241© Springer Science+Business Media LLC 2017 P. Malik, J. Tisdale (eds.), Gene and Cell Therapies for Beta- Globinopathies, Advances in Experimental Medicine and Biology 1013, DOI 10.1007/978-1-4939-7299-9

AAAV vectors, 205AB0 incompatibility, 98Acute anemic events (AAEs), 80Acute chest syndrome (ACS), 17, 78, 79Acute splenic sequestration crisis (ASSC), 18,

19, 74Adenoviral vectors, 223Adult, HSCT, 92Adult red blood cells, 180Adult β globin gene, 27Allelic HLA matching, 137Allogeneic gene therapy, 205Allogeneic hematopoietic stem cell

transplantation (allo-HSCT), 91–100, 105–113, 204, 205

beta-TM, 90blood transfusion and iron chelation, 91chronic transfusions, 91in developed countries, 90fertility preservation, 90myeloablative CR, 90non-myeloablative (see Non-myeloablative

HSCT)oral iron-chelators, 91risk factors, 90in SCA

barriers, 111–113costs, 110, 111geno-identical indications, 107–110myeloablative, 105, 106non-myeloablative, 106, 107

sibling cord blood cryopreservation, 90

in thalassemiaAB0 incompatibility, 98adults, 92children, 91, 92, 94, 95conditioning regimen, 92–96cost-effectiveness, 99, 100DLI, 98growth and pubertal anomalies, 98, 99GVHD prophylaxis, 96mixed chimerism, 97and spleen, 97stem cell source, 96–97

α-Globin genes, 178, 204Alpha and beta thalassemia, 180

clinical severity, 61, 62deletions, 61gene clusters, 60genotypes and classification, 60, 61

Alternative donors, 145, 147Anticancer drug, 182Antiproliferative agent, 184Antiprotozoan, 184Apicidin, 184, 185Avascular necrosis (AVN), 205-Azacytidine (5-Aza), 182, 186

BBacterial infection, 73β-Globin (HBB) gene, 2, 3, 5, 6, 8, 10, 28, 29,

159–161, 178, 211–213adult, 180allo-HSCT, 204, 205

Index

242

β-Globin (HBB) gene (cont.)codon 6 glutamic acid, 204de-repressing γ−globin, 209–211disorders, 183dysfunctional protein, 203engineered nucleases, 206–209gene correction, 211–213genetic analyses, 180genome editing, 205–206insufficient production, 204mutations, 203pathologic dysfunction, 204protein, 182RBCs, 204SCD, 204sickle trait, 204stiff polymers disrupt, 204transcription, 180

β-Globin locus control region (β-LCR), 28, 29β-Hemoglobinopathies, 35–38, 203, 211–213

de-repressing γ−globin, 209–211engineered nucleases, 206–209gene correction, 212, 213Genome editing, 205–206HBB gene (see β-Globin (HBB) gene)

βT87Q LentiGlobin vector, 164β-Thalassemia (BT). See also Allogeneic

hematopoietic stem cell transplantation (allo-HSCT)

chronic anemia, 6curative treatment, 204epidemiology, 5and gamma-globin gene, 156, 157gene mutations, 6gene therapy, 157history, 5HSC transplantation, 156and iron, 7ineffective erythropoiesis, 156non-pathologic heterozygous, 204palliative therapeutic options, 156pathophysiology, 6therapy, 186TI, 10TM (see β-Thalassemia major (TM))

β-Thalassemia intermedia (TI), 10β-Thalassemia major (TM), 90, 130–135

cardiac complications, 9donor transplantation, 141endocrine complications, 9hematological feature, 8hepatitis complications, 9haploidentical and mismatch

transplantation, 139–141

skeletal abnormalities, 8transplants, 145, 146umbilical cord blood transplantation

related, 130–134unrelated, 134, 135

β-Thalassemia minor, 10, 11β-Thalassemia trait, 204Blood disorders, 181Bone marrow transplantation (BMT), 182Bone mineral density, 65Butyrate response elements (BREs), 184

CCardiac pathology, 65CBT. See Cord blood transplantation (CBT)Cell banks, 230Cell-cycle specific agents, 183Center for International Blood and Marrow

Transplant Research (CIBMTR), 125Cerebral vasculopathy, 103, 106, 107,

109–111Children, HSCT, 91, 92Chimerism, 97Chromosomal abnormalities, 229Chromosomal deletion, 210Chronic transfusion therapy, 10, 81cis-regulatory DNA elements, 178Clinical-grade gene therapy vector, 157Clustered Regularly Interspaced Short

Palindromic Repeat (CRISPR)-Cas9 system, 226

Conventional plasmids, 223Cooley’s Anemia, 181Cooperative Study of Sickle Cell Disease

(CSSCD), 100Copy and paste mechanism, 205Copy number variants (CNVs), 229Cord blood transplantation (CBT)

allelic HLA matching, 137double transplant, 137ex vivo expansion, 137, 138supportive care and conditioning

regimen, 136Cre-excisable vectors, 223CSA. See Cyclosporine (CSA)CSSCD. See The Cooperative Study of Sickle

Cell Disease (CSSCD)Cyclosporine (CSA), 96

DDecitabine (5-aza-2’-deoxycytidine), 186Deletional forms of HPFH, 211

Index

243

Demethylating agents, 67Dimeric CRISPR nucleases, 208Disease-free survival (DFS), 96DLI. See Donor lymphocyte infusions (DLI)DNA-binding drugs, HbF, 187DNA-free RNA-based genetic methods,

223, 229DNMT inhibitors, HbF, 185–186Donor lymphocyte infusions (DLI), 98Double cord blood transplant, 137Double stand breaks (DSBs), 205, 206, 221

EEchocardiography, 75Electrocardiogram, 75Embryoid bodies (EBs), 231Embryonic-fetal-adult, 179–180Engineered nucleases, 205, 210

hybrid nuclease platforms, 208, 209meganucleases (homing endonucleases), 206RNA guided endonucleases

(CRISPR/Cas9), 208TALENs, 207, 208ZFNs, 206, 207

Erythroid cells, 179, 233Erythroid Krüppel-like factor (EKLF), 29, 188Erythroid lineage, 226Erythropoiesis, 187Erythropoietin, 68Excisable systems, 223

FFertility

ovarian cryopreservation, 99preservation, 90TRM, 107

Fetal hemoglobin (HbF), 178in adult hematopoietic cells, 178BCL11A, 39chemical inducers, 178, 183DNA-binding drugs, 187DNMT inhibitors, 185–186fetal γ-globin induction, 182–183globin gene clusters, 178, 179HBS1L-MYB, 40HDAC inhibitors, 184–185hemoglobinopathies, 181HU, 183–184KLF1, 39MAOI inhibitors, 186mTOR Inhibitors, 187MYB transcription factor, 40

production, 178regulation, 40switching, 179–180transcriptional regulation, γ-globin genes,

187–189variants, 181Xmn1-HBG2, 38β-globin gene (see β-Globin gene)

Fetal γ-globin induction, 182SCD and BT

BMT, 182characterization, 182clinical data, 182complications, 182epigenetic chromatin modifications, 182HPFH, 182HU, 182red cell membrane, 182

FokI Type II S restriction endonuclease (Fn), 206

F reticulocytes, 183

Gγ-Globin genes, 187–189

fetal, 182–183de-repressing

BCL11A, 210epidemiologic studies, 209HPFH genotypes, 210, 211Knocking Out BCL11A, 209, 210

transcriptional regulationBCL11A, 188, 189co-activators and co-repressors, 187EKLF, 188elucidation, molecular basis, 187erythropoiesis, 187GATA1, 188Ikaros, 188NF-E2, 188NF-E4, 188Sox6, 188TR2/TR4, 189

Gamma-retroviral/lentiviral vectors, 161, 221, 222

G-CSF. See Granulocyte colony-stimulating factor (G-CSF)

Gene addition strategy, 232G-CSF, 165 (see also Genome toxicity)HMGA2 overexpression, 165lentiviral vectors, 158–162 (see also Mixed

chimerism)non-globin genetic elements, 162–166oncoretroviral vectors, 157, 158

Index

244

Gene addition strategy (cont.)splenectomized and non-splenectomized

patients, 165therapeutic Hb-βT87Q LentiGlobin, 165trials, 165, 166βT87Q LentiGlobin vector, 164

Gene disruption, 226Gene dysregulation, 226Genetic engineering, 230Genetic modifiers, 41–47

β-thalassemiaα-globin genotype, 46β chain deficiency, 45clinical studies, 44complications, 47HBB, 45iron homeostasis, 47non-α globin chain imbalance, 47QTLs, 45, 46

SCDα-thalassemia, 43HbF, 41, 42HbS polymerization and sickling, 42HbSS, 41qualitative and quantitative, 41sub-phenotypes and complications,

42–44Genetics, β-thalassemia

causes, 34deletions, 33, 34mutations, 30, 31non-deletion, 30, 31RNA processing, 32, 33transcriptional mutations, 31–33

Genome editingHBB gene, 205–206HgbF, 209, 210HPFH genotypes, 210, 211

Genome toxicityAAVS1, 169HSVtk and iCasp9, 169insulators, 168iPSCs, 169non-integrating episomal technology, 169random transgene integration, 169safe harbor sites, 169SB system, 168self-inactivating design, 168vector-transduced cells, 169zinc finger nucleases, 169

Genome wide association studies (GWAS), 29, 209

Genotypes, 36, 37Gilbert’s syndrome, 47

Globin gene clusters, HbF, 178, 179Glutamine 87, 209Good manufacturing practice (GMP), 230Graft vs. host disease (GvHD), 205Granulocyte colony-stimulating factor

(G-CSF), 165

HHaploidentical and mismatch related

transplantation, 138–143description, 138SCD patients, 138–140TM patients, 139–143

Hb Gower-1 (ζ2ε2), 179Hb Gower-2 (α2ε2), 179Hb Portland-1 (ζ2γ2), 179Hb Portland-2 (ζ2β2), 180HbF quantitative trait loci (QTLs), 45HbS mutation, 12HDAC inhibitors, 184, 185

HbFapicidin, 184, 185butyrate, 184chromatin structure, 184histone acetylation, 184pomalidomide, 185scriptaid, 185synthesis, 184thalidomide, 185transcriptional regulation, gene

expression, 184TSA, 185

Hematopoietic stem cell (HSC), 156, 159, 161, 162, 165, 212, 213, 220, 231

Hematopoietic system, 205, 220, 231, 232Hemoglobin (Hb) disorders. See also

Haploidentical and mismatch related transplantation. See also Matched unrelated donor transplantation. See also Umbilical cord blood transplantation

beta-globin gene, 156non-thalassemic controls, 160qualitative, 5quantitative, 4structure and function, 2–4and transfusion independence, 167types, 4

Hemoglobin beta pseudogene 1 (HBBP1), 178Hemoglobin E (α2βE2), 181Hemoglobin H (β4), 181Hemoglobin S (α2βS2), 181Hemoglobin switching, HbF, 179–180

Index

245

Hemoglobinopathies, 62, 224βmajor form, 181βminor form, 181blood disorders, 181hemoglobin C (α2βC2), 181hemoglobin E (α2βE2), 181hemoglobin H (β4), 181hemoglobin S (α2βS2), 181milder forms, 181RBCs lyse, 181sickle cell disease, 181

Hereditary persistence of fetal hemoglobin (HPFH), 182, 210, 211

Histone deacetylase (HDAC) inhibitors, 67Homologous recombination (HR), 205, 206HR-mediated gene repair, 232HSCT. See Allogeneic hematopoietic stem cell

transplantation (allo-HSCT)HSPC-based gene therapy

BT and SCDadvantages, 220blood diseases, 220ex vivo, 221hematopoietic system, 220protocols and standardized practices, 220quality control, 221target cells, 221transplantation, 220viral vectors, 221

HU. See Hydroxyurea (HU) therapyHuman embryonic stem cells (hESCs), 222Human immunodeficiency virus 1 (HIV-1), 221Human leukocyte antigen (HLA), 230Human pluripotent stem cells (hPSCs), 231

characteristics, 222features, 222

Human ε-globin, 179Hybrid nuclease platforms, 208, 209Hydrops fetalis, 180Hydroxyurea (HU) therapy, 68, 80–82, 103,

182–184Hypogonadotropic hypogonadism (HH), 99

IIkaros, 188Induced pluripotent cells (iPSCs)

allo-HSCT, 212pre-implantation embryos, 211

Induced pluripotent stem cells (iPSCs), 211, 224–228

advantages, 221allo-HSCT, 212β-hemoglobinopathies, 211

blood and HSC-like cells, 211characteristics, 223derive transgene-free mouse/human, 223disadvantages, 223DNA-free methods, 223excisable systems, 223gene and cell therapy

advantages, 226autologous, 224BT, 224, 226cell culture conditions, 226cell types, 224donor DNA design, 227endogenous genes, 226engineering, site-specific

endonucleases, 226erythroid lineage, 226gene delivery methods, 226gene targeting, 226genetic correction, 225–228hemoglobinopathies, 224in situ genetic correction in human

cells, 226multiplicity of infection, 225restoration of expression, 228safe harbor criteria, 225SCD, 224, 226

gene correction, 211, 212generation, 222genetic manipulation, 222genetic modification, 222pluripotency characteristics, 222quality control, 221, 222reprogramming process, 211, 222, 223somatic HSCs, 212transcription factors, 211

Intrafemoral injection, 231iPSC-based cell therapy, 229, 230, 233, 234Iron chelation therapy, 68, 69Iron chelators, 69Iron monitoring, 63Iron overload, 7, 9IVS2–745 pre-mRNA splicing, 164

KKnocking Out BCL11A, γ−Globin, 209, 210

LLentiviral vector, 221, 225

AnkT9W expression, 162beta-globin vectors, 159–161Cooley’s anemia model, 159

Index

246

Lentiviral vector (cont.)cPPT/CTS element, 159gamma-globin vectors, 161gene therapy, 159genetic elements, 160GLOBE vector, 161HbF synthesis, 162HIV-1, 158human beta-thalassemic cells, 160lentiviruses, 158, 159SAD and BERK model, 159vector silencing, 160

Lentivirus. See Lentiviral vectorsLiver iron concentration (LIC), 64Locus control regions (LCRs), 178–180

MMammalian hematopoiesis, 187Mammalian target of rapamycin (mTOR)

inhibitors, 187Matched sibling donor (MSD), 92Matched unrelated donor (MUD), 125, 126, 141Meganucleases (homing endonucleases), 206MegaTALs, 208mESCs, 226Mild anemia, 181Milder forms, 181Minicircles, 223Misshapen blood cells, 181Mitogen-activated protein (MAP) kinase

signaling, 184–185Mixed chimerism

description, 166dogs and non-human primates, 168hepato- and splenomegaly, 167human multidrug resistance 1 (MDR1)

gene, 167iron chelation, 167mutated MGMT gene, 167myeloablation, 166reduced-intensity conditioning regimen, 167researchers, 168resistant genes, 168TNS9 trial, vector-transduced cells, 167

Monoallelic correction, 233Monoamine oxidases inhibitors (MAOI), 186MSD. See Matched sibling donor (MSD)MUD. See Matched unrelated donor (MUD)Multiplicity of infection (MOI), 225Murine leukemia viruses (MLVs), 221Myeloablative chemotherapy, 204Myeloablative HSCT

arterial velocities, 106

ATG dose, 105erythroblasts, 105PBCs, 105prenatal/pre-implantation diagnosis, 106preventive measures, 106SCA patients, 101, 102, 105transplantation risks, 105

NNational Marrow Donor Program (NMDP), 126Non-deletional forms, HPFH, 211Non-homologous end-joining (NHEJ), 205Non-integrating DNA vectors, 223Non-myeloablative HSCT

calcineurin inhibitors, 107and immunosuppression, 107reduced-intensity conditioning, 107RIC matched/related, 106–108

OOncoretroviral vectors

beta-globin expression, 158LCR, 158oncoretroviruses, 157therapeutic levels of expression, 158

Ophthalmologic and auditory screening, 65oriP/EBNA1 episomes, 223

PPGD. See Preimplantation genetic diagnosis

(PGD)PH. See Pulmonary hypertension (PH)Pomalidomide, 185Pre-implantation diagnosis

and sibling cord blood cryopreservation, 106double selection HLA-disease, 112

Pre-implantation embryos, 211Preimplantation genetic diagnosis (PGD), 112premature stop codons (PTCs), 33Priapism, 80Pulmonary hypertension (PH), 18, 104

RReactivation of HbF. See Fetal hemoglobin (HbF)Red blood cells (RBCs), 179, 204, 233Reduced intensity conditioning (RIC)

regimens, 96Related umbilical cord blood transplantation

SCD, 127–130TM, 130–134

Index

247

Repeat variable diresidue (RVD), 207, 208Reprogrammed cells, 211, 222, 224Reprogramming method, iPSC line, 223,

224, 229Retroviral vector, 221RIC. See Reduced intensity conditioning

(RIC) regimensRight heart catheterization (RHC), 18RNA Guided Endonucleases

(CRISPR/Cas9), 208RNA-mediated delivery, 223

SSCA. See Sickle cell anemia (SCA)Scriptaid, 185Sendai viruses, 223, 224Sibling cord blood cryopreservation, 90Sicke cell anemia (SCA), 204. See also

Allogeneic hematopoietic stem cell transplantation (allo-HSCT)

cerebral vasculopathy, 103cognitive functioning and quality of life, 104CSSCD, 100gene therapy, 157hemoglobin S (HbS) formation, 156hereditary hemoglobinopathies, 90high-risk complications, 100HU therapy, 103inherited blood disorders, 156morbidity and early death risks, 100periodic blood transfusions, 156PH, 104polymerized HbS, 100recessive genetic disorder, 100TCD, 103TRJV, 104TWITCH, 103

Sickle cell disease (SCD), 78–80, 127–130, 181–183, 204

acute splenic sequestration, 74and BTfetal γ-globin induction, 182–183AVN, 20bacterial infection, 73blood counts and serum, 75cardiac complications, 17cerebrovascular disease, 16classification, 14clinical phenotype, 14complications management

AAEs, 80ACS, 78, 79acute painful episodes, 78

priapism, 80stroke, 79

curative treatment, 204educational attainment, 77electrocardiogram, 75epidemiology, 12genetics, 35genotype, 72growth and development, 74HbS, 2haploidentical and mismatch

transplantation, 138, 139HBB gene, 204health maintenance and screening, 76, 77hematologic parameters, 15hepatobiliary, 19history, 11humanized mouse model, 224infectious complication, 16MRI and SCI, 77mouse model, 212outcomes, 83pain, 15pathophysiology, 14, 72, 73potential therapy, 182pulmonary complications, 17, 18renal complications, 19spleen, 18, 19stroke prevention, 74TCD ultrasonography, 75transplants, 145, 146treatment, 20umbilical cord blood transplantation

related, 127–130unrelated, 130

vaso-occlusive, 15Sickle cell unrelated transplant (SCURT)

trial, 141Sickle hemoglobin (HbS), 2Sickle trait, 204Silent cerebral infarction (SCI), 16, 75–77Single nucleotide polymorphisms (SNPs), 39Single-nucleotide variants (SNVs), 229Site-specific endonucleases, 221Sleeping beauty transposase (SB) system, 168Somatic HSCs

advantages, 213developmental and regulatory, 212frequency of gene correction, 212, 213non-sickle RBC and RBC progenitors, 212SCD, 213single engineered nuclease, 213TALENs and CRISPR/Cas9 nucleases, 213transplantation, 212

Index

248

Spleen and HSCT, 97Splenectomy, 70Standard care and screening guidelines

bone mineral density, 65cardiac pathology, 65endocrine studies, 63growth abnormalities, 63infection, 65intermedia and major, thalassemia, 64iron monitoring, 63ophthalmologic and auditory screening, 65

Strokescerebral ischemic lesions, 109cerebral vasculopathy, 107high-risk complications, SCA, 100macrovasculopathy, 103microvasculopathy, 104prevention, 74

TTAL effector code, 208TAL effector nucleases (TALENs),

207, 208TCD. See Transcranial Doppler

ultrasonography (TCD)Teratogenicity, 182Tetrameric hemoglobin molecule, 179Thalassemia

alpha and beta, 60, 61characterization, 60diagnosis, 63HbF, 66hereditary hemoglobinopathies, 90origins of, 90outcomes, 70, 71preventive care (see Standard care and

screening guidelines)transfusion therapy, 66

Thalidomide, 185TR2/TR4, 189Transcranial Doppler ultrasonography (TCD),

11, 75, 103Transcription activator-like effector nucleases

(TALENs), 163, 226Transcriptional regulation, γ-globin genes,

187–189Transfusion therapy, 66Transplant-related mortality (TRM), 90Tranylcypromine (TCP), 186

Trichostatin A (TSA), 185Tricuspid regurgitant velocity (TRV), 75Tricuspid valve regurgitant jet velocity

(TRJV), 104

UUmbilical cord blood transplantation,

127–135CB grafts, 127immunological differences, 126malignant and nonmalignant hematologic

diseases, 126SCD

patients with, 127–129related, 127–130unrelated, 130

TMpatients with, 131–133related, 130–134unrelated, 134, 135

Universal donor iPSC lines, 230Unrelated umbilical cord blood transplantation

SCD, 130TM, 134, 135

VValproic acid, 185Vaso-occlusive crisis (VOC), 15, 78Vaso-occlusive event (VOE), 15Viral vectors, 221

WWhole-genome sequencing, 230

XXenograft models, 232

YYolk-sac derived hematopoietic progenitors,

232

ZZinc finger nucleases (ZFNs), 163, 206,

207, 233

Index