 3379-3392
        • Traxler E.A.
        • Yao Y.
        • Wang Y.D.
        • Woodard K.J.
        • Kurita R.
        • Nakamura Y.
        • et al.
        A genome-editing strategy to treat beta-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition.
        Nat Med. 2016; 22: 987-990
        • Tuschl T.
        • Zamore P.D.
        • Lehmann R.
        • Bartel D.P.
        • Sharp P.A.
        Targeted mRNA degradation by double-stranded RNA in vitro.
        Genes Dev. 1999; 13: 3191-3197
        • Hammond S.M.
        • Bernstein E.
        • Beach D.
        • Hannon G.J.
        An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.
        Nature. 2000; 404: 293-296
        • Caplen N.J.
        • Parrish S.
        • Imani F.
        • Fire A.
        • Morgan R.A.
        Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
        Proc Natl Acad Sci U S A. 2001; 98: 9742-9747
        • Elbashir S.M.
        • Harborth J.
        • Lendeckel W.
        • Yalcin A.
        • Weber K.
        • Tuschl T.
        Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
        Nature. 2001; 411: 494-498
        • Lee R.C.
        • Feinbaum R.L.
        • Ambros V.
        The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.
        Cell. 1993; 75: 843-854
        • Bofill-De Ros X.
        • Gu S.
        Guidelines for the optimal design of miRNA-based shRNAs.
        Methods. 2016; 103: 157-166
        • Fire A.
        • Xu S.
        • Montgomery M.K.
        • Kostas S.A.
        • Driver S.E.
        • Mello C.C.
        Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
        Nature. 1998; 391: 806-811
        • Rao D.D.
        • Vorhies J.S.
        • Senzer N.
        • Nemunaitis J.
        siRNA vs. shRNA: similarities and differences.
        Adv Drug Deliv Rev. 2009; 61: 746-759
        • Grimm D.
        • Wang L.
        • Lee J.S.
        • Schurmann N.
        • Gu S.
        • Borner K.
        • et al.
        Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.
        J Clin Invest. 2010; 120: 3106-3119
        • Bethune J.
        • Artus-Revel C.G.
        • Filipowicz W.
        Kinetic analysis reveals successive steps leading to miRNA-mediated silencing in mammalian cells.
        EMBO Rep. 2012; 13: 716-723
        • Eichhorn S.W.
        • Guo H.
        • McGeary S.E.
        • Rodriguez-Mias R.A.
        • Shin C.
        • Baek D.
        • et al.
        mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues.
        Mol Cell. 2014; 56: 104-115
        • Denli A.M.
        • Tops B.B.
        • Plasterk R.H.
        • Ketting R.F.
        • Hannon G.J.
        Processing of primary microRNAs by the Microprocessor complex.
        Nature. 2004; 432: 231-235
        • Gregory R.I.
        • Yan K.P.
        • Amuthan G.
        • Chendrimada T.
        • Doratotaj B.
        • Cooch N.
        • et al.
        The microprocessor complex mediates the genesis of microRNAs.
        Nature. 2004; 432: 235-240
        • Bourlioux P.
        • Botto H.
        • Karam D.
        • Amgar A.
        • Camey M.
        Inhibition of bacterial adherence by nitroxoline on cellular adhesion and on urinary catheter surfaces.
        Pathol Biol (Paris). 1989; 37: 451-454
        • Bernstein E.
        • Caudy A.A.
        • Hammond S.M.
        • Hannon G.J.
        Role for a bidentate ribonuclease in the initiation step of RNA interference.
        Nature. 2001; 409: 363-366
        • Liu J.
        • Carmell M.A.
        • Rivas F.V.
        • Marsden C.G.
        • Thomson J.M.
        • Song J.J.
        • et al.
        Argonaute2 is the catalytic engine of mammalian RNAi.
        Science. 2004; 305: 1437-1441
        • Esrick E.B.
        • Lehmann L.E.
        • Biffi A.
        • Achebe M.
        • Brendel C.
        • Ciuculescu M.F.
        • et al.
        Post-transcriptional genetic silencing of BCL11A to treat sickle cell disease.
        N Engl J Med. 2021; 384: 205-215
        • Li 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. 2021; 29: 822-837
        • Cornetta K.
        • Duffy L.
        • Turtle C.J.
        • Jensen M.
        • Forman S.
        • Binder-Scholl G.
        • et al.
        Absence of Replication-competent Lentivirus In The Clinic: Analysis Of Infused T cell products.
        Mol Ther. 2018; 26: 280-288