Granted Patent
B2
US 12,606,848 · App. 18/355,575 · Granted Apr 21, 2026
Globin gene therapy for treating hemoglobinopathies
Assignee:
MEMORIAL SLOAN-KETTERING CANCER CENTER
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Abstract
The presently disclosed subject matter provides for expression cassettes that allow for expression of a globin gene or a functional portion thereof, vectors comprising thereof, and cells transduced with such expression cassettes and vectors. The presently disclosed subject matter further provides methods for treating a hemoglobinopathy in a subject comprising administering an effective amount of such transduced cells to the subject.
Claims (27)
1 . A method of treating a hemoglobinopathy in a subject, comprising administering an effective amount of an autologous cell transduced with an expression cassette to the subject, wherein the expression cassette comprises:
a) a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of between 600 bp and 900 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50; or
b) a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of between 600 bp and 900 bp does not comprise the nucleotide sequence set forth in SEQ ID NO:50.
2 . The method of claim 1 , wherein the β-globin LCR comprises:
a) a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6;
b) a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35;
c) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6;
d) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35;
e) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6; or
f) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
3 . The method of claim 1 , wherein the expression cassette, further comprising at least one erythroid-specific enhancer.
4 . The method of claim 3 , wherein the at least one erythroid-specific enhancer is positioned within the β-globin LCR.
5 . The method of claim 3 , wherein the at least one erythroid-specific enhancer is positioned between the HS1 region and the HS3 region of the β-globin LCR.
6 . The method of claim 3 , wherein the at least one erythroid-specific enhancer consists of the nucleotide sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
7 . The method of claim 1 , wherein the expression cassette further comprising at least one insulator that comprises the CTCF binding site sequence set forth in SEQ ID NO:18.
8 . The method of claim 7 , wherein the at least one insulator consists of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, or a fragment thereof.
9 . The method of claim 1 , wherein the β-globin gene is a wild-type human β-globin gene or a non-wild-type human β-globin gene.
10 . The method of claim 9 , wherein non-wild-type human β-globin gene is selected from a human β-globin gene comprising one or more deletions of intron sequences, a human β-globin gene encoding at least one anti-sickling amino acid residue, and a human β-globin gene comprising one or more deletions of intron sequences and encoding at least one anti-sickling amino acid residue.
11 . The method of claim 10 , wherein the non-wild-type human β-globin gene encoding at least one anti-sickling amino acid residue is selected from the group consisting of a human β A -globin gene encoding a threonine to glutamine mutation at codon 87 (β A-T87Q ), a human β A -globin gene encoding a glutamic acid to alanine mutation at codon 22 (β A-E22A ), a human β A -globin gene encoding an asparagine to lysine mutation at codon 80 (B −N80K ), a human β A -globin gene encoding a glutamic acid to alanine mutation at codon 22 and an asparagine to lysine mutation at codon 80, and a human β A -globin gene encoding a glutamic acid to alanine mutation at codon 22 and a threonine to glutamine mutation at codon 87.
12 . The method of claim 10 , wherein the one or more deletions of intron sequences comprise a deletion in intron 2.
13 . The method of claim 1 , wherein the expression cassette further comprises a β-globin promoter.
14 . The method of claim 13 , wherein said β-globin promoter is positioned between the globin gene or functional portion thereof and the β-globin LCR.
15 . The method of claim 14 , wherein said β-globin promoter is a human β-globin promoter that is about 265 bp in length, and/or consists of the nucleotide sequence set forth in SEQ ID NO:11.
16 . The method of claim 1 , wherein the expression cassette further comprises a human β-globin 3′ enhancer.
17 . The method of claim 16 , wherein said human β-globin 3′ enhancer is positioned in the upstream of the globin gene or functional portion thereof.
18 . The method of claim 16 , wherein said human β-globin 3′ enhancer is about 880 bp in length and/or consists of the nucleotide sequence set forth in SEQ ID NO:12.
19 . The method of claim 1 , wherein the cell is selected from the group consisting of a hematopoietic stem cell, an embryonic stem cell, and an induced pluripotent stem cell, optionally wherein the hematopoietic stem cell is a CD34+ hematopoietic stem cell.
Continuity (4)
Continuation
PCTUS2018064256
· Dec 6, 2018
Provisional Application
62595277
· Dec 6, 2017
References Cited (248)
US 4873192A
· Kunkel
· 1989
[cited by applicant]
US 5399346A
· Anderson et al.
· 1995
[cited by applicant]
US 5399363A
· Liversidge et al.
· 1995
[cited by applicant]
US 5543158A
· Gref et al.
· 1996
[cited by applicant]
US 5641515A
· Ramtoola
· 1997
[cited by applicant]
US 5725871A
· Illum
· 1998
[cited by applicant]
US 5756353A
· Debs
· 1998
[cited by applicant]
US 5780045A
· McQuinn et al.
· 1998
[cited by applicant]
US 5804212A
· Illum
· 1998
[cited by applicant]
US 6797494B1
· Antoniou et al.
· 2004
[cited by applicant]
US 7541179B2
· Sadelain et al.
· 2009
[cited by applicant]
US 20090156534A1
· Lisowski et al.
· 2009
[cited by applicant]
WO WO2015138852A1
· 2015
[cited by applicant]
WO WO2016037138A1
· 2016
[cited by applicant]
Petrus-Reurer et al. Commuication Biology 4:798 pp. 1-16 (Year: 2021).
[cited by examiner]
Yao et al. Molecular Therapy 32, pp. 103-123, 2024 (Year: 2024).
[cited by examiner]
Lange et al. Cell Mol Life Sci., 78:4143-4160 (Year: 2021).
[cited by examiner]
U.S. Appl. No. 16/890,436 (US 2020/0291433), filed Jun. 2, 2020 (Sep. 17, 2020).
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Jul. 20, 2023 Issue Fee Payment.
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Apr. 20, 2023 Notice of Allowance.
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Mar. 9, 2023 Response to Non-Final Rejection.
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Dec. 9, 2022 Non-Final Rejection.
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Oct. 7, 2022 Response to Restriction Requirement.
[cited by applicant]
U.S. Appl. No. 16/890,436, filed Jun. 30, 2022 Restriction Requirement.
[cited by applicant]
Adams et al., “Binding of Disparate Transcriptional Activators to Nucleosomal DNA Is Inherently Cooperative,” Molecular and Cellular Biology 15(3):1405-1421 (1995).
[cited by applicant]
Aker et al., “Extended Core Sequences from the cHS4 Insulator Are Necessary for Protecting Retroviral Vectors from Silencing Position Effects,” Hum Gene Ther 18:333-343 (2007).
[cited by applicant]
Anderson, “Prospects for Human Gene Therapy,” Science 226(4673):401-409 (1984).
[cited by applicant]
Antoniou et al., “The human β-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers,” EMBO J., 7(2):377-384 (1988).
[cited by applicant]
Armstrong et al., “NF-E2 Disrupts Chromatin Structure at Human β-Globin Locus Control Region Hypersensitive Site 2 In Vitro,” Mol. Cell. Biol. 16(10):5634-5644 (1996).
[cited by applicant]
Arumugam et al., “Genotoxic Potential of Lineage-specific Lentivirus Vectors Carrying the β-Globin Locus Control Region,” Mol Ther 17(11):1929-1937 (2009).
[cited by applicant]
Arumugam et al., “Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element,” Mol Ther 15(10):1863-1871 (2007).
[cited by applicant]
Atweh et al., “Pharmacological Induction of Fetal Hemoglobin in Sickle Cell Disease and β-Thalassemia,” Semin Hematol 38:367-373 (2001).
[cited by applicant]
Bank et al., “A Phase I/II Clinical Trial of β-Globin Gene Therapy for β-Thalassemia,” Ann N.Y. Acad. Sci. 1054:308-316 (2005).
[cited by applicant]
Barski et al., “High-Resolution Profiling of Histone Methylations in the Human Genome,” Cell 129:823-837 (2007).
[cited by applicant]
Baum et al., “Mutagenesis and Oncogenesis by Chromosomal Insertion of Gene Transfer Vectors,” Hum Gene Ther 17:253-263 (2006).
[cited by applicant]
Belfort et al., “Homing endonucleases: keeping the house in order,” Nucleic Acids Res., 25(17):3379-3388 (1997).
[cited by applicant]
Bell et al., “The Protein CTCF Is Required for the Enhancer Blocking Activity of Vertebrate Insulators,” Cell 98:387-396 (1999).
[cited by applicant]
Benton et al., “Screening lambdagt recombinant clones by hybridization to single plaques in situ,” Science, 196(4286):180-182 (1977).
[cited by applicant]
Blomer et al., Highly Efficient and Sustained Gene Transfer in Adult Neurons J Virol 6641-9, 1997.
[cited by applicant]
Borgna-Pignatti et al., “Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine,” Haematologica 89:1187-1193 (2004).
[cited by applicant]
Boulad et al., “Bone Marrow Transplantation for Homozygous β-Thalassemia. The Memorial Sloan-Kettering Cancer Center Experience,” Ann NY Acad Sci 850:498-502 (1998).
[cited by applicant]
Braun et al., “Gene Therapy for Wiskott-Aldrich Syndrome-Long-Term Efficacy and Genotoxicity,” Sci Transl Med 6:227ra33 (2014).
[cited by applicant]
Bregni, et al., “Human Peripheral Blood Hematopoietic Progenitors Are Optimal Targets of Retroviral-Mediated Gene Transfer,” Blood 80(6):1418-1422 (1992).
[cited by applicant]
Brigham et al., “Rapid Communication: In vivo Transfection of Murine Lungs with a Functioning Prokaryotic Gene Using a Liposome Vehicle,” Am. J. Med. Sci. 298(4):278-281 (1989).
[cited by applicant]
Brownell et al., “Special HATs for special occasions: linking histone acetylation to chromatin assembly and gene activation,” Curr Opin Genet Dev 6:176-184 (1996).
[cited by applicant]
Bulger et al., “Conservation of sequence and structure flanking the mouse and human β-globin loci: The β-globin genes are embedded within an array of odorant receptor genes,” PNAS 1999, 5129-34.
[cited by applicant]
Bungert et al., “Hypersensitive Site 2 Specifies a Unique Function within the Human β-Globin Locus Control Region To Stimulate Globin Gene Transcription,” Mol. and Cell Biol. 19(4):3062-3072 (1999).
[cited by applicant]
Burgess-Beusse et al., “The insulation of genes from external enhancers and silencing chromatin,” PNAS USA 99(Suppl 4):16433-16437 (2002).
[cited by applicant]
Caterina et al., “Multiple elements in human β-globin locus control region 5′ HS 2 are involved in enhancer activity and position-independent, transgene expression,” Nucleic Acids Res. 22(6):1006-1011 (1994).
[cited by applicant]
Cavazzana-Calvo et al., “Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia,” Nature 467:318-322 (2010).
[cited by applicant]
Cayouette et al., “Adenovirus-Mediated Gene Transfer of Ciliary Neurotrophic Factor Can Prevent Photoreceptor Degeneration in the Retinal Degeneration (rd) Mouse,” Human Gene Therapy 8:423-430 (1997).
[cited by applicant]
Chang et al., “Correction of the sickle cell mutation in embryonic stem cells,” Proc Natl Acad Sci 103(4):1036-1040 (2006).
[cited by applicant]
Chang et al., “Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development,” Stem Cell Rev and Rep 9:397-407 (2013).
[cited by applicant]
Chang et al., “Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1,” Gene Ther 12:1133-1144 (2005).
[cited by applicant]
Chang et al., “The Genetic Engineering of Hematopoietic Stem Cells: the Rise of Lentiviral Vectors, the Conundrum of the LTR, and the Promise of Lineage-Restricted Vectors,” Mol Ther 15(3):445-456 (2007).
[cited by applicant]
Charache et al., “Hydroxyurea: Effects on Hemoglobin F Production in Patients With Sickle Cell Anemia,” Blood 79(10):2555-2565 (1992).
[cited by applicant]
Chung et al. “A 5′ Element of the Chicken β-Globin Domain Serves as an Insulator in Human Erythroid Cells and Protects against Position Effect in
[cited by applicant]
Chung et al., “Characterization of the chicken β-globin insulator,” PNAS USA 94:575-580 (1997).
[cited by applicant]
Collis et al., “Definition of the minimal requirements within the human β-globin gene and the dominant control region for high level expression,” The EMBO Journal 9(1):233-240 (1990).
[cited by applicant]
Cooley, T.B. & Lee, P., “A Series of Cases of Splenomegaly in Children with Anemia and Peculiar Bone Changes,” Trans. Am. Pediatr. Soc. 37:29 (1925).
[cited by applicant]
Cornetta et al., “Gene Transfer into Primates and Prospects for Gene Therapy in Humans,” Nucleic Acid Research and Molecular Biology 36:311-322 (1987).
[cited by applicant]
Danos, et al., “Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges,” Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988).
[cited by applicant]
Dayhoff et al., “A Model of Evolutionary Change in Proteins,” Atlas of Protein Sequence and Structure, pp. 345-352 (1978).
[cited by applicant]
Dickson et al., “VEZF1 Elements Mediate Protection from DNA Methylation,” PLoS Genet 6:e1000804 (2010).
[cited by applicant]
Dorschner et al., “High-throughput localization of functional elements by quantitative chromatin profiling,” Nat Methods 1(3):219-225 (2004).
[cited by applicant]
Dull et al., “A Third-Generation Lentivirus Vector with a Conditional Packaging System,” Journal of Virology 72(11):8463-8471 (1998).
[cited by applicant]
Eglitis et al., “Retroviral Vectors for Introduction of Genes into Mammalian Cells,” BioTechniques 6(7):608-614 (1988).
[cited by applicant]
Elgin, “DNAase I-Hypersensitive Sites of Chromatin,” Cell 27:413-415 (1981).
[cited by applicant]
Elgin, S.C., “Molecular Biology. Anatomy of hypersensitive sites,” Nature 309:213-214 (1984).
[cited by applicant]
Ellis et al., “A Dominant chromatin-opening activity in 5′ hypersensitive site 3 of the human β-globin locus control region,” The EMBO Journal 15(3):562-568 (1996).
[cited by applicant]
Elnitski et al., “Conserved E Boxes Function as Part of the Enhancer in Hypersensitive Site 2 of the β-Globin Locus Control Region,” The Journal of Biological Chemistry 272(1):369-378 (1997).
[cited by applicant]
Emery et al., “A chromatin insulator protects retrovirus vectors from chromosomal position effects,” PNAS USA 97(16):9150-9155 (2000).
[cited by applicant]
Emery et al., “Development of a Condensed Locus Control Region Cassette and Testing in Retrovirus Vectors for Aγ-Globin,” Blood Cells, Molecules, and Diseases 24(16):322-339 (1998).
[cited by applicant]
Emery et al., “Development of virus vectors for gene therapy of β chain hemoglobinopathies: flanking with a chromatin insulator reduces γ-globin gene silencing in vivo,” Blood 100:2012-2019 (2002).
[cited by applicant]
Emery, “The Use of Chromatin Insulators to Improve the Expression and Safety of Integrating Gene Transfer Vectors,” Hum Gene Ther 22:761-774 (2011).
[cited by applicant]
Evans-Galea et al., “Suppression of Clonal Dominance in Cultured Human Lymphoid Cells by Addition of the cHS4 Insulator to a Lentiviral Vector,” Mol Ther 15(4):801-809 (2007).
[cited by applicant]
Felgner et al., “Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure,” Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987).
[cited by applicant]
Felsenfeld et al., “Chromatin structure and gene expression,” PNAS USA 93:9384-9388 (1996).
[cited by applicant]
Felsenfeld et al., “Controlling the double helix,” Nature 421:448-453 (2003).
[cited by applicant]
Felsenfeld, “Chromatin as an essential part of the transcriptional mechanism,” Nature 355:219-224 (1992).
[cited by applicant]
Follenzi et al., “Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences,” Nature Genetics 25:217-222 (2000).
[cited by applicant]
Fraser et al., “Each hypersensitive site of the human β-globin locus control region confers a different developmental pattern of expression on the globin genes,” Genes & Development 7:106-113 (1993).
[cited by applicant]
Friedmann, “Progress Toward Human Gene Therapy,” Science 244(4910):1275-1281 (1989).
[cited by applicant]
Gaszner et al., “Insulators: exploiting transcriptional and epigenetic mechanisms,” Nat Rev Genet 7:703-713 (2006).
[cited by applicant]
Giardina et al., “Chelation Therapy in β-Thalassemia: An Optimistic Update,” Semin Hematol 38:360-366 (2001).
[cited by applicant]
Giardini et al., “Bone marrow transplantation in the treatment of thalassemia,” Current Opinion in Hematology 1:170-176 (1994).
[cited by applicant]
Giles et al., “Chromatin Boundaries, Insulators, and Long-Range Interactions in the Nucleus,” Cold Spring Harbor Symposia on Quantitative Biology 75:79-85 (2010).
[cited by applicant]
Gross et al., “Nuclease Hypersensitive Sites in Chromatin,” Ann Rev Biochem 57:159-197 (1988).
[cited by applicant]
Grosveld et al., “Position-Independent, High-Level Expression of the Human β-Globin Gene in Transgenic Mice,” Cell 51:975-985 (1987).
[cited by applicant]
Grunstein et al., “Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene,” Proc. Natl. Acad. Sci. USA 72(10):3961-3965 (1975).
[cited by applicant]
Hanawa et al., “Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken β-Globin Locus HS4 Insulator Element,” Mol Ther 17(4):667-674 (2009).
[cited by applicant]
Hanna et al., Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin, Science 318:1920-1923 (2007).
[cited by applicant]
Hardison et al., “Locus control regions of mammalian β-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights,” Gene 205:73-94 (1997).
[cited by applicant]
Hino et al., “Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure,” Gene Ther 11:819-828 (2004).
[cited by applicant]
Horak et al., “GATA-1 binding sites mapped in the β-globin locus by using mammalian chIp-chip analysis,” PNAS 99(5):2924-2929 (2002).
[cited by applicant]
Hug et al., “Analysis of Mice Containing a Targeted Deletion of β-Globin Locus Control Region 5′ Hypersensitive Site 3,” Mol. and Cell Biol. 16(6):2906-2912 (1996).
[cited by applicant]
Hughes et al., “Retroviral Gene Transfer to Primitive Normal and Leukemic Hematopoietic Cells Using Clinically Applicable Procedures,” J. Clin. Invest. 89:1817-1824 (1992).
[cited by applicant]
International Search Report dated May 14, 2019 in International Application No. PCT/US18/64256.
[cited by applicant]
Jakobsson et al., “Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator,” Experimental Cell Research 298:611-623 (2004).
[cited by applicant]
Jinek et al., A Programmable Dual-RNA—Guided DNA Endonuclease in Adaptive Bacterial Immunity, Science 337:816-821 (2012).
[cited by applicant]
Johnson et al., “Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain,” Molecular and Cellular Biology 23(18):6484-6493 (2003).
[cited by applicant]
Johnson, “Gene Therapy for Cystic Fibrosis,” Chest 107(2):77S-83S (1995).
[cited by applicant]
Kadonaga, “Eukaryotic Transcription: An Interlaced Network of Transcription Factors and Chromatin-Modifying Machines,” Cell 92:307-313 (1998).
[cited by applicant]
Kido et al., “Use of retroviral vector with an internal opsin promoter to direct gene expression to retinal photoreceptor cells,” Current Eye Research 15:833-844 (1996).
[cited by applicant]
Kim et al., “Analysis of the Vertebrate Insulator Protein CTCF-Binding Sites in the Human Genome,” Cell 128:1231-1245 (2007).
[cited by applicant]
Kim et al., “Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain” Proc. Natl. Acad. Sci. USA 93:1156-1160 (1996).
[cited by applicant]
Kimmel, “[54] Identification and Characterization of Specific Clones: Strategy for Confirming the Validity of Presumptive Clones,” Methods in Enzymology 152:507-511 (1987).
[cited by applicant]
Kingston et al., “ATP-dependent remodeling and acetylation as regulators of chromatin fluidity,” Genes & Development 13:2339-2352 (1999).
[cited by applicant]
Kunkel et al., “Rapid and Efficient Site-Specific Mutagenesis without Phenotypic Selection,” Methods in Enzymol, 154:367-382 (1987).
[cited by applicant]
Kunkel, “Rapid and efficient site-specific mutagenesis without phenotypic selection,” Proc. Natl. Acad. Sci. USA 82:488-492 (1985).
[cited by applicant]
Ladis et al., “Survival in a large cohort of Greek patients with transfusion-dependent beta thalassaemia and mortality ratios compared to the general population,” European Journal of Haematology 86:332-338 (2011).
[cited by applicant]
Le Gal La Salle et al., “An Adenovirus Vector for Gene Transfer into Neurons and Glia in the Brain,” Science 259:988-990 (1993).
[cited by applicant]
Leboulch et al., “Mutagenesis of retroviral vectors transducing human β-globin gene and β-globin locus control region derivatives results in stable transmission of an active transcriptional structure,” EMBO J 13(13):306…
[cited by applicant]
Levasseur et al., Blood, vol. 102, pp. 4312-4319 (2003).
[cited by applicant]
Levings et al., “Recruitment of transcription complexes to the β-globin locus control region and transcription of hypersensitive site 3 prior to erythroid differentiation of murine embryonic stem cells,” The FEBS Journa…
[cited by applicant]
Ley et al., “Reduced β-Globin Gene Expression in Adult Mice Containing Deletions of Locus Control Region 5′ HS-2 or 5′ HS-3a,” Ann. N.Y. Acad. Sci. 850:45-53 (1998).
[cited by applicant]
Li et al., “Evidence that DNase I hypersensitive site 5 of the human β-globin locus control region functions as a chromosomal insulator in transgenic mice,” Nucleic Acids Res 30(11):2484-2491 (2002).
[cited by applicant]
Li et al., “Genomic and Functional Assays Demonstrate Reduced Gammaretroviral Vector Genotoxicity Associated With Use of the cHS4 Chromatin Insulator,” Mol Ther 17(4):716-724 (2009).
[cited by applicant]
Li et al., “Hypersensitive Site 5 of the Human β Locus Control Region Functions as a Chromatin Insulator,” Blood 84(5):1399-1401 (1994).
[cited by applicant]
Li et al., “Nucleotide Sequence of 16-Kilobase Pairs of DNA 5′ to the Human ϵ-Globin Gene,” J. Biol. Chem. 260(28):14901-14910 (1985).
[cited by applicant]
Li et al., “Primary Structure of the Goat β-Globin Locus Control Region,” Genomics 9:488-499 (1991).
[cited by applicant]
Li et al., “The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus,” Gene Ther 15:49-53 (2008).
[cited by applicant]
Li et al., “β-Globin locus activation regions: Conservation of organization, structure, and function,” Proc. Natl. Acad. Sci. USA 87:8207-8211 (1990).
[cited by applicant]
Lisowski et al., “Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in β-thalassemic mice,” Blood 110(13):4175-4178 (2007).
[cited by applicant]
Lowrey et al., “Mechanism of DNase I hypersensitive site formation within the human globin locus control region,” PNAS USA 89:1143-1147 (1992).
[cited by applicant]
Lucarelli et al., “Bone Marrow Transplantation in Adult Thalassemic Patients,” Blood 93(4):1164-1167 (1999).
[cited by applicant]
Luzzatto et al., “Sickle cell anaemia. A simple disease with no cure,” Nature 337:17-18 (1989).
[cited by applicant]
Ma et al., “High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors,” Stem Cells 21:111-117 (2003).
[cited by applicant]
Mancuso et al., “A Prospective Study of Hepatocellular Carcinoma Incidence in Thalassemia,” Hemoglobin 30(1):119-124 (2006).
[cited by applicant]
Margot et al., “Complete Nucleotide Sequence of the Rabbit β-like Globin Gene Cluster: Analysis of Intergenic Sequences and Comparison with the Human β-like Globin Gene Cluster,” J. Mol. Biol. 205:15-40 (1989).
[cited by applicant]
Maurano et al., “Systematic Localization of Common Disease-Associated Variation in Regulatory DNA,” Science 337:1190-1195 (2012).
[cited by applicant]
May et al., “Successful treatment of murine β-thalassemia intermedia by transfer of the human β-globin gene,” Blood 99:1902-1908 (2002).
[cited by applicant]
May et al., “Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin,” Nature 406:82-86 (2000).
[cited by applicant]
McArthur et al., “Quantification of DNasel-sensitivity by Real-time PCR: Quantitative Analysis of DNasel-hypersensitivity of the Mouse β-Globin LCR,” J Mol Biol 313:27-34 (2001).
[cited by applicant]
McGhee et al., “A 200 Base Pair Region at the 5′ End of the Chicken Adult β-Globin Gene Is Accessible to Nuclease Digestion,” Cell 27:45-55 (1981).
[cited by applicant]
Meissner et al., “Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells,” Nature Biotechnology 25:1177-1181 (2007).
[cited by applicant]
Miccio et al., “In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of β-thalassemia,” PNAS USA 105(30):10547-10552 (2008).
[cited by applicant]
Miller et al., “Generation of Helper-Free Amphotropic Retroviruses That Transduce a Dominant-Acting, Methotrexate-Resistant Dihydrofolate Reductase Gene,” Mol. Cell. Biol. 5(3):431-437 (1985).
[cited by applicant]
Miller et al., “Improved Retroviral Vectors for Gene Transfer and Expression,” Biotechniques 7(9):980-990 (1989).
[cited by applicant]
Miller et al., “Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production,” Mol. Cell. Biol. 6(8):2895-2902 (1986).
[cited by applicant]
Miller, “Retrovirus Packaging Cells,” Human Gene Therapy 1:5-14 (1990).
[cited by applicant]
Miyoshi et al., “Development of a Self-Inactivating Lentivirus Vector,” J. Virol. 72(10):8150-8157 (1998).
[cited by applicant]
Miyoshi et al., “Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector,” Proc. Natl. Acad. Sci. USA 94:10319-10323 (1997).
[cited by applicant]
Moen, “Directions in Gene Therapy,” Blood Cells 17:407-416 (1991).
[cited by applicant]
Moi et al., “Synergistic enhancement of globin gene expression by activator protein-1-like proteins,” PNAS USA 87:9000-9004 (1990).
[cited by applicant]
Moi et al., “Towards the genetic treatment of β-thalassemia: new disease models, new vectors, new cells,” Haematologica 93(3):325-330 (2008).
[cited by applicant]
Nagel et al., “Structural bases of the inhibitory effects of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S,” PNAS USA 76(2):670-672 (1979).
[cited by applicant]
Nakagawa et al., Generation of induced pluripotent stem cells without Myc from mouse or human fibroblasts Nat Biotechnol 2008, 26, 101-6.
[cited by applicant]
Naldini et al., “In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector,” Science 272:263-267 (1996).
[cited by applicant]
Navas et al., “Developmental specificity of the Interaction between the Locus Control Region and Embryonic or Fetal Globin Genes in Transgenic Mice with an HS3 Core Deletion,” Molecular and Cellular Biology 18(7):4188-4…
[cited by applicant]
Neph et al., “An expansive human regulatory lexicon encoded in transcription factor footprints,” Nature 489:83-90 (2012).
[cited by applicant]
Neph et al., “Circuitry and Dynamics of Human Transcription Factor Regulatory Networks,” Cell 150:1274-1286 (2012).
[cited by applicant]
Ney et al., “Tandem AP-1-binding sites within the human β-globin dominant control region function as an inducible enhancer in erythroid cells,” Genes & Dev. 4:993-1006 (1990).
[cited by applicant]
Nienhuis et al., “Genotoxicity of Retroviral Integration In Hematopoietic Cells,” Mol Ther 13(6):1031-1049 (2006).
[cited by applicant]
Nienhuis, “Development of gene therapy for blood disorders: an update,” Blood 122(9):1556-1564 (2013).
[cited by applicant]
Nishino et al. “Partial correction of murine β-thalassemia with a gammaretrovirus vector for human γ-globin,” Blood Cells Mol Dis 37:1-7 (2006).
[cited by applicant]
Okita et al., “Generation of germline-competent induced pluripotent stem cells,” Nature 448:313-317 (2007).
[cited by applicant]
Ono et al., “Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells,” Neuroscience Letters 117:259-263 (1990).
[cited by applicant]
Papapetrou et al., “Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells,” Nat Biotechnol. 29(1):73-78 (2011).
[cited by applicant]
Papayannopoulou et al., “Hemopoietic lineage commitment decisions: in vivo evidence from a transgenic mouse model harboring μLCR-βpro-LacZ as a transgene,” Blood 95:1274-1282 (2000).
[cited by applicant]
Park et al., “Reprogramming of human somatic cells to pluripotency with defined factors,” Nature 451:141-146 (2007).
[cited by applicant]
Pauling et al., “Sickle Cell Anemia, a Molecular Disease,” Science 110:543-548 (1949).
[cited by applicant]
Pawliuk et al., “Correction of Sickle Cell Disease in Transgenic Mouse Models by Gene Therapy,” Science 294:2368-2371 (2001).
[cited by applicant]
Perrine et al., “Induction of Fetal Globin in β-Thalassemia: Cellular Obstacles and Molecular Progress,” Ann. N.Y. Acad. Sci. 1054:257-265 (2005).
[cited by applicant]
Persons et al., “Gene Therapy for the Hemoglobin Disorders,” Semin Hematol 41:279-286 (2004).
[cited by applicant]
Persons, “The challenge of obtaining therapeutic levels of genetically modified hematopoietic stem cells in β-thalassemia patients,” Ann NY Acad Sci 1202:69-74 (2010).
[cited by applicant]
Perumbeti et al., “Therapy for β-globinopathies: a brief review and determinants for successful and safe correction,” Ann NY Acad Sci 1202:36-44 (2010).
[cited by applicant]
Pestina et al., “Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-Level Expression of Fetal Hemoglobin,” Molecular Therapy 17(2):245-252 (2009).
[cited by applicant]
Phillips et al., “CTCF: Master Weaver of the Genome,” Cell 137:1194-1211 (2009).
[cited by applicant]
Pikaart et al., “Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators,” Genes & Development 12:2852-2862 (1998).
[cited by applicant]
Platt et al., “Hydroxyurea Enhances Fetal Hemoglobin Production in Sickle Cell Anemia,” J. Clin. Invest. 74:652-656 (1984).
[cited by applicant]
Pluta et al., “Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters,” J Gene Med 7:803-817 (2005).
[cited by applicant]
Prioleau et al., “An insulator element and condensed chromatin region separate the chicken β-globin locus from an independently regulated erythroid-specific folate receptor gene,” EMBO J. 18(14):4035-4048 (1999).
[cited by applicant]
Proc. Natl. Acad. Sci. 96:5129-5134 (1999).
[cited by applicant]
Pruzina et al., “Hypersensitive site 4 of the human β globin locus control region,” Nucleic Acids Research 19(7):1413-1419 (1991).
[cited by applicant]
Puthenveetil et al., “Successful correction of the human β-thalassemia major phenotype using a lentiviral vector,” Blood 104:3445-3453 (2004).
[cited by applicant]
Ramezani et al., “Combinatorial Incorporation of Enhancer-Blocking Components of the Chicken β-Globin 5′HS4 and Human T-cell Receptor α/δ BEAD-1 Insulators in Self-Inactivating Retroviral Vectors Reduces Their Genotoxic…
[cited by applicant]
Ramezani et al., “Performance-and safety-enhanced lentiviral vectors containing the human interferon-β scaffold attachment region and the chicken β-globin insulator,” Blood 101:4717-4724 (2003).
[cited by applicant]
Ramezani et al., “Stable Gammaretroviral Vector Expression during Embryonic Stem Cell-Derived In Vitro Hematopoietic Development,” Mol Ther 14(2):245-254 (2006).
[cited by applicant]
Recillas-Targa et al., “Position-effect protection and enhancer blocking by the chicken β-globin insulator are separable activities,” PNAS USA 99(10):6883-6888 (2002).
[cited by applicant]
Renda et al., “Critical DNA Binding Interactions Of The Insulator Protein CTCF: A Small Number of Zinc Fingers Mediate Strong Binding, and a Single Finger-DNA Interaction Controls Binding at Imprinted Loci,” J Biol Chem…
[cited by applicant]
Rivella et al., “A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human β-globin gene transfer,” Blood 101:2932-2939 (2003).
[cited by applicant]
Rivella et al., “The cHS4 Insulator Increases the Probability of Retroviral Expression at Random Chromosomal Integration Sites,” J Virol 74(10):4679-4687 (2000).
[cited by applicant]
Rosenberg et al., “Gene Transfer into Humans—Immunotherapy of Patients with Advanced Melanoma, Using Tumor-Infiltrating Lymphocytes Modified by Retroviral Gene Transduction,” N. Engl. J. Med 323(9):570-578 (1990).
[cited by applicant]
Ryu et al., “A chromatin insulator blocks interactions between globin regulatory elements and cellular promoters in erythroid cells,” Blood Cells Mol Dis 39:221-228 (2007).
[cited by applicant]
Ryu et al., “An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation,” Blood 111(4):1866-1875 (2008).
[cited by applicant]
Sabo et al., “Discovery of functional noncoding elements by digital analysis of chromatin structure,” PNAS USA 101(48):16837-16842 (2004).
[cited by applicant]
Sabo et al., “Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays,” Nat Methods 3(7):511-518 (2006).
[cited by applicant]
Sabo et al., “Genome-wide identification of DNasel hypersensitive sites using active chromatin sequence libraries,” PNAS USA 101(13):4537-4542 (2004).
[cited by applicant]
Sadelain et al., “Safe Harbours for the integration of new DNA in the human genome,” Nature Reviews 12:51-58 (2012).
[cited by applicant]
Sadelain et al., “Generation of a high-titer retroviral vector capable of expressing high levels of the human β-globin gene,” PNAS USA 92:6728-6732 (1995).
[cited by applicant]
Sadelain et al., “Stem Cell Engineering for the Treatment of Severe Hemoglobinopathies,” Curr Mol Med 8:690-697 (2008).
[cited by applicant]
Sadelain et al., “Therapeutic Options for Patients with Severe β-Thalassemia: The Need for Globin Gene Therapy,” Hum Gene Ther 18:1-9 (2007).
[cited by applicant]
Sadelain, “Genetic Treatment of the Haemoglobinopathies: Recombinations and New Combinations,” Br J Haematol 98:247-253 (1997).
[cited by applicant]
Sadelain, “Recent advances in globin gene transfer for the treatment of beta-thalassemia and sickle cell anemia,” Current Opinion in Hematology 13:142-148 (2006).
[cited by applicant]
Samakoglu et al., “A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference,” Nat. Biotechnol 24:89-94 (2006).
[cited by applicant]
Schmidt et al., “Waves of Retrotransposon Expansion Remodel Genome Organization and CTCF Binding in Multiple Mammalian Lineages,” Cell 148:335-348 (2012).
[cited by applicant]
Sharp, The Lancet 337:1277-1278 (1991).
[cited by applicant]
Shehee et al., “Nucleotide Sequence of the BALB/c Mouse β-Globin Complex,” J. Mol. Biol. 205:41-62 (1989).
[cited by applicant]
Shimotsuma et al., “DNase I Hypersensitivity and e-Globin Transcriptional Enhancement Are Separable in Locus Control Region (LCR) HS1 Mutant Human β-Globin YAC Transgenic Mice,” Journal of Biological Chemistry 285(19):1…
[cited by applicant]
Shivdasani et al., “Transcription Factor NF-E2 is Required for Platelet Formation Independent of the Actions of Thrombopoietin/MGDF in Megakaryocyte Development,” Cell 81:695-704 (1995).
[cited by applicant]
Stamatoyannopoulos, “Prospects for developing a molecular cure for thalassemia,” Hematology 10(Suppl 1):255-257 (2005).
[cited by applicant]
Stergachis et al., “Developmental Fate and Cellular Maturity Encoded in Human Regulatory DNA Landscapes,” Cell 154:888-903 (2013).
[cited by applicant]
Stergachis et al., “Exonic Transcription Factor Binding Directs Codon Choice and Affects Protein Evolution,” Science 342:1367-1372 (2013).
[cited by applicant]
Straubinger et al., “[32] Liposomes as Carriers for Intracellular Delivery of Nucleic Acids,” Methods in Enzymology 101:512-527 (1983).
[cited by applicant]
Struhl, “Histone acetylation and transcriptional regulatory mechanisms,” Genes & Development 12:599-606 (1998).
[cited by applicant]
Swank et al., “Fetal gene reactivation,” Curr Opin Genet Dev 8:366-370 (1998).
[cited by applicant]
Taboit-Dameron et al., “Association of the 5′ HS4 sequence of the chicken β-globin locus control region with human EFlα gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rab…
[cited by applicant]
Takahashi et al., “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors,” Cell 131:861-872 (2007).
[cited by applicant]
Takahashi et al., “Induction of pluripotent stem cells from fibroblast cultures,” Nature Protocols 2(12):3081-3089 (2007).
[cited by applicant]
Takahashi et al., Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126:663-676 (2006).
[cited by applicant]
Talbot et al., “A dominant control region from the human β-globin locus conferring integration site-independent gene expression,” Nature 338:352-355 (1989).
[cited by applicant]
Talbot et al., “The 5′HS2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites,” EMBO J 10(6):1391-1398 (1991…
[cited by applicant]
Telfer et al. “Improved survival in thalassemia major patients on switching from desferrioxamine to combined chelation therapy with desferrioxamine and deferiprone,” Haematologica 94(12):1777-1778 (2009).
[cited by applicant]
Thurman et al., “The accessible chromatin landscape of the human genome,” Nature 489:75-82 (2012).
[cited by applicant]
Tisdale et al., “Toward Gene Therapy for Disorders of Globin Synthesis,” Semin Hematol 38(4):382-392 (2001).
[cited by applicant]
Tolstoshev et al., Gene expression using retroviral vectors, Current Opinion in Biotechnology 1:55-61 (1990).
[cited by applicant]
Trudel et al., “A 3′ enhancer contributes to the stage-specific expression of the human β-globin gene,” Genes & Development 1:954-961 (1987).
[cited by applicant]
Trudel et al., “Upstream Gγ-Globin and Downstream β-Globin Sequences Required for Stage-Specific Expression in Transgenic Mice,” Molecular and Cellular Biology 7(11):4024-4029 (1987).
[cited by applicant]
Tsukiyama et al., “Chromatin remodeling and transcription,” Curr Opin Genet Dev 7:182-191 (1997).
[cited by applicant]
Vermylen et al., “Haematopoietic stem cell transplantation for sickle cell anaemia: the first 50 patients transplanted in Belgium,” Bone Marrow Transplant 22:1-6 (1998).
[cited by applicant]
Vieira et al., “Recruitment of Transcription Complexes to the β-Globin Gene Locus in Vivo and in Vitro,” J Biol Chem 279(48):50350-50357 (2004).
[cited by applicant]
Wahl et al., Molecular Hybridization of Immobilized Nucleic Acids: Theoretical Concepts and Practical Considerations, Methods in Enzymology 152:399-407 (1987).
[cited by applicant]
Wallace et al., “We gather together: insulators and genome organization,” Curr Opin Genet Dev 17:400-407 (2007).
[cited by applicant]
Wang et al., “Ligand-inducible and liver-specific target gene expression in transgenic mice,” Nature Biotechnology 15:239-243 (1997).
[cited by applicant]
Wang et al., “Widespread plasticity in CTCF occupancy linked to DNA methylation,” Genome Research 22:1680-1688 (2012).
[cited by applicant]
Weatherall, “Phenotype—Genotype Relationships In Monogenic Disease: Lessons From The Thalassaemias,” Nature Reviews Genetics 2:245-255 (2001).
[cited by applicant]
Wernig et al., “In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state,” Nature 448:318-324 (2007).
[cited by applicant]
Wilber et al., “Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities,” Blood 117(15):3945-3953 (2011).
[cited by applicant]
With a Lentivirus Vector, Journal of Virology 71:6641-6649 (1997).
[cited by applicant]
Wolff et al., “Direct Gene Transfer into Mouse Muscle in Vivo,” Science 247(4949):1465-1468 (1990).
[cited by applicant]
Wolffe et al., “Activators and repressors: making use of chromatin to regulate transcription,” Genes to Cells 2:291-302 (1997).
[cited by applicant]
Wu et al., “Receptor-mediated Gene Delivery and Expression in Vivo,” The Journal of Biological Chemistry 263(29):14621-14624 (1988).
[cited by applicant]
Wu et al., “Targeting Genes: Delivery and Persistent Expression of a Foreign Gene Driven by Mammalian Regulatory Elements in Vivo,” The Journal of Biological Chemistry 264(29):16985-16987 (1989).
[cited by applicant]
Wu, “The 5′ ends of
[cited by applicant]
Xu et al., Correction of the enzyme deficiency in hematopoietic cells of Gaucher patients using a clinically acceptable retroviral supernatant transduction protocol, Experimental Hematology 22:223-230 (1994).
[cited by applicant]
Yannaki et al., “Gene therapy for β-thalassaemia: the continuing challenge,” Expert Reviews in Molecular Medicine 12:e31 (2010).
[cited by applicant]
Yannaki et al., “Topological Constraints Governing the Use of the Chicken HS4 Chromatin Insulator in Oncoretrovirus Vectors,” Mol Ther 5(5):589-598 (2002).
[cited by applicant]
Yao et al., “Retrovirus silencer blocking by the cHS4 insulator is CTCF independent,” Nucleic Acids Res 31(18):5317-5323 (2003).
[cited by applicant]
Yu et al., “Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells,” Science 318:1917-1920 (2007).
[cited by applicant]
Yusufzai et al., “The 5′-HS4 chicken β-globin insulator is a CTCF-dependent nuclear matrix-associated element,” PNAS USA 101(23):8620-8624 (2004).
[cited by applicant]
Zufferey et al., “Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery,” Journal of Virology 72(12):9873-9880 (1998).
[cited by applicant]