BINDING AGENTS FOR BCL11A AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63/339,193 filed May 06, 2022, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on May 3, 2023, is named “701039-191690WOPT_SL.xml” and is 135,192 bytes in size. BACKGROUND [0003] BCL11A (B-cell lymphoma/leukemia 11A) is a repressor of γ-globin gene transcription that is important in the fetal-to-adult hemoglobin switch that begins shortly before birth in humans. BCL11A represses fetal hemoglobin expression by binding to recognition sites in the promoters for the γ-globin genes HBG1 and HBG2, which encode the γ-globin fetal hemoglobin (HbF, α2 ^2). Reversal of this repression induces expression of HbF in adult erythroid cells, and provides an avenue for treating hemoglobinopathies, including sickle cell-disease and thalassemias, in which the adult form of the hemoglobin protein is defective or inadequately expressed. As such, methods of inhibiting BCL11A function or expression are being investigated as approaches for treatment of such hemoglobinopathies. SUMMARY [0004] Described herein is the development of polypeptides that specifically bind to BCL11A. More specifically, described herein are antibody polypeptides that specifically bind to BCL11A, and that discriminate for binding between BCL11A and the structurally- similar factor BCL11B. Described more particularly are single domain antibodies that specifically bind BCL11A, including single domain antibodies that bind BCL11A at epitopes comprised by or abutting Zinc Fingers 2/3 and 4-6. Compositions and methods of using the antibody polypeptides for the inhibition of BCL11A activity, for inducing HbF expression, and for treating hemoglobinopathy disorders are described. Also described are methods of using the BCL11A binding polypeptides, e.g., in combination with BCL11A, to screen for and identify small molecule agents that bind to and inhibit the function of BCL11A. [0005] In one aspect, described herein are single domain antibody polypeptides that specifically bind to BCL11A and do not cross-react with BCL11B. [0006] In one embodiment of this and all other aspects described herein, the single domain antibody polypeptide is a camelid or cartilaginous fish single domain antibody polypeptide, a humanized version of a camelid or cartilaginous fish single domain antibody polypeptide, or a human single domain antibody polypeptide. [0007] In one aspect, described herein are single domain antibody polypeptides that specifically binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [0008] In another embodiment of this and all other aspects described herein, the single domain antibody of specifically binds BCL11A at an epitope abutting by zinc finger 23 (ZNF23) of the BCL11A polypeptide. With regard to nomenclature, as used herein, “ZNF 23” is shorthand for zinc fingers 2 and 3, or a polypeptide fragment of BCL11A including those zinc fingers, and “ZNF456” is shorthand for zinc fingers 4, 5, and 6, or a polypeptide fragment of BCL11A including those zinc fingers. [0009] In another embodiment of this and all other aspects described herein, the single domain antibody has an amino acid sequence at least 90% identical to SEQ ID NO: 4. In another embodiment, the single domain antibody has an amino acid sequence at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. [0010] In another embodiment of this and all other aspects described herein, amino acid sequence variation relative to SEQ ID NO: 4 occurs at one or more of amino acids according to Table 2. In some embodiments, variation relative to SEQ ID NO: 4 occurs only at only one site, two sites, three sites, four sites, five sites, six sites, seven sites, eight sites, 9 sites, 10 sites, 11 sites of 12 sites as set out in Table 2. [0011] In another embodiment of this and all other aspects described herein, the single domain antibody has an amino acid sequence at least 90% identical to SEQ ID NO: 26. In another embodiment, the single domain antibody has an amino acid sequence at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 26. [0012] In another embodiment of this and all other aspects described herein, amino acid sequence variation relative to SEQ ID NO: 26 occurs at one or more of amino acids at sites 102 or 108. In some embodiments, variation relative to SEQ ID NO: 26 occurs only at sites 102 or 108. [0013] In another embodiment of this and all other aspects described herein, the single domain antibody has 3 CDRs, wherein: CDR1 has an amino acid sequence selected from SEQ ID NOs: 41-48; CDR2 has an amino acid sequence selected from SEQ ID NOs: 49-57; and CDR3 has an amino acid sequence selected from SEQ ID NOs: 58-63. [0014] In another embodiment of this and all other aspects described herein, the single domain antibody has 3 CDRs, wherein: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 29); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 30); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 31), ADQDGYPYEYW (SEQ ID NO: 32) and ADQDVYPYEYL (SEQ ID NO: 33). [0015] In another embodiment of this and all other aspects described herein, the single domain antibody comprises the amino acid sequence of any one of SEQ ID NOs: 4-28 or 34. [0016] In another aspect, described herein is a fusion polypeptide comprising a single domain antibody polypeptide as described herein. [0017] In one embodiment of this and any other aspect described herein, the fusion polypeptide comprises a single domain antibody polypeptide as described herein and an Fc domain. [0018] In another embodiment of this and any other aspect described herein, the Fc domain is a human Fc domain. [0019] In another embodiment of this and any other aspect described herein, the fusion polypeptide comprises a protease polypeptide. [0020] In another embodiment of this and any other aspect described herein, the fusion polypeptide comprises an E3 ubiquitin-protein ligase. In another embodiment, the E3 ubiquitin ligase comprises a TRIM21 polypeptide. [0021] In another embodiment of this and any other aspect described herein, the fusion polypeptide comprises a cell-penetrating peptide. [0022] In another aspect, described herein is an isolated nucleic acid encoding a single domain antibody or a fusion polypeptide as described herein. [0023] In another aspect, described herein is a vector comprising a nucleic acid encoding a single domain antibody or a fusion polypeptide as described herein. [0024] In one embodiment of this and any other aspect described herein, the vector comprises a plasmid or a viral vector. In another embodiment, the viral vector is an adeno-associated virus (AAV) vector. [0025] In another aspect, described herein is a nanoparticle comprising a nucleic acid encoding a single domain antibody polypeptide or a fusion polypeptide as described herein. In one embodiment, the nanoparticle is a lipid nanoparticle. [0026] In another embodiment of this or any other aspect described herein, the nucleic acid is DNA or RNA. [0027] In another aspect, described herein is a method of inhibiting BCL11A activity, the method comprising introducing a single domain antibody polypeptide as described herein, a fusion polypeptide as described herein, a nucleic acid as described herein, a vector as described herein or a nanoparticle as described herein to a cell that expresses BCL11A, wherein BCL11A activity is inhibited by the introducing. [0028] In one embodiment of this and any other aspect described herein, the cell is an erythroid cell. [0029] In another embodiment of this and any other aspect described herein, the cell is a stem cell. In another embodiment, the stem cell is an hematopoietic stem cell. [0030] In another aspect, described herein is a method of treating hemoglobinopathy disorders, the method comprising administering a construct comprising or encoding the expression of a single domain antibody polypeptide or fusion polypeptide as described herein, or administering a nucleic acid, vector or nanoparticle as described herein to a subject in need thereof, whereby the single domain antibody polypeptide is introduced to or expressed in an erythroid cell, and whereby BCL11A expression or activity is inhibited, and expression of fetal hemoglobin (HbF) is induced, thereby treating the hemoglobinopathy disorders. [0031] In another aspect, described herein is a method of screening for small molecules that bind to and inhibit BCL11A, the method comprising contacting a complex comprising BCL11A polypeptide and a single domain antibody polypeptide or fusion polypeptide as described herein with members of a small molecule library and detecting disruption of the complex, wherein identification of a small molecule that disrupts the complex identifies the small molecule as a candidate BCL11A inhibitor. [0032] Unless otherwise defined herein, scientific, and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [0033] The terms “increased”, “increase”, “enhance”, “activate” are all used herein to refer to an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. [0034] The term “improve” or “improvement,” when applied to a score in a standardized scale or rating, e.g., for disease symptoms or severity, means a statistically significant, favorable change in the scale or rating on that scale. [0035] The term “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “decrease”, “reduced”, “reduction”, or “inhibit” typically means a decrease by at least 10% as compared to a reference level, for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% as compared to a reference level. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. [0036] As used herein, a “reference level” refers to the level or value for a given parameter against which one compares the level or value in a given sample or situation to determine whether the level or value has changed in a meaningful way. A reference level can be a level in or from a sample that is not treated to change the parameter. A reference level can alternatively be a level in or from a normal or otherwise unaffected sample. A reference level can alternatively be a level in or from a sample obtained from a subject at a prior time point, for example, prior to a given treatment. [0037] As used herein, an “appropriate control” refers to an untreated, otherwise identical cell or population (e.g., a subject who was not administered an agent described herein, or was administered by only a subset of agents described herein, as compared to a non-control cell). [0038] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not. [0039] As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment. The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. [0040] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."   BRIEF DESCRIPTION OF THE DRAWINGS [0041] Figs.1A-1D show the screen for nanobodies directed to BCL11A (Fig.1A) Flowchart of procedures for Nb selection. For the first round of MACS, SBP-tagged ZF456-mediated target yeast cells were isolated with streptavidin microbeads. For the second round of MACS, the cells were labeled with a mixture of flag-tagged ZF456 and anti-flag-FITC antibody, followed by selection with magnetic anti-FITC microbeads. For the third round selection, the fluorophore FITC and AF647-labeled cells were sorted by flow cytometry. (Fig.1B) Flow cytometry plot indicating that an increased proportion of yeast interacted with ZF456 following FACS selection. (Fig.1C) Pull-down assays confirmed four nanobodies targeting ZF456. (Fig.1D) NMR assay that confirms binding of nanobody to ZNF456 protein. [0042] Fig. 2 NMR peak intensity ratios between spectra of 1:1 ZF456:Nb61 complex and ZF456 control showing tight binding at residues 800 to 829 (values of 0 indicate unassigned residues). [0043] Figs.3A-3E show affinity maturation of Nb61 and Nb53 by error-prone PCR. (Fig 3A) Scheme for mutagenesis and successive enrichment of positive nanobody clones. (Figs.3B and 3C) SPR data showing affinity-matured variants Nb6101, Nb6102, and Nb6160 exhibited a higher affinity than Nb61. (Fig.3D) Co-elution on the SEC column indicating Nb6101 formed a stable complex with ZF456. (Fig. 3E) Gel shift showing ZF6, but not ZF4, was essential for Nb6101 and Nb6160 binding. [0044] Figs. 4A-4G shows further evolution of nanobodies based on structural determination (Fig.4A) X-ray structure of nanobody, Nb6101, in complex with ZNF456 protein, showing that the nanobody binds to ZNF6. (Fig.4B) X-ray structure highlighting residues that were mutated to enhance interactions. Methionine 45 in Nb6101 close to Lysine 806 in ZF6 could be replaced for potential binding enhancement. (Fig. 4C) Affinity measurements of evolved antibodies. Single-cycle SPR indicates substitution of methionine 45 with aspartic improved the binding affinity. (Fig. 4D) Additional structural relationships of nanobodies and ZNF6. Loops built before and after ZF6 for Nb6101(M45D) computational evolution. (Fig.4E) Binding affinity of nanobodies as determined by AlphaScreen with negative control included. (Fig.4F) Summary of enhanced nanobody affinities. (Fig.4G) Structure showing that DNA binding is incompatible with nanobody binding. [0045] Fig 5. shows the amino acid sequences of evolved nanobodies specific for BCL11A (SEQ ID NOs: 8-11). [0046] Figs.6A-6D shows nanobodies promote degradation of BCL11A and bind BCL11A and not BCL11B. (Fig.6A) Transfection experiment in 293T cells in which nanobodies fused with an Fc-domain promote degradation of BCL11A via TRIM-Away. (Fig.6B) Nb6101-19 fused to the Fc fragment did not induce degradation of BCL11B (Left), whereas BCL11A was degraded (Right). (Fig. 6C) Transfection experiment in 293T cells in which nanobodies fused with TRIM21 promote degradation of BCL11A. (Fig.6D) AlphaScreen assay demonstrating binding of nanobody to BCL11A (circle) and not to BCL11B (square) ZNF456 region. [0047] Figs.7A-7B shows reactivation of HbF by nanobody-mediated degradation of BCL11A (Fig.7A) Western blot showing HUDEP-2 erythroid cells infected with lentiviruses expressing BCL11A nanobodies, either alone or fused to an Fc domain had decreased BCL11A in presence of Fc-fused nanobodies. (Fig. 7B) γ-globin RNA levels seven days after HUDEP-2 cells were infected with lentiviruses expressing BCL11A nanobodies alone or fused with an Fc domain. [0048] Fig 8. shows development of nanobodies targeting ZNF6 (from top to bottom: SEQ ID NOs: 64-76, SEQ ID NO: 19, SEQ ID NOs: 77-80, SEQ ID NO: 8, SEQ ID NO: 25, SEQ ID NOs: 81-85, SEQ ID NO: 24, and SEQ ID NOs 86-89). Nanobodies were modified or designed with reasonable distance from the Nb6101-M45D using the Rosetta software. Mutations were computationally introduced into the interaction interface while the ZNF6 domain was fixed. The top 30 models with low energy were selected for protein expression and purification. [0049] Fig.9A-9C shows the evolution of nanobodies targeting ZNF456 of BCL11A. (Fig.9A) Amino acid sequences of nanobodies targeting ZNF456 of BCL11A and the mutations that occur during the nanobody evolution (from top to bottom: SEQ ID NOs: 12-17, SEQ ID NO: 4, SEQ ID NO: 18, SEQ ID NOs: 5-6, SEQ ID NO: 9, SEQ ID NOs: 19-21, SEQ ID NOs: 10-11, SEQ ID NOs: 22-24, SEQ ID NO: 8, and SEQ ID NO: 25). (Fig.9B) KDs and source of the evolved nanobodies. (Fig. 9C) Structure and interactions of ZNF6-Nb6101 complex. The methionine 45 of the nanobody locates closely to lysine 806 of BCL11A. [0050] Figs.10A-10D shows purification and properties of initial nanobody that is targeted to a region just downstream of ZNF23 of BCL11A. (Fig.10A) Amino acid sequences (from top to bottom: SEQ ID NOs: 102-103) of BCL11A and BCL11B proteins including ZNF23 region (dotted) and “extended region” to the right. The sequence alignment shows that the “extended region” is divergent between BCL11A and BCL11B. This is the area to which the nanobodies bind. (Fig.10B) Purification of the extended ZNF23 of BCL11A, used to screen for nanobodies. (Fig.10C) Complex formation, co-migration, of nanobody with target protein. (Fig.10D) Two difference methods of assessment of binding affinity of 2D9 nanobody to target protein. The methods do not give same affinity measurement but indicate that the nanobody has only modest affinity (i.e. Kd > 139 nM). [0051] Figs. 11A-11B shows error-prone mutagenesis of 2D9 nanobody to evolve to more favorable binding affinity. (Fig. 11A) The initial nanobody 2D9 was subjected to error-prone mutagenesis in bacteria to alter the sequence of the CDRs and individual clones were sequenced. Two nanobodies shown here V102G and W108L had amino acid substitutions that appeared to enhance complex formation, and hence were likely to exhibit improved binding affinity. (Fig. 11B) Following error-prone mutagenesis of 2D9, complex formation with the extZNF2/3 protein was assessed via pulldown assay and SDS-PAGE. The extended region of ZNF23 of BCL11A was immobilized on nickel beads for pull down. Two nanobodies shown here V102G and W108L had amino acid substitutions that appeared to enhance complex formation, and hence were likely to exhibit improved binding affinity. [0052] Figs. 12A-12E shows improved binding affinity of evolved nanobodies V102G and W108L. (Figs. 12A-12B) affinity measurements by MST. (Fig.12C) affinity measurement by AlphaScreen method. (Fig. 12D) Summary of AlphaScreen measurements and MST measurements as shown in Fig.12A-12C. (Fig.12E) Gel panel reveals that V102G and W108L form protein complex with the extended form of extZNF23 and do not form complex with ZNF23 itself or with the extended region of BCL11B. [0053] Figs. 13A-13C shows the sequence of evolved nanobodies V102G and W108L. (Fig. 13A) Schematic of nanobody structure and CDRs of initial 2D9 and evolved nanobodies V102G and W108L (SEQ ID NOs: 29-33). (Fig.13B) Structure of nanobodies, which was taken from McMahon et al. Nat Struct Mol Biol (2018). (Fig.13C) amino acid sequences of original 2D9, and evolved nanobodies V102G and W108L (SEQ ID NOs: 26-28). [0054] Figs. 14A-14B show degradation of BCL11A by nanobodies V102G and W108L and induction of HbF in HUDEP-2 cells. (Fig.14A) Western blots of 2 independent experiments in which HUDEP-2 cells were infected with lentiviruses expressing control (GFP) or nanobodies fused to Fc domains. (Fig. 14B) Following infection cells were differentiated in culture to generate more mature cells (which express hemoglobin). HbF, as scored by %HBG, is elevated in differentiated cells (days 4 and 7). [0055] Figs. 15A-15D show reactivation of HbF upon TRIM21 fused nanobody-mediated degradation of BCL11A. (Fig.15A) Western blot of BCL11A in which HEK293T cells were co- transfected with BCL11A and V102G or W108L fused to TRIM21 or to mutant TRIM21. BCL11A protein was degraded in the presence of V102G or W108L fused to wild-type TRIM21 but not with the nanobodies fused to mutant TRIM21. (Fig.15B) Western blot of BCL11A in which HUDEP-2 cells were co-transfected with BCL11A and V102G or W108L fused to TRIM21 or to mutant TRIM21. BCL11A protein was degraded in the presence of V102G or W108L fused to wild-type TRIM21 but not with the nanobodies fused to mutant TRIM21. (Fig. 15C) HBG% in HUDEP-2 cells on d0 and following differentiation (d7), showing that expression of nanobodies fused to TRIM21 but not mutant TRIM21 reactivate HbF (%HBG). [0056] Figs. 16A-16D show nanobody specific for ZNF4 of BCL11A. (Fig. 16A) amino acid sequence of nanobody from alpaca immunized with BCL11A (SEQ ID NO: 34). (Fig. 16B) Pulldown assay and SDS-PAGE confirming protein complex formation of Nb12 with ZNF456 of BCL11A. In vitro purified His-tagged ZNF456 of BCL11A was immobilized to nickel beads and pulled down Strep-tagged Nb12. (Fig.16C) Domain structure of ZNF456 of BCL11A and NMR experiment demonstrating Nb12 binds to ZNF4. (Fig.16D) Affinity measurement of Nb12 to ZNF456 by MicroScale Thermophoresis (MST). Estimated binding affinity is 86.3± 7.0nM. [0057] Fig.17 shows sequence analysis of randomly picked yeast colonies following MACS and FACS affinity enrichment. Mutations that were enriched in a yeast display library generated by error-prone PCR. [0058] Figs.18A-18C show structural basis of Nb6101 interaction with ZF6. (Figs.18A-18C) Key residues forming interaction between Nb6101 and ZF6. [0059] Figs.19A-19B show measurments of nanobody interaction with ZF6. (Fig.19A) Alpha- screen measurement revealed that Nb6101 bound to ZF456 of BCL11A but not that of BCL11B. (Fig.19B) Negative control Nb58 did not interact with ZF456, as confirmed by the alpha-screen. [0060] Figs.20A-20C show further evolution of Nb6101-M45D by protein design. (Fig.20A) Valuation of designed models with RMSD and total score. (Fig. 20B) Valuation of designed models with the solvent accessible surface area (dSASA) buried in contact and the change in energy of the resulting complex (dG_separated). (Fig. 20C) Binding affinities of designed Nb6101 variants determined by alpha-screen. [0061] Figs.21A-21G show nanobody-mediated BCL11A degradation in HEK293T, HUDEP2 and CD34+ cells and induction of HbF expression. (Fig.21A) Western blot indicating Nb6101- 14, Nb6101-19, Nb6101-20, and Nb6101-22 fused to TRIM21 reduced the level of BCL11A protein in HEK293T cells. (Fig. 21B) Nb6101-19 fused with Fc domain reduced level of BCL11A protein in HUDEP2 cells. Deletion of ZF456 abolished nanobody-mediated degradation of BCL11A. (Fig.21C) Induction of HBG expression by Nb6101-19 fused with Fc domain in differentiating HUDEP2 cells. (Fig.21D) HbF protein level increased in presence of Nb6101-19 fused with Fc domain on day 7 of HUDEP2 cells differentiation compared to GFP and nanobody alone. (Fig.21E) Nb6101-19 fused with Fc domain induced BCL11A degradation in CD34+ cells on day 0. (Fig.21F) Induction of HBG expression by Nb6101-19 fused with Fc domain in differentiating CD34+ cells on day 7, 9, and 12. (Fig. 21G) HbF protein level dramatically increased in presence of Nb6101-19 fused with Fc domain on day 12 of CD34+ cells differentiation compared to GFP and nanobody alone. [0062] Figs. 22A-22B show schematic diagram of BCL11A and family tree of evolved nanobodies. (Fig 22A) Schematic diagram of BCL11A. ZF456 domain highlighted by the dotted rectangle was expressed and purified for bait in the nanobody screen. (Fig.22B) Family tree of evolved nanobodies [0063] Figs. 23A-23C show validation of primary nanobody candidates. (Fig. 23A) ¹ H- ¹⁵ N- HSQC spectrum of ZF456 (open circle) overlaid with that of 1:1 ZF456:Nb14/15/53/61 complex (filled circle). Reduction in peak intensity indicating slow exchange and tight binding of Nb14, Nb15, Nb53, and Nb61. (Fig.23B) Intensity ratio reduction of residues 800-829 reflected binding of Nb14, Nb15, and Nb53 to residues of ZF6. (Fig. 23C) Co-elution of Nb53 and Nb61 with eZF456 (residue 606-835) on the SEC column confirmed stable complex formation. [0064] Figs.24A-24C show nanobody maturation by error-prone PCR. (Fig.24A) Matured yeast library was assessed by monitoring the frequency of mutated residues (Fig.24B) Binding affinity measurement of Nb6101 with substitutions at Met 45. (Fig.24C) Gel-shift confirmed enhanced binding affinity of Nb6101 with M45D replacement. [0065] Figs. 25A-25D show Nb5344 maturation and crystal structure. (Fig. 25A) CDRs that contact ZF6 of BCL11A. (Fig. 25B) SPR of Nb5344 and Nb5344-N74D was employed for measurement of binding affinity. (Fig. 25C) Summary of binding affinities of Nb5344 with substitutions observed at a high rate in the selected library. (Fig.25D) Superposition of structures of ZF6-Nb6101 and ZF6-Nb5344(N74D) exhibited a high degree of similarity. [0066] Figs.26A-26C show affinity maturation of Nb6101-M45D by protein design. (Fig.26A) Valuation of designed models with Lennard-Jones attractive and the change in energy of the forming complex (dG_separated). (Fig.26B) Alpha screen measurement of the binding affinity of Nb6101-22. (Fig.26C) Sequence alignment of Nb6101(M45D) with designed variants (from top to bottom: SEQ ID NO: 11, SEQ ID NO: 8, SEQ ID NO: 82, SEQ ID NO: 19, and SEQ ID NO: 25). [0067] Figs.27A-27B show schematic diagrams of nanobody mediated degradation of BCL11A and induction of HbF expression. (Fig. 27A) Schematic diagrams of fusion protein TRIM21- Nanobody, Nanobody-Fc, and HA-tagged BCL11A. (Fig.27B) Schematic diagrams of HUDEP2 and CD34+ cell differentiation protocols. [0068] Figs. 28A-28C show identification and construction of cell permeant Nb ligands for BCL11A. (Fig. 28A) Disorder probability of BCL11A revealing predicted order in the ZnF domains (0−6) and the sequence divergence from BCL11B in the extended ZnF23 region. The ZnF23 domains of the paralogs are 93.2% identical and 96.6% similar; in the exZnF23 fragment, the sequence identity and similarity are 69.3% and 78.1%, respectively. (Fig. 28B) Flowchart summarizing Nb selection. (Fig. 28C) SDS-PAGE analysis showing that Nb 2D9 and 2D9_W108L interact with exZnF23 of BCL11A (exZnF23A) but not ZnF23 and exZnF23 of BCL11B (exZnF23B). [0069] Figs. 29A-29H show delivery of ZF5.3-2D9 to erythroid precursor cells. (Fig. 29A) Schematic description of the cell-permeant Nb ZF5.3-2D9 created from the individual structures of ZF5.3 (modeled secondary structure) and 2D9 (PDB 7UTG). (Fig.29B) MST analysis of the binding of 2D9 and ZF5.3-2D9 to exZnF23 of BCL11A (mean ± SD, n = 3). (Fig. 29C) Immunoprecipitation revealing that 2D9 and ZF5.3-2D9 bind to endogenous BCL11A. (Fig. 29D) Confocal microscopy images of HUDEP-2 cells revealing ZF5.3-2D9 entered HUDEP-2 cells and show significant colocalization with the nucleus. (Figs. 29E-29F) Immunoblots revealing a (Fig.29E) concentration- and (Fig.29F) time-dependent cell penetration by ZF5.3- 2D9. (Fig.29G) Immunoblot showing increased accumulation of ZF5.3-2D9 in the nucleus. (Fig. 29H) Co-immunoprecipitation of endogenous BCL11A using delivered ZF5.3-2D9. GAPDH and BCL11A were used as loading controls for the cytosolic and nuclear fractions, respectively. [0070] Figs. 30A-30D show Nb 2D9 and 2D9_W108L fused with Fc or Trim21 induced the degradation of BCL11A but not BCL11B. (Fig.30A) Degradation of BCL11A upon lentiviral transduction of Nb-Fc fusion in differentiated HUDEP-2 cells. (Fig.30B) Lentiviral transduction of Nb-wtTrim21 but not Nb-mutTrim21 led to degradation of BCL11A in HUDEP-2 cells. (Fig. 30C) Nb-wtTrim21 but not Nb-mutTrim21 led to degradation of overexpressed BCL11A in HEK293T cells. (Fig. 30D) Neither Nb-Fc nor Nb-Trim21 mediated degradation of overexpressed BCL11B in HEK293T cells. [0071] Figs. 31A-31E show nanobody-mediated degradation of BCL11A in HUDEP-2 cells. (Fig.31A) Schematic depiction of BCL11A degraders. The individual structures of the RNF4 RING domain (4PPE), the BTB domain of SPOP (3HTM), ZF5.3 (modeled secondary structure), and 2D9 (7UTG) were retrieved from the Protein Data Bank. (Fig. 31B) Representative immunoblot showing loss of BCL11A in HUDEP-2 cells treated with ZF5.3-2D9-tRNF4 or ZF5.3-2D9-tSPOP. (Fig.31C) Quantification of BCL11A loss by immunoblots (mean ± SD, n = 3). Immunoblots revealing that (Fig.31D) loss of BCL11A requires the presence of 2D9 and (Fig.31E) is prevented by addition of 5 μM of the proteasome inhibitor MG-132. Lamin B1 was used as a loading control for the immunoblots. [0072] Figs.32A-32I show HbF induction in HUDEP-2 and CD34 ⁺ cells. (Fig.32A) Schematic depiction of the HUDEP-2 cells differentiation and treatment. (Fig.32B) qRT-PCR showing an increase in γ-globin mRNA after treatment with ZF5.3-2D9-tSPOP in HUDEP-2 cells (mean ± SD, n = 3, ****P < 0.0001). (Fig.32C) Immunoblot revealing the loss of BCL11A and γ-globin increase after treatment with ZF5.3-2D9-tSPOP in HUDEP-2 cells. (Fig. 32D) Representative flow cytometric analysis of immunostained HUDEP-2 cells from Day 7 of differentiation showing an increase in the population of HbF ⁺ cells following the degradation of BCL11A (n = 2). (Fig. 32E) Schematic depiction of the CD34 ⁺ cell differentiation and treatment. (Fig. 32F) qRT-PCR showing an increase of the γ-globin mRNA level after treatment with ZF5.3-2D9- tSPOP but not ZF5.3-tSPOP or ZF5.3-GNb-tSPOP in CD34 ⁺ cells (mean ± SD, n = 3, ****p < 0.0001). (Fig. 32G) Immunoblots revealing the loss of BCL11A and γ-globin increase after treatment with ZF5.3-2D9-tSPOP in CD34 ⁺ cells. GAPDH was used as a loading control in all immunoblots. (Fig. 32H) qRT-PCR showing an increase of γ-globin mRNA levels in CD34 ⁺ cells from three different donors after treatment with ZF5.3-2D9-tSPOP (mean ± SD, n = 3, ****p < 0.0001). (Fig. 32I) The population of CD36 ⁺ and CD235a ⁺ in differentiating CD34 ⁺ cells treated with or without ZF5.3-2D9-tSPOP; two repeats in cells from different donors were performed. [0073] Figs. 33A-33C show purification of BCL11A ZnF23 nanobodies (Fig. 33A) Size- exclusion chromatogram and SDS-PAGE analysis of purified wt2D9 (arrow pointing to fraction with pure protein). (Fig.33B) SDS-PAGE gel showing the pull-down of BCL11A exZnF23 by wt2D9. (Fig.33C) MST binding curves of wt2D9 to BCL11A ZnF23 (left), BCL11A exZnF23 (exZnF23A, middle) and BCL11B exZnF23 (exZnF23B, right) [0074] Figs. 34A-34B show characterization of BCL11A ZnF23 nanobodies. (Fig. 34A) Identification of nanobodies targeting exZnF23 of BCL11A. Sequence analysis of 96 randomly picked yeast colonies following MACS and FACS enrichment reveals mutations that favor binding to exZnF23 of BCL11A. (Fig. 34B) The binding affinities of selected nanobodies to exZnF23 of BCL11A were measured by AlphaScreen. [0075] Figs. 35A-35D show chromatography of BCL11A ZnF23 and nanobodies (Fig. 35A) Analytical size-exclusion chromatography (SEC) of BCL11A exZnF23, 2D9, and the complex they form.100 nM of purified exZnF23, 2D9, and the mixture of the two at a 1:1 molar ratio (pre-incubated for 0.5 h on ice) was injected into Superdex 20010/300GL and eluted in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 100 µM ZnSO4 and 7 mM b- mercaptoethanol. SEC-MALS analysis of (Fig.35B) 2D9, (C) exZnF23, and (D) their complex revealed that exZnF23 is monomeric and forms a stable complex with 2D9. [0076] Fig. 36 shows a cartoon depiction of the crystal structure of 2D9. Mutations of the Nb (A97, Q100, G102and W108) that produced proteins with improved binding to BCL11A are highlighted in the CDR3 region. [0077] Fig.37 shows SDS-PAGE gels of purified proteins. [0078] Fig.38 shows immunoblotting of HUDEP-2 cell lysate reveals the cellular uptake of both of ZF5.3-2D9-tSPOP and ZF5.3-2D9-tRNF4. [0079] Fig.39 shows an immunoblot showing loss of BCL11A in HUDEP-2 cells treated with ZF5.3-2D9-tRNF4 or ZF5.3-2D9-tSPOP. This is a second biological replicate of the data shown in Fig.3. [0080] Fig.40 shows the viability of HUDEP-2 cells measured 24 h after protein delivery. For protein delivery, cells were incubated with serum free medium containing 10 μM ZF5.3-2D9- tSPOP for 45 min. After that, cells were cultured in full medium for 24h. [0081] Fig. 41 shows persistence of BCL11A degradation in HUDEP-2 cells. HUDEP-2 cells were incubated with 10 μM ZF5.3-2D9-tSPOP for 45 min, washed, and cultured in fresh medium without ZF5.3-2D9-tSPOP for the times as indicated. [0082] Fig. 42 shows BCL11A levels in differentiated HUDEP-2 cells. Upon differentiation, BCL11A levels gradually increase and peak at Day 7 before decreasing to basal levels. [0083] Figs.43A-43B show Nb 2D9 and 2D9_W108L fused with Fc or Trim21 induced fetal hemoglobin induction in Day 7 differentiated HUDEP-2 cells. (Fig.43A) Nb-Fc fusions but not Nb itself increased the level of γ-globin transcripts. (Fig. 43B) Nb-wtTrim21 but not Nb- mutTrim21 increased the level of γ-globin transcripts. [0084] Fig. 44 shows BCL11A and β-hemoglobin levels in differentiated CD34 ⁺ cells. Upon differentiation, BCL11A levels gradually increased, peaked at Day 8, and then decreased to basal levels. [0085] Fig.45 shows representative analytical flow cytometry showing that the differentiation of CD34 ⁺ cells was unaffected by the treatment of ZF5.3-2D9-tSPOP. CD34 ⁺ cells were treated on Day 8. Before treatment, cells were collected on days 4, 6, and 8; after treatment, cells from control group and ZF5.3-2D9-tSPOP treated group were collected on days 9, 10 and 11. [0086] Figs. 46A-46B show (Fig. 46A) cell proliferation and (Fig. 46B) viability in differentiating CD34 ⁺ cells with or without treatment of ZF5.3-2D9-tSPOP.   DETAILED DESCRIPTION [0087] The technology described herein relates to the development of polypeptides that specifically bind BCL11A and uses thereof. The polypeptides described herein discriminate for binding to BCL11A relative to the structurally-related BCL11B polypeptide and are therefore well-suited for specific targeting of BCL11A, e.g., for the induction of HbF expression, the treatment of hemoglobinopathies or for use in screening assays to identify further agents that specifically bind to BCL11A. [0088] The following describes the considerations involved in the preparation and uses of polypeptides that specifically bind BCL11A as developed.   BCL11A [0089] B-cell lymphoma/leukemia 11A (BCL11A), also known as Evi9, CTIP1, or ZNF856 is a Kruppel-like sequence specific C2H2 type zinc-finger transcription factor located on chromosome 2. See, e.g., Liu et al., Cell.173, 430-442 (2018) and Satterwhite et al., Blood. 98, 3413-3420 (2001), which are incorporated herein by reference in their entireties. BCL11A functions mainly as a transcriptional repressor that is involved in brain and hematopoietic system development, as well as fetal-to-adult hemoglobin switching. Sequences of BCL11A are known for a number of species, e.g., human BCL11A (NCBI GeneID: 53335), mRNA (e.g., BCL11A isoform 1 NCBI Ref Seq NM_00022893.4, SEQ ID NO: 1) and polypeptide (e.g., BCL11A isoform 1: NCBI Ref Seq: NP_075044.2, SEQ ID NO: 2). BCL11A can refer to human BCL11A, including naturally occurring variants and alleles thereof. For example, [0090] SEQ ID NO: 1 is a mRNA sequence for Isoform 1 of human BCL11A. Note: U’s replaced with T’s in this representation. [0091] SEQ ID NO: 2 is an amino acid sequence of Isoform 1 of human BCL11A. [0092] Alternative splicing of BCL11a leads to four isoforms containing 1, 3, or 6 C2H2 zinc- finger domains required for DNA-binding. Isoform 1, also known as BCL11A-XL, contains 6 C2H2 zinc-finger domains, and is the most abundant isoform in erythroid cells. Zinc finger domains are located at the following amino acid sequences: Zinc finger domain 1 amino acids: 170-193 (SEQ ID NO: 104: ytcttckqpftsawfllqhaqnth); Zinc finger domain 2 amino acids: 377-399 (SEQ ID NO: 105: kscefcgktfkfqsnlvvhrrsh); Zinc finger domain 3 amino acids: 405-429 (SEQ ID NO: 106: ykcnlcdhactqasklkrhmkthmh); Zinc finger domain 4 amino acids: 742-764 (SEQ ID NO: 107: dtceycgkvfkncsnltvhrrsh); Zinc finger domain 5 amino acids: 770-792 (SEQ ID NO: 108: ykcelcnyacaqsskltrhmkth) Zinc finger domain 6 amino acids: 800-823 (SEQ ID NO: 109:   ykceickmpfsvystlekhmkkwh). [0093] BCL11B is closely related to BCL11A in structure, but is not involved in repression of fetal hemoglobin expression. Among other functions, BCL11B is involved in normal T cell development. As such, it is desirable that an inhibitor targeted to BCL11A should not cross- react with or inhibit BCL11B. By “does not cross-react with or inhibit BCL11B” is meant that a BCL11A-specific binding moiety binds to BCL11B, if at all, with a dissociation constant (Kd) at least 1,000 times greater than its Kd for BCL11A binding. Sequences of BCL11B are known for a number of species, e.g., human BCL11B (NCBI GeneID: 53335), mRNA (e.g., BCL11B isoform 1 NCBI Ref Seq NM_138576.4) and polypeptide (e.g., BCL11B isoform 1: NCBI Ref Seq: NP_612808.1, SEQ ID NO: 3). BCL11B can refer to human BCL11B, including naturally occurring variants and alleles thereof. [0094] SEQ ID NO: 3 is an amino acid sequence of Isoform 1 of human BCL11B (amino acids identical to BCL11A isoform 1 are in bold) [0095] Alternative splicing of BCL11B leads to two human isoforms containing 6 C2H2 zinc- finger domains required for DNA-binding. Zinc finger domains are located at the following amino acid sequences: ZF1 a.a.221-251 (SEQ ID NO: 110: yicttckqpfnsawfllqhaqnthgfriyle); ZF2 a.a.427-454 (SEQ ID NO: 111: ksce fcgktfkfqs nlivhrrsht gekp); ZF3 a.a.455-482 (SEQ ID NO: 112: ykcqlcdhacsqasklkrhmkthmhkag); ZF4 a.a.796-823 (SEQ ID NO: 113: dtceycgkvfkncsnltvhrrshtgerp); ZF5 a.a.824-853 (SEQ ID NO: 114: ykcelcnyacaqsskltrhmkthgqigkev); and ZF6 a.a.854-884 (SEQ ID NO: 115: yrcdicqmpfsvystlekhmkkwhgehlltn). [0096] For the development of a BCL11A-specific, non-BCL11B cross-reactive binding moiety, a peptide or polypeptide fragment of BCL11A including one or more regions with greater amino acid sequence divergence relative to BCL11B can be used as an antigen (e.g., where an animal is to be immunized) or as a probe of a polypeptide library (e.g., using phage display or yeast display as described elsewhere herein or as known in the art). Binding moieties identified through their binding to BCL11A in a given approach should be analyzed in a BCL11B binding assay to be sure they do not substantially cross-react with BCL11B. [0097] Zinc finger domains or motifs are common in transcription factors, where they can mediate sequence-specific DNA binding. BCL11A binds the promoter sequence TGACCA in the γ-globin gene promoter via its zinc finger domains. See, e.g., Liu et al., Cell 173: 430-442 (2018). Where the zinc finger domains or motifs of BCL11A play an integral role in DNA binding by the polypeptide, it is reasonable to expect that binding of a moiety that specifically binds BCL11A at one or more of the zinc finger motifs could inhibit BCL11A repressor function and de-repress γ-globin expression, leading to an increase in fetal hemoglobin. Thus, polypeptide, antibody or small molecule agents identified through their ability to bind a peptide or polypeptide including one or more of the BCL11A zinc fingers would reasonably be expected to inhibit the DNA binding and repressor activities of BCL11A. Indeed, described herein are single domain antibody polypeptides that specifically bind epitopes comprised by the zinc finger domains of the BCL11A polypeptide. Such single domain antibody polypeptides can directly interfere with BCL11A repressor activity or, as part of various constructs or fusion polypeptides, can target BCL11A for degradation.   ANTIBODIES [0098] As discussed above, in various embodiments, described herein are antibodies or antigen-binding fragments thereof that specifically bind to BCL11A, and do not cross-react with BCL11B. There are a wide variety of antibodies and constructs based upon them, but each generally includes an antigen-binding structure that includes so-called complementarity determining regions (CDRs) of an immunoglobulin polypeptide separated by so-called “framework regions” of the immunoglobulin. The CDRs are highly variable between antibodies that bind different antigens, while the framework regions tend to be more conserved. Most naturally-occurring antibodies include six CDRs, with three contributed by a so-called heavy chain variable domain, V _H , and three contributed by a so-called light chain variable domain, VL. In these antibodies, the VH and VL domains form a complex in which residues in the CDRs of each chain are configured to make contact with an epitope on a given antigen, thereby conferring binding specificity for that epitope of that antigen. While the majority of naturally-occurring antibodies include the V _H /V _L , six CDR configuration, several classes of animals, including camelids and cartilaginous fishes, produce antibodies that include only a VH domain, with three CDRs. As discussed further below, the discovery of such antibodies, and the recognition that they can bind to target antigens with specificity and avidity closely comparable to that of antibodies with six CDRs has spawned a movement to isolate so-called single domain antibodies that are highly specific for target antigens. The movement has developed approaches for the generation or selection of single domain antibodies based on immunization of camelid species, selection from libraries generated from pre-immune and immune camelid genes, humanized camelid-based antibodies, as well as approaches for the selection of human single domain antibodies from libraries of pre-immune or immune V region human genes. Among the benefits of single domain antibodies are their smaller size, which makes it easier to introduce them to cells, better suited to the packaging constraints of, e.g., viral vectors and better suited as fusion partners in bi- or multifunctional constructs. The following provides additional detail in regard to antibodies generally and single domain antibodies more specifically. [0099] As used herein, the term “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-binding fragments thereof (including, but not limited to, a Fab, F(ab')2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria. [0100] As described herein, an "antigen" is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term "antigenic determinant" refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen- binding site of said molecule. An epitope is that portion of an antigen molecule with which an antibody makes direct physical contact via its antigen-binding site when the antibody specifically binds the antigen. [0101] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen- binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions, as occurs, for example, in an IgG immunoglobulin. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and single domain antibodies (sdAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol.1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. [0102] As noted above, the V _H and V _L regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). The term “complementarity determining region” or “CDR” refers to variable regions in antibody polypeptides and contains the amino acid sequences that mediate specific binding to antigenic targets. These CDR regions account for the basic specificity of the antibody or antigen-binding fragment thereof for a particular antigenic determinant structure. Such regions are also referred to as “hypervariable regions.” Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Single domain antibodies have three CDR regions, each non- contiguous with the others (termed CDR1, CDR2, CDR3) and separated by framework regions. [0103] The terms "antigen-binding fragment" or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the V _H and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a V _H or V _L domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. [0104] In some embodiments, a target ligand-binding recognition domain is a single-domain antibody. By the term "single-domain antibody" or "sdAb", it is meant an antibody fragment comprising a single protein domain that specifically binds a target antigen. A single domain antibody requires only three CDRs to specifically bind its target antigen. Single domain antibodies can comprise any variable fragment, including VL, VH, VHH (camelid), and VNAR (shark), and can be naturally- occurring or produced by recombinant technologies. For example, VH, VL, VHH, and VNAR domains can be generated by techniques well known in the art (Holt, et al., 2003; Jespers, et al., 2004a; Jespers, et al., 2004b; Tanha, et al., 2001; Tanha, et al., 2002; Tanha, et al., 2006; Revets, et al., 2005; Holliger, et al., 2005; Harmsen, et al., 2007; Liu, et al., 2007; Dooley, et al., 2003; Nuttall, et al., 2001; Nuttall, et al., 2000; Hoogenboom, 2005; Arbabi-Ghahroudi et al., 2008). In the recombinant DNA technology approach, libraries of sdAbs can be constructed in a variety of ways, "displayed" in a variety of formats such as phage display, yeast display, ribosome display, and subjected to selection to isolate binders to the targets of interest (panning). Examples of libraries include immune libraries derived from llama, shark or human immunized with the target antigen; non- immune/naïve libraries derived from non-immunized llama, camel, shark or human; or synthetic or semi-synthetic libraries such as VH, VL, VHH or VNAR libraries. In one embodiment, the sdAb can be a heavy variable domain (VH). The term includes single domain antibodies as initially identified by selection/isolation of clones, as well as affinity-matured versions prepared by mutagenesis of isolated candidates. [0105] In some embodiments, the target ligand-binding recognition domain is a nanobody. A “nanobody” (Nb) is a single variable domain (V _H H) single domain antibody generally derived, whether via immunization or via recombinant techniques, from for example, camelids, alpacas, llamas, and sharks. Nanobodies generally comprise a single amino acid chain that can be considered to comprise four framework regions and three complementarity determining regions. The term “camelids” refers to old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Lama paccos, Lama glama, Lama guanicoe and Lama vicugna). The small size and unique biophysical properties of Nbs exceed conventional antibody fragments for the recognition of uncommon or hidden epitopes and for binding into cavities or active sites of protein targets. Further, Nbs can be designed as multi- specific and multivalent antibodies or attached to reporter molecules. Certain Nbs and Nb variants can survive the gastro-intestinal system and Nbs can easily be manufactured. Therefore, Nbs can be used in many applications including drug discovery and therapy, but also as a versatile and valuable tool for purification, functional study, and crystallization of proteins. [0106] As used herein, the term “specific binding” refers to a physical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. The specificity of an antibody or antibody fragment thereof can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation (KD) of an antigen with an antigen-binding protein, is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein, such as an antibody or antigen-binding fragment thereof: the less the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule. Alternatively, the affinity can also be expressed as the affinity constant (K _A ), which is 1/ K _D ). Accordingly, an antibody or antigen-binding fragment thereof as described herein is said to be "specific for" or to “specifically bind” or “selectively bind” a first target or antigen compared to a second target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed, for example as a K _D value) that is at least 1000 times, 10000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to another given polypeptide. Generally, a molecule that “specifically binds,” “selectively binds” or “is specific for” a given target will bind with a K _D of 10 ^-5 M (10000 nM) or less, e.g., 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^-12 M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which polypeptide agents as described herein selectively bind the target using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay. [0107] It should be understood in this context that the specific binding is mediated by the CDRs of the antibody polypeptide, as opposed to any other portion of the antibody polypeptide. Antibody dissociation constants and affinities can be determined, for example, by a surface plasmon resonance based assay (such as the BIACORE assay described in PCT Application Publication No. WO2005/012359); Forte Bio Octet ^TM analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s), for example. [0108] As used herein, “avidity” is a measure of the strength of binding between an antigen- binding molecule (such as an antibody or antibody fragment thereof described herein) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antigen-binding proteins (such as an antibody or portion of an antibody as described herein) will bind to their cognate or specific antigen with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 moles/liter or less, such as 10 ^-7 to 10- ¹² moles/liter or less, or 10 ^-8 to 10 ^-12 moles/liter (i.e., with an association constant (KA) of 10 ⁵ to 10 ¹² liter/moles or more, such as 10 ⁷ to 10 ¹² liter/moles or 10 ⁸ to 10 ¹² liter/moles). Any K _D value greater than 10 ^-4 mol/liter (or any KA value lower than 10 ⁴ M ^-1 ) is generally considered to indicate non-specific binding. The KD for biological interactions which are considered meaningful (e.g., specific) are typically in the range of 10 ^-10 M (0.1 nM) to 10 ^-5 M (10000 nM). The stronger an interaction, the lower is its K _D . For example, a binding site on an antibody or portion thereof described herein will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be determined in any suitable manner, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art; as well as other techniques as known in the art and/or mentioned herein. The person of ordinary skill in the art can determine appropriate conditions under which polypeptide agents as described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable binding assay. [0109] As used herein, the term “selectively inhibits” means that an agent inhibits, as that term is used herein, the function or activity of a given target but does not substantially inhibit the function or activity of a relevant non-target. Thus, for example, an antibody polypeptide that selectively inhibits the binding or function of BCL11A will not substantially inhibit the binding or function of the structurally-related BCL11B polypeptide. [0110] As used herein, the term "target" refers to a biological molecule (e.g., peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, etc.) to which a polypeptide domain which has a binding site can selectively bind. The target can be, for example, an intracellular target (e.g., an intracellular protein target) or a cell surface target (e.g., a membrane protein, a receptor protein). Exemplary “target” biological molecules for the purposes of the methods and compositions described herein include BCL11A. [0111] As used herein, an antibody reagent (e.g., an antibody or antigen-binding domain thereof) that specifically binds to BCL11A, binds BCL11A with a dissociation constant (K _D) of 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less and binds to that target at least 100x, 1000x, or 10,000x more strongly than it binds to an off-target protein or distinct cell-surface or intracellular marker. An antibody reagent that specifically binds BCL11A will bind BCL11B, if at all, with a K _D at least 100X, or at least 1000X greater than the KD with which it binds to BCL11A. [0112] Additionally, and as described herein, a recombinant antibody or antigen-binding domain thereof can be further optimized to decrease potential immunogenicity, while maintaining functional activity, e.g., for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with an antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to specifically bind to a target. One approach for decreasing potential immunogenicity of an antibody or antigen-binding fragment thereof is referred to as “humanizing” the antibody or antigen-binding fragment thereof. The term "humanized antibody" refers to forms of antibodies (or an antigen-binding fragment thereof) that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody or antigen-binding fragment will ideally comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992)). The constant region, can if desired, include one or more modifications that modify or disrupt interaction of the human or humanized antibody with an Fc receptor. Humanization can essentially be performed following the method of Winter and co-workers (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-3'27 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)), by substituting rodent, camelid or shark framework sequences with the corresponding sequences of a human antibody. [0113] As discussed above, antibody polypeptides are provided herein that specifically bind to BCL11A and do not substantially cross-react with BCL11B. Non-limiting examples include the following nanobodies that target different zinc finger domains of the BCL11A polypeptide. Nanobodies that target BCL11A ZNF6 [0114] The following provides amino acid sequence for nanobodies that specifically bind human BCL11A. Bold indicates sites with conserved amino acids across ZNF6 nanobodies. Table 1: CDR’s for Nanobodies directed to ZNF6. Bold indicates conserved amino acids.

  Nanobodies directed to ZNF23 “extended region” Bold indicates sites with amino acids conserved across ZNF23 nanobodies. CDR’s for Nanobodies directed to ZNF23 extended region Nanobodies directed to BCL11A ZNF4 CDR’s for Nb12   [0115] As a means of describing structure involved in or necessary for binding to BCL11A, the nanobody NB14 of SEQ ID NO: 4 is used as a reference herein. It should be understood that other nanobodies described herein can be used as a reference in a similar manner. The following Table 2 shows the amino acid sequence of NB14, with variations at each site that can permit specific binding of the nanobody to the same epitope of BCL11A. In some embodiments, variation relative to SEQ ID NO: 4 occurs only at the sites listed in Table 2. Table 2: BCL11A ZNF6 nanobody amino acid variations. Compared to Nb14.

  HEMOGLOBINOPATHIES [0116] Fetal hemoglobin (HbF) is a tetramer of two adult α-globin polypeptides and two fetal β-like γ-globin polypeptides. During gestation, the duplicated γ-globin genes constitute the predominant genes transcribed from the β-globin locus. Following birth, γ-globin becomes progressively replaced by adult β-globin, a process referred to as the "fetal switch.” In humans, the developmental switch from production of predominantly fetal hemoglobin or HbF ( α2 γ2) to production of adult hemoglobin or HbA ( α2 β2) begins at about 28 to 34 weeks of gestation and continues shortly after birth at which point HbA becomes predominant. This switch results primarily from decreased transcription of the gamma-globin genes and increased transcription of beta-globin genes. On average, the blood of a normal adult contains only about 2% HbF, though residual HbF levels have a variance of over 20 fold in healthy adults (Atweh, Semin. Hematol.38(4):367-73 (2001)). [0117] Hemoglobinopathies encompass a number of anemias of genetic origin in which there are insufficient amounts of hemoglobin capable of carrying oxygen in red blood cells (RBCs). These disorders include genetic defects that result in the production of abnormal hemoglobins with a concomitant impaired ability to maintain oxygen concentration. Some such disorders involve the failure to produce normal β-globin in sufficient amounts, while others involve the failure to produce normal β-globin entirely. Those disorders specifically associated with the β-globin protein are referred to generally as β-hemoglobinopathies. For example, β-thalassemias result from a partial or complete defect in the expression of the β- globin gene, leading to deficient or absent HbA. Sickle cell anemia or sickle cell disease (SCD) results from a point mutation in the β-globin structural gene, leading to the production of an abnormal hemoglobin (HbS) that results in deformed (sickled) RBCs. HbS RBCs are more fragile than normal RBCs and undergo hemolysis more readily, leading eventually to anemia (Atweh, Semin. Hematol.38(4):367-73 (2001)). [0118] The search for treatment aimed at reduction of globin chain imbalance in patients with β-hemoglobinopathies has focused on the manipulation of fetal hemoglobin (α2γ2; HbF). The important therapeutic potential of such approaches is indicated by observations of the mild phenotype of individuals with co-inheritance of both homozygous β-thalassemia and hereditary persistence of fetal hemoglobin (HPFH), as well as by those patients with homozygous β-thalassemia who synthesize no adult hemoglobin, but in whom a reduced requirement for transfusions is observed in the presence of increased concentrations of fetal hemoglobin. Furthermore, it has been observed that certain populations of adult patients with β chain abnormalities have higher than normal levels of fetal hemoglobin (HbF), and have been observed to have a milder clinical course of disease than patients with normal adult levels of HbF. For example, a group of Saudi Arabian sickle-cell anemia patients who express 20-30% HbF have only mild clinical manifestations of the disease (Pembrey, et al., Br. J. Haematol.40: 415-429 (1978)). Thus, it is now accepted that β-hemoglobinopathies, such as sickle cell anemia and the β-thalassemias, can be ameliorated by increased HbF production. (Reviewed in Jane and Cunningham Br. J. Haematol.102: 415-422 (1998) and Bunn, N. Engl. J. Med.328: 129-131 (1993)). [0119] As used herein, treating or reducing a risk of developing a hemoglobinopathy in a subject means to ameliorate at least one symptom of hemoglobinopathy. In one aspect, the methods described herein feature methods of treating, e.g., reducing severity or progression of, a hemoglobinopathy in a subject. In another aspect, the methods can also be used to reduce a risk of developing a hemoglobinopathy in a subject, delaying the onset of symptoms of a hemoglobinopathy in a subject, or increasing the longevity of a subject having a hemoglobinopathy. In one aspect, the methods can include selecting a subject on the basis that they have, or are at risk of developing a hemoglobinopathy, but do not yet have symptoms of a hemoglobinopathy. Selection of a subject can include detecting symptoms of a hemoglobinopathy, a blood test, genetic testing, or clinical recordings. If the results of the test(s) indicate that the subject has a hemoglobinopathy, the methods can also include administering a composition as described herein, thereby treating, or reducing the risk of developing, a hemoglobinopathy in the subject. As non-limiting examples, a subject with a diagnosis of SCD with genotype HbSS, HbS/β0 thalassemia, HbSD, or HbSO, and/or HbF <10% by electrophoresis is indicated for treatment using compositions and methods as described herein. By the phrase "risk of developing disease" is meant the relative probability that a subject will develop a hemoglobinopathy in the future as compared to a control subject or population (e.g., a healthy subject or population). For example, an individual carrying the genetic mutation associated with SCD, an A to T mutation of the β-globin gene, and whether the individual in heterozygous or homozygous for that mutation increases that individual's risk. Methods that promote the de-repression or re-expression of fetal hemoglobin in an individual diagnosed with or suffering from a hemoglobinopathy can be effective for treatment of the disease or disorder. [0120] As used herein, the term "hemoglobinopathy" refers to a condition involving the presence of an abnormal hemoglobin molecule or insufficient levels of hemoglobin capable of carrying oxygen in the blood and releasing the oxygen in tissues of the body. The term refers to a condition involving any defect in the structure, function or amount of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of a globin gene, or mutations in, or deletions of, the promoters or enhancers of such genes that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition. The term further includes any decrease in the amount or effectiveness of hemoglobin, whether normal or abnormal, caused by external factors such as disease, chemotherapy, toxins, poisons, or the like. [0121] The term “sickle cell disease” or "SCD" is defined herein to include any symptomatic anemic condition which results from sickling of red blood cells. Manifestations of SCD include: anemia; pain; and/or organ dysfunction, such as renal failure, retinopathy, acute-chest syndrome, ischemia, priapism, and stroke. The term refers to a variety of clinical problems attendant upon SCD, especially in those subjects who are homozygotes for the sickle cell substitution in HbS. Among the constitutional manifestations referred to herein by use of the term of SCD are delay of growth and development, an increased tendency to develop serious infections, particularly due to pneumococcus, marked impairment of splenic function, preventing effective clearance of circulating bacteria, with recurrent infarcts and eventual destruction of splenic tissue. Also involved in SCD are acute episodes of musculoskeletal pain, which affect primarily the lumbar spine, abdomen, and femoral shaft, and which are similar in mechanism and in severity. In adults, such attacks commonly manifest as mild or moderate bouts of short duration every few weeks or months interspersed with agonizing attacks lasting 5 to 7 days that strike on average about once a year. Among events known to trigger such crises are acidosis, hypoxia, and dehydration, all of which potentiate intracellular polymerization of HbS (J. H. Jandl, Blood: Textbook of Hematology, 2nd Ed., Little, Brown and Company, Boston, 1996, pages 544-545). [0122] As used herein, "THAL" or “thalassemia” refers to a hereditary disorder characterized by defective production of hemoglobin. In one embodiment, the term encompasses hereditary anemias that occur due to mutations affecting the synthesis of hemoglobins. In other embodiments, the term includes any symptomatic anemia resulting from thalassemic conditions such as severe or β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemias such as hemoglobin H disease. β-thalassemias are caused by a mutation in the β-globin chain, and can occur in a major or minor form. In the major form of β-thalassemia, children are normal at birth, but develop anemia during the first year of life. The mild form of β -thalassemia produces small red blood cells. Alpha-thalassemias are caused by deletion of a gene or genes from the globin chain. [0123] Where HbF is functional for carrying and delivering oxygen to tissues, the re- induction or de-repression of HbF expression provides an avenue for treating β- hemoglobinopathies. One approach as described herein targets BCL11A, which is responsible for repression of HbF expression. In various embodiments, an antibody or nucleic acid or vector encoding an antibody that specifically binds BCL11A and does not substantially cross-react with BCL11B, can be used to de-repress expression of the HbF subunit genes. Inhibition of BCL11A [0124] In various embodiments, the activity of BCL11A is inhibited or decreased, e.g., by binding of an antibody or antigen-binding fragment thereof. By “decreases BCL11A activity” or “inhibits BCL11A activity” is meant that the amount of functional activity of BCL11A is at least 5% lower in a cell or cell population treated with the methods described herein, than a comparable, control cell or population, wherein no BCL11A inhibitor is present. It is preferred that the BCL11A activity in a treated population is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5- fold lower, at least 10 fold lower, at least 100 fold lower, at least 1000-fold lower, or less relative to a control treated population in which no BCL11A inhibitor is present. At a minimum, BCL11A activity can be assayed by determining the amount of BCL11A at the protein level, using techniques standard in the art. Alternatively, or in addition, BCL11A activity can be determined using a reporter construct, wherein the reporter construct is sensitive to BCL11A activity. [0125] Alternatively, or in addition, BCL11A activity can be assayed by measuring fetal hemoglobin expression at the mRNA or protein level following treatment with a BCL11A inhibitor. Increased expression of endogenous HbF in adult cells treated or contacted with a BCL11A inhibitor is indicative of de-repression, and therefore reduced activity of BCL11A. Treatment of hemoglobinopathies by targeting BCL11A [0126] BCL11A activity and/or expression has been shown to repress expression of fetal hemoglobin isoforms. Thus, as noted above, inhibition or degradation of BCL11A removes or reduces this repression and permits fetal hemoglobin isoforms to be re-induced, for example, in an adult. Increasing expression of the γ-globin genes can ameliorate hemoglobinopathies. Thus, in some embodiments, single domain antibody polypeptides that specifically bind and inhibit BCL11A are used to treat subjects with a hemoglobinopathy. In some embodiments, the single domain antibodies bind to zinc-finger domain 4 of the BCL11A polypeptide. In some embodiments, the single domain antibodies bind to zinc- finger domain 6 of the BCL11A polypeptide. In other embodiments, the single domain antibodies bind to zinc-finger domains 2-3 of the BCL11A polypeptide. [0127] For the treatment of hemoglobinopathies, the primary function of BCL11A to inhibit is its repressive activity on HbF expression. BCL11A interacts directly with DNA in a sequence- specific manner via its zinc finger domains, but also interacts physically and functionally with SOX6. BCL11A and SOX6 co-occupy the human β-globin cluster along with GATA1, and BCL11A participates in long-range interactions that modulate chromosomal loop formation (see, e.g., Xu et al., Genes Dev. 24: 783-798 (2010)). Thus, while the effect of a BCL11A inhibitor that is most particularly relevant is the derepression of HbF expression (evidenced, for example by increased γ-globin HBG1 and/or HGB2 mRNAs as measured by PCR, or increased HBG1 and/or HBG2 proteins as measured by immunoassay), it is contemplated that a BCL11A-binding polypeptide such as an antibody polypeptide as described herein can also inhibit the interaction of BCL11A with protein binding partners including SOX6 and GATA1, among others (BCL11A has also been reported to interact with FOG-1, components of the NuRD complex, matrin-3, MTA2 and RBBP7), and thereby modulate BCL11A function. [0128] In connection with contacting a cell with an inhibitor of BCL11A, “increasing the fetal hemoglobin levels” in a cell indicates that HbF is at least 5% higher in populations treated with a BCL11A inhibitor (e.g., BCL11A-specific antibody or antigen-binding fragment or domain thereof, including but not limited to a BCL11A-specifi single-domain antibody or nanobody), than in a comparable, control population, wherein no BCL11A inhibitor is present. It is preferred that the percentage of HbF expression in a BCL11A inhibitor-treated population is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10 fold higher, at least 100 fold higher, at least 1000-fold higher, or more than a control treated population of comparable size and culture conditions. The term “control treated population” is used herein to describe a population of cells that has been treated with identical media, viral induction, nucleic acid sequences, temperature, confluency, flask size, pH, etc., without the BCL11A inhibitor. In one embodiment, any method known in the art can be used to measure an increase in fetal hemoglobin expression, e. g. Western Blot analysis of fetal γ-globin protein and PCR quantification of mRNA encoding fetal γ-globin (e.g., HBG1 or HBG2 mRNA). [0129] It should be understood that complete inhibition of BCL11A activity is not required to derepress HbF expression enough for therapeutic benefit. As discussed above, HbF makes up about 2% of hemoglobin in adult humans, although the percentage varies. In one embodiment, derepression of HbF expression such that the amount of HbF expressed in adult erythroid cells is increased to at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the total hemoglobin can provide therapeutic benefit. In another embodiment, an increase in HbF expression by at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50 fold or more relative to baseline without a BCL11A inhibitor can provide therapeutic benefit. FUSION PROTEINS [0130] In some embodiments, an antibody or antigen-binding fragment thereof, e.g., a single domain antibody or nanobody, is fused to another polypeptide sequence to provide additional functionality or, for example, to modify stability of the antibody or antigen-binding fragment thereof. [0131] While a BCL11A-specific antibody or antigen-binding domain thereof can exert inhibitory activity on the function of BCL11A simply by binding to BCL11A and interfering with its normal functional interactions, in some embodiments fusion of the BCL11A antigen- binding fragment or domain to a polypeptide that targets the BCL11A for degradation is also of particular interest. In some embodiments, a fusion partner that targets BCL11A for degradation comprises a ubiquitin ligase, e.g., an E3 ubiquitin ligase. By expressing a fusion polypeptide comprising an antigen-binding domain or fragment that binds BCL11A, e.g., a single domain antibody or a nanobody that specifically binds BCL11A, and a ubiquitin ligase, e.g., an E3 ubiquitin ligase in a cell, the ubiquitin ligase is brought into close proximity with BCL11A in the cell, thereby resulting in ubiquitination of the BCL11A polypeptide, which marks the BCL11A for targeted, ubiquitin-mediated degradation. [0132] In some embodiments of any of the aspects, the E3 ubiquitin ligase is TRIM 21. TRIM21 mediates ubiquitin-mediated proteasomal degradation as well as ER-associated degradation. Sequences of TRIM21 are known for a number of species, e.g., human TRIM21 (NCBI GeneID: 6737), mRNA (e.g., NCBI Ref Seq NM_003131.4, SEQ ID NO: 35) and polypeptide (e.g., NCBI Ref Seq: NP_003132.2, SEQ ID NO: 36). TRIM21 can refer to human TRIM21, including naturally occurring variants and alleles thereof. For example, [0133] SEQ ID NO: 35 is a mRNA sequence for TRIM21. Note: U’s replaced with T’s in this representation. [0134] SEQ ID NO: 36 is an amino acid sequence for TRIM21 [0135] In some embodiments of any of the aspects, the E3 ubiquitin ligase is TRIM10. TRIM10 is an E3 ubiquitin ligase that plays an essential role in the differentiation and survival of terminal erythroid cells. Sequences of TRIM10 are known for a number of species, e.g., human TRIM10 (NCBI GeneID: 10107), mRNA (e.g., Isoform 1: NCBI Ref Seq NM_006778.4, SEQ ID NO: 37) and polypeptide (e.g., Isoform 1: NCBI Ref Seq: NP_006769.2, SEQ ID NO: 38). TRIM10 can refer to human TRIM10, including naturally occurring variants, molecules, and alleles thereof. For example, [0136] SEQ ID NO: 37 is a mRNA sequence for TRIM10. Note: U’s replaced with T’s in this representation. [0137] SEQ ID NO: 38 is an amino acid sequence of TRIM10. [0138] In some embodiments of any of the aspects, the E3 ubiquitin ligase is TRIM58. TRIM58 is an E3 ubiquitin ligase that plays a role during late erythropoiesis and erythroblast enucleation. Sequences of TRIM58 are known for a number of species, e.g., human TRIM58 (NCBI GeneID: 25893), mRNA (e.g., NCBI Ref Seq NM_015431.4, SEQ ID NO: 39) and polypeptide (e.g., NCBI Ref Seq: NP_056246.3, SEQ ID NO: 40). TRIM58 can refer to human TRIM58, including naturally occurring variants, molecules, and alleles thereof. For example, [0139] SEQ ID NO: 39 is a mRNA sequence for TRIM58. Note: U’s replaced with T’s in this representation. [0140] SEQ ID NO: 40 is an amino acid sequence of TRIM58. [0141] In some embodiments of any of the aspects, the E3 ubiquitin ligase is Speckle Type BTB/POZ Protein (SPOP). SPOP is an E3 adaptor protein that functions in complex with cullin-3, and is composed of a substrate binding MATH domain and a CUL3-binding BTB domain. Sequences of SPOP are known for a number of species, e.g., human SPOP (NCBI GeneID: 8405), mRNA (e.g., NCBI Ref Seq NM_001007226.1, SEQ ID NO: 93) and polypeptide (e.g., NCBI Ref Seq: NP_001007227.1, SEQ ID NO: 94) SPOP can refer to human SPOP, including naturally occurring variants and alleles thereof. For example, SEQ ID NO: 93 is a mRNA sequence for SPOP. Note: U’s replaced with T’s in this representation. [0142] SEQ ID NO: 94 is an amino acid sequence for SPOP [0143] In some embodiments of any of the aspects, the E3 ubiquitin ligase is Ring Finger Protein 4 (RNF4). RNF4 is an E3 ligase that contains an N-terminal SUMO substrate binding site and a C-terminal RING domain responsible for dimerization and E2 binding. Sequences of RNF4 are known for a number of species, e.g., human RNF4 (NCBI GeneID: 6047), mRNA (e.g., NCBI Ref Seq NM_001185009.3, SEQ ID NO: 95) and polypeptide (e.g.,

    Attorney Docket No.701039-191690WOPT NCBI Ref Seq: NP_001171938.1, SEQ ID NO: 96) RNF4 can refer to human RNF4, including naturally occurring variants and alleles thereof. For example, SEQ ID NO: 95 is a mRNA sequence for RNF4. Note: U’s replaced with T’s in this representation.   45 4  875-5586-5185.3 [0144] SEQ ID NO: 96 is an amino acid sequence for SPOP Expression and Delivery of BCL11A-specific antibodies or antigen-binding fragments [0145] In various embodiments, the BCL11A-specific antibodies or antigen-binding fragments thereof, e.g., a single domain antibody or nanobody as described herein, or a fusion polypeptide including such antibody or antigen-binding fragment thereof as described herein, can be expressed from a vector as a recombinant polypeptide. [0146] Sequences encoding a BCL11A-specific antibody or antigen-binding fragment thereof can be contained in or expressed by a desired vector. Many such vectors useful for transferring exogenous genes into target mammalian cells are available. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. [0147] An expression vector can direct expression of a polypeptide (e.g., a BCL11A-specific antibody or antigen-binding fragment thereof) from nucleic acid sequences contained therein linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector can comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human or mammalian cells for expression and in a prokaryotic host for cloning and amplification. Expression refers to the cellular processes involved in producing RNA and/or proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene or gene construct. [0148] Thus, in some embodiments, provided herein is a vector comprising a nucleic acid encoding a BCL11A-specific antibody or antigen-binding fragment thereof, e.g., a single domain antibody or nanobody as described herein. In other embodiments, provided herein is a vector comprising a nucleic acid encoding a BCL11A-specific antibody or antigen-binding fragment thereof, e.g., a single domain antibody or nanobody fused to a heterologous polypeptide as described herein. Typically, where introduction of the sequence encoding the antibody construct to a cell is desired, the vector is a viral vector which is an adeno- associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus or a polyoma virus, among others. [0149] In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. [0150] Retroviruses can be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. The medium containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos.6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of viral vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No.5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector as described herein includes "expression control sequences", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. A promoter sequence is a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is typically derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence. Transcription promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. In some embodiments, the promoter can be a promoter for an erythroid-enriched or erythroid-specific gene, e.g., a hemoglobin gene. [0151] In some embodiments, provided herein is a host cell transformed with a nucleic acid molecule encoding a BCL11A-specific antibody or antigen-binding fragment thereof as described herein, or with a nucleic acid molecule encoding a fusion polypeptide thereof. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed." Where the sequence was introduced via a viral vector, the host cell can also be said to have been “transduced” with the sequence. [0152] In some embodiments, for expressing and producing the BCL11A-specific antibody or antigen-binding fragment thereof, e.g., the single domain antibody or nanobody polypeptide, prokaryotic cells and, in particular E. coli cells, can be chosen. In instances where the polypeptide will not be used for in vivo therapy, but rather, for example as part of a screening assay for agents that compete for binding with the antibody polypeptide or antigen- binding fragment thereof, prokaryotic expression can be advantageous. [0153] In other instances, e.g., where the polypeptide will be used in vivo, e.g., therapeutically (or where mammalian post-translational modifications are or may be beneficial) it can be advantageous to produce the polypeptide in mammalian cell culture, in order to, for example, produce a protein with mammalian-type post translational modifications, such as glycosylation patterns. In such embodiments, the host cells can be isolated from a mammalian subject selected from a group consisting of: a human, a horse, a dog, a cat, a mouse, a rat, a cow, a pig and a sheep. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a cell in culture. The cells can be obtained directly from a mammal (preferably human), or from a commercial source, or from tissue, or in the form for instance of cultured cells, prepared on site or purchased from a commercial cell source and the like. The cells can come from any organ including but not limited to the blood or lymph system, e.g., an hematopoietic cell, an erythroid cell or erythroid precursor, from muscles, any organ, gland, the skin, brain, lung, liver, kidney, etc. In some embodiments, the cells are selected from the group consisting of epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, hepatocytes, B-cells, T-cells, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, vascular smooth muscle cells, splenocytes, pancreatic β cells, among others. [0154] While introduction of nucleic acid encoding the BCL11A-specific antibody or antigen-binding fragment polypeptides as described herein to prokaryotic or mammalian cells can be used to prepare and isolate the polypeptides or fusion polypeptides for various uses, introduction to mammalian cells, e.g., via viral vectors can also be used therapeutically. In therapeutic embodiments, expression from a vector, e.g., a viral vector, e.g., an AAV vector, can be used to introduce sequence encoding the antibody polypeptide or fusion thereof into a target cell expressing BCL11A to thereby inhibit the action of the BCL11A expressed therein. Such cells include, for example, erythroid cells or erythroid progenitor cells in which inhibition of BCL11A can induce expression of HbF. [0155] In some embodiments, the host cell is a stem cell. As used herein, the term "stem cell" refers to an undifferentiated cell that can be induced to proliferate. The stem cell is capable of self-maintenance or self-renewal, meaning that with each cell division, one daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, post-natal, juvenile, or adult tissue. Stem cells can be pluripotent or multipotent. The term "progenitor cell," as used herein, refers to an undifferentiated cell derived from a stem cell, and is not itself a stem cell. Some progenitor cells can produce progeny that are capable of differentiating into more than one cell type. Stem cells include pluripotent stem cells, which can form cells of any of the body's tissue lineages: mesoderm, endoderm and ectoderm. Therefore, for example, stem cells can be selected from a human embryonic stem (ES) cell; a human inner cell mass (ICM)/epiblast cell; a human primitive ectoderm cell, a human primitive endoderm cell; a human primitive mesoderm cell; and a human primordial germ (EG) cell. Stem cells also include multipotent stem cells, which can form multiple cell lineages that constitute an entire tissue or tissues, such as but not limited to hematopoietic stem cells or neural precursor cells. Stem cells also include totipotent stem cells, which can form an entire organism. In some embodiment, the stem cell is a mesenchymal stem cell. The term "mesenchymal stem cell" or "MSC" is used interchangeably for adult cells which are not terminally differentiated, which can divide to yield cells that are either stem cells, or which, irreversibly differentiate to give rise to cells of a mesenchymal cell lineage, e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines. In some embodiments, the stem cell is a partially differentiated or differentiating cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC), which has been reprogrammed or de-differentiated. Stem cells can be obtained from embryonic, fetal or adult tissues. [0156] In the context of cell ontogeny, the adjective "differentiated", or "differentiating" is a relative term. A "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with. Thus, stem cells can differentiate to lineage-restricted precursor cells (such as a hematopoietic progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an erythrocyte precursor), and then to an end-stage differentiated cell, such as an erythrocyte, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further. [0157] If so desired, viral vectors can also be targeted, e.g. to an erythroid cell or erythroid progenitor cell by manipulating the viral capsid to comprise or display a ligand for a myeloid cell-specific cell-surface molecule as known in the art. [0158] “Hematopoietic stem or progenitor cell” as the term is used herein, refers to cells of a stem cell lineage that give rise to all the blood cell types including the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and the lymphoid lineages (T-cells, B-cells, NK-cells). A “cell of the erythroid lineage” indicates that the cell being contacted with a vector is a cell that undergoes erythropoiesis such that upon final differentiation it forms an erythrocyte or red blood cell (RBC). Such cells belong to one of three lineages, erythroid, lymphoid, and myeloid, originating from bone marrow hematopoietic progenitor cells. Upon exposure to specific growth factors and other components of the hematopoietic microenvironment, hematopoietic progenitor cells can mature through a series of intermediate differentiation cellular types, all intermediates of the erythroid lineage, into RBCs. Thus, cells of the “erythroid lineage”, as the term is used herein, comprise hematopoietic progenitor cells, rubriblasts, prorubricytes, erythroblasts, metarubricytes, reticulocytes, and erythrocytes. [0159] In some embodiments, the hematopoietic stem or progenitor cell has at least one of the cell surface markers characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thy1/CD90+, CD38lo/-, and C-kit/CD117+. Preferably, the hematopoietic progenitor cells have several of these markers. [0160] In some embodiments, the hematopoietic progenitor cells of the erythroid lineage have the cell surface marker characteristic of the erythroid lineage: CD71 and Ter119. [0161] It is contemplated that a construct or vector encoding a BCL11A-specific antibody or antigen-binding fragment thereof can be introduced to target cells ex vivo, which are then re- introduced to a subject, either with or without expansion or selection for transformed cells prior to re-introduction. In some embodiments, the cells can be autologous to the subject. In others, the cells can be allogeneic to the subject. In such embodiments, it is optional to enrich a population of target cells, whether from peripheral blood or, for example, from bone marrow, for target hematopoietic cells or target erythroid progenitor or erythroid cells. Such enrichment can be performed on the basis of cell surface expression of hematopoietic progenitor cell markers or erythroid progenitor cell or erythroid cell markers, e.g., as noted above. In one embodiment of this aspect, and all other aspects, the hematopoietic progenitor is a cell of the erythroid lineage. Methods of isolating hematopoietic progenitor cells are well known in the art, e.g., by flow cytometric purification of CD34+ or CD133+ cells, microbeads conjugated with antibodies against CD34 or CD133, markers of hematopoietic progenitor cells. Commercial kits are also available, e.g., MACS® Technology CD34 MicroBead Kit, human, and CD34 MultiSort Kit, human, and STEMCELL™ Technology EasySep™ Mouse Hematopoietic Progenitor Cell Enrichment Kit. Similar approaches can be applied to isolate other sub-populations based on the specific markers they express. Cells transduced or transformed with vectors as described herein can be cultured for expansion and/or subjected to selection for expression of the exogenous sequence prior to (re)introduction to a subject. In other embodiments, such expansion or selection is not performed, and cells contacted with a vector are (re)introduced to a subject after contacting in vitro. [0162] In other embodiments, the construct or vector, e.g., a viral vector (e.g., an AAV vector, among others) can be introduced to a subject to thereby deliver an expression construct to cells in vivo. Such delivery can inhibit the function of BCL11A in the target hematopoietic progenitor, erythroid progenitor or erythroid cells and thereby increase HbF expression in the cells and/or their progeny. [0163] In other embodiments, BCL11A-specific antibody or an antigen-binding fragment thereof can be produced, e.g., as a fusion with a cell-penetrating polypeptide to thereby introduce the antibody polypeptide to a target cell. [0164] In some embodiments, the hematopoietic stem or progenitor cells described herein are derived from isolated pluripotent stem cells. An advantage of using iPSCs is that the cells can be derived from the same subject to which the progenitor cells are to be administered. That is, a somatic cell can be obtained from a subject, reprogrammed to an induced pluripotent stem cell, and then re-differentiated into a hematopoietic progenitor cell to be administered to the subject (e.g., autologous cells). Since the progenitors are essentially derived from an autologous source, the risk of engraftment rejection or allergic responses is reduced compared to the use of cells from another subject or group of subjects. In some embodiments, the hematopoietic progenitors are derived from non-autologous sources. In addition, the use of iPSCs negates the need for cells obtained from an embryonic source. Thus, in one embodiment, the stem cells used in the disclosed methods are not embryonic stem cells. [0165] Although differentiation is generally irreversible under physiological contexts, several methods have been recently developed to reprogram somatic cells to induced pluripotent stem cells. Exemplary methods are known to those of skill in the art and are described briefly herein below. [0166] As used herein, the term “reprogramming” refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g., a somatic cell). Stated another way, reprogramming refers to a process of driving the differentiation of a cell backwards to a more undifferentiated or more primitive type of cell. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. Thus, simply culturing such cells included in the term differentiated cells does not render these cells non- differentiated cells (e.g., undifferentiated cells) or pluripotent cells. The transition of a differentiated cell to pluripotency requires a reprogramming stimulus beyond the stimuli that lead to partial loss of differentiated character in culture. Reprogrammed cells also have the characteristic of the capacity of extended passaging without loss of growth potential, relative to primary cell parents, which generally have capacity for only a limited number of divisions in culture. [0167] The cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming. In some embodiments, reprogramming encompasses complete reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent state or a multipotent state. In some embodiments, reprogramming encompasses complete or partial reversion of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in expression of particular genes by the cells, the expression of which further contributes to reprogramming. In certain embodiments described herein, reprogramming of a differentiated cell (e.g., a somatic cell) causes the differentiated cell to assume an undifferentiated state (e.g., is an undifferentiated cell). The resulting cells are referred to as “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs or iPS cells).” [0168] Reprogramming can involve alteration, e.g., reversal, of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation. Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a hematopoietic stem cell). Reprogramming is also distinct from promoting the self-renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods described herein can also be of use for such purposes, in some embodiments. [0169] The specific approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as “reprogramming”) is not critical to the claimed invention. Thus, any method that re-programs a somatic cell to the pluripotent phenotype would be appropriate for use in the methods described herein. Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes including, for example Oct-4 (also known as Oct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. In one embodiment, reprogramming using the methods and compositions described herein can further comprise introducing one or more of Oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family to a somatic cell. In one embodiment, the methods and compositions described herein further comprise introducing one or more of each of Oct 4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from the reprogrammed cells are to be used in, e.g., human therapy, in one embodiment the reprogramming is not effected by a method that alters the genome. Thus, in such embodiments, reprogramming is achieved, e.g., without the use of viral or plasmid vectors. [0170] Somatic cells, as that term is used herein, refer to any cells forming the body of an organism, excluding germline cells. Every cell type in the mammalian body—apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells—is a differentiated somatic cell. For example, internal organs, skin, bones, blood, and connective tissue are all made up of differentiated somatic cells. [0171] Additional somatic cell types for use with the compositions and methods described herein include: a fibroblast (e.g., a primary fibroblast), a muscle cell (e.g., a myocyte), a cumulus cell, a neural cell, a mammary cell, an hepatocyte and a pancreatic islet cell. In some embodiments, the somatic cell is a primary cell line or is the progeny of a primary or secondary cell line. In some embodiments, the somatic cell is obtained from a human sample, e.g., a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy or an adipose biopsy), a swab sample (e.g., an oral swab sample), and is thus a human somatic cell. [0172] When reprogrammed cells are used for generation of hematopoietic progenitor cells to be used in the therapeutic treatment of disease, it is desirable, but not required, to use somatic cells isolated from the patient being treated. For example, somatic cells involved in diseases, and somatic cells participating in therapeutic treatment of diseases and the like can be used. In some embodiments, a method for selecting the reprogrammed cells from a heterogeneous population comprising reprogrammed cells and somatic cells they were derived or generated from can be performed by any known means. For example, a drug resistance gene or the like, such as a selectable marker gene can be used to isolate the reprogrammed cells using the selectable marker as an index. [0173] Reprogrammed somatic cells as disclosed herein can express any number of pluripotent cell markers, including: alkaline phosphatase (AP); ABCG2; stage specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49/6E; ERas/ECAT5, E-cadherin; fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); (ES cell associated transcript 1 (ECAT1); ESG1/DPPA5/ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; undifferentiated embryonic cell transcription factor (Utf1); Rex1; p53; G3PDH; telomerase, including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box containing protein 15 (Fbx15); Nanog/ECAT4; Oct3/4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency- associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3/Stella; DPPA4; other general markers for pluripotency, etc. Other markers can include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by the down-regulation of markers characteristic of the somatic cell from which the induced pluripotent stem cell is derived. [0174] To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for the expression of a stem cell marker. Such expression in a cell derived from a somatic cell identifies the cells as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cell that expresses Oct4 or Nanog is identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of the encoded polypeptides, such as Western blots or flow cytometric analyses. In some embodiments, detection does not involve only RT-PCR, but also includes detection of protein markers. Intracellular markers may be best identified via RT-PCR, while cell surface markers are readily identified, e.g., by immunocytochemistry. [0175] The pluripotent stem cell character of isolated cells can be confirmed by tests evaluating the ability of the iPSCs to differentiate to cells of each of the three germ layers. As one example, teratoma formation in nude mice can be used to evaluate the pluripotent character of the isolated clones. The cells are introduced to nude mice and histology and/or immunohistochemistry is performed on a tumor arising from the cells. The growth of a tumor comprising cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells. [0176] In some embodiments, cell-penetration peptides or cell-penetrating peptides (CPPs) can be used as a transmembrane drug delivery agent for delivery of BCL11A-specific antibody polypeptide constructs as described herein. CPPs are a class of small cationic peptides of at least 10, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15, or at least 15, or at least 20, or at least 25, or at least 30 amino acids that can be used as transmembrane drug delivery agents through various forms of endocytosis for compounds including drugs, imaging agents, oligonucleotides, peptides and proteins. CPPs are also known as “protein transduction domains.” CPPs include but are not limited to the peptides Tat (e.g., HIV-derived CPP TAT (48-60)) and penetratin. Addition of a CPP to a fusion protein provides an option for introducing the fusion protein to a target cell. [0177] Delivery of BCL11A-specific antibody polypeptides as described herein, or nucleic acids encoding them can include the use of lipid complexes or lipid nanoparticles complexed or loaded with the antibody, antibody fusion polypeptide or nucleic acid encoding same. As used herein, the term “nanoparticle” refers to particles that are on the order of about 10 ^-9 or one to several billionths of a meter. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork. The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. Non-limiting examples of lipid-based nanoparticles include, but are not limited to: a solid lipid nanoparticle (SLN; e.g., a nanoparticle comprising a single outer phospholipid layer and an inner core comprising a lipophilic substance, such as a therapeutic agent); a nanostructured lipid carrier (NLC; e.g., which comprises a mixture of solid crystalline lipids and liquid lipids); a microemulsion or a nanoemulsion, e.g., comprising a liquid lipid droplet; a cubosome (e.g., a liquid crystalline nano-structure formed from the cubic phase of lipids, such as monooleate, or any other amphiphilic macromolecules with the property to be dispersed into particles; such cubosomes can further comprise a stabilizer); a non-lamellar lipid nanoparticle, referring to a nanoparticle that does not comprise a lipid bilayer, but rather comprises non-lamellar liquid crystalline structures, such as cubic, hexagonal, and sponge phases (such a non-lamellar lipid nanoparticle can be particularly useful for controlled release formulations, e.g., for delivering inhaled drugs); or any combination thereof, or any other known structures in the art such as an ethasome, which is a lipid vesicular carrier comprising a relatively high percentage of ethanol. In some embodiments, the lipid-based nanoparticle comprises at least one phospholipid, at least one charged lipid, cholesterol, at least one membrane protein, and/or at least one nucleic acid or polypeptide construct for delivery to a cell. In some embodiments, the polypeptide or nucleic acid construct is linked to the at least one phospholipid, the at least one charged lipid, the cholesterol, or the at least one membrane protein of the lipid-based nanoparticle. See e.g., Naseri et al., Adv. Pharm. Bull.2015 Sep, 5(3): 305–313; Montenegro et al., Journal of Drug Delivery Science and Technology, Volume 32, Part B, April 2016, Pages 100-112; Barriga et al., Angew Chem Int Ed Engl.2019 Mar 4, 58(10):2958-2978; Chang et al., Advances in Colloid and Interface Science, Volume 222, August 2015, Pages 135-147; Abdulbaqi et al., Int J Nanomedicine.2016 May 25, 11:2279-304; the contents of which are incorporated herein by reference in their entireties.   Administration & Efficacy [0178] As used herein, the terms "administering," "introducing" and "transplanting" are used interchangeably in the context of the placement of an agent, e.g. a BCL11A single domain antibody or fusion protein or compositions thereof, as described herein into a subject, by a method or route which results in at least partial localization of the introduced agent (i.e., BCL11A single domain antibody or fusion protein or composition described herein) at a desired site, such as an hematopoietic progenitor cell, erythroid progenitor cell or erythroid cell, among others, such that a desired effect(s) is produced. Where cells are introduced, the cells, e.g. hematopoietic progenitor cells, or their differentiated progeny can be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. [0179] Modes of administration include injection, infusion and instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. [0180] In one embodiment, the agent (e.g., BCL11A single domain antibody or fusion protein or composition described herein) as described herein is administered systemically. The phrases “systemic administration," “administered systemically", “peripheral administration" and “administered peripherally" as used herein refer to the administration of a formulation (whether including cells expressing a BCL11A single domain antibody or fusion protein or composition described herein, or including nucleic acid or a vector encoding a BCL11A single domain antibody or fusion protein or composition described herein, or including an isolated BCL11A single domain antibody or fusion protein or composition described herein) other than directly into a target site, tissue, or organ, such that it enters, instead, the subject’s circulatory system and, thus, is subject to metabolism and other like processes. [0181] When provided prophylactically, an agent (e.g., BCL11A single domain antibody or fusion protein or composition described herein) can be administered to a subject in advance of any symptom of a hemoglobinopathy, e.g., perinatally, prior to the switch or prior to the completion of the switch from fetal γ-globin to predominantly β-globin. Accordingly, the prophylactic administration of, e.g., a modified hematopoietic progenitor cell population can serve to prevent a hemoglobinopathy, as described herein. [0182] When provided therapeutically, the agent is provided at (or after) the onset of a symptom or indication of a hemoglobinopathy, e.g., upon the onset of sickle cell disease or thalassemia. [0183] In one embodiment, the term “effective amount" as used herein refers to the amount of an agent (e.g., BCL11a single domain antibody or fusion protein or composition described herein) needed to alleviate at least one or more symptom or marker of a hemoglobinopathy, and relates to a sufficient amount of a composition to provide the desired effect, e.g., treat a subject having a hemoglobinopathy. The term "therapeutically effective amount" therefore refers to an amount of an agent that is sufficient to promote a particular effect when administered to a typical subject, such as one who has or is at risk for a hemoglobinopathy. An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using routine experimentation. [0184] The efficacy of a treatment comprising an agent (e.g., BCL11a single domain antibody or fusion protein or composition described herein) as described herein for the treatment of a hemoglobinopathy can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if any one or more of the signs or symptoms of a hemoglobinopathy is altered in a beneficial and/or statistically significant manner. For the avoidance of doubt, an improvement of at least 10% or more in a given sign, symptom or marker after treatment is considered effective treatment, and preferably improvement by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more. Effective treatment expressly includes an increase in levels of HbF as that term is defined herein. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. [0185] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, an effective dose can be formulated in an appropriate animal model. The effects of any particular dosage can be monitored by a suitable bioassay, including, but not limited to measurement of HbF expression. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. [0186] The agents described herein can be formulated, in some embodiments, with one or more additional therapeutic agents currently used to prevent or treat hemoglobinopathy, for example, hydroxyurea. The effective amount of such other agents depend on the amount of the agent/compositions provided herein in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used herein before or about from 1 to 99% of the heretofore employed dosages. [0187] The dosage ranges for the agents or pharmaceutical compositions provided herein depend upon the potency, and encompass amounts large enough to produce the desired effect. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication. In some embodiments, the dosage ranges from 0.001 mg/kg body weight to 100 mg/kg body weight. In some embodiments, the dose range is from 5 μg/kg body weight to 100 μg/kg body weight. For systemic administration, subjects can be administered a therapeutic amount, such as, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. These doses can be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until, for example, the hemoglobinopathy is treated, as measured by the methods described above or known in the art. [0188] However, other dosage regimens can be useful. The duration of a therapy using the methods described herein will continue for as long as medically indicated or until a desired therapeutic effect (e.g., those described herein) is achieved. In certain embodiments, the administration of the pharmaceutical composition described herein is continued for 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or for a period of years up to the lifetime of the subject. [0189] As will be appreciated by one of skill in the art, appropriate dosing regimens for a given composition can comprise a single administration or multiple ones. Subsequent doses may be given repeatedly at time periods, for example, about two weeks or greater up through the entirety of a subject's life, e.g., to provide a sustained therapeutic or preventative effect. The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the practitioner or physician will decide the amount of the agent or composition thereof to administer to particular subjects. [0190] The treatment as described herein ameliorates one or more symptoms associated with a β-globin disorder by increasing the amount of fetal hemoglobin in the individual. Symptoms typically associated with a hemoglobinopathy include, for example, anemia, tissue hypoxia, organ dysfunction, abnormal hematocrit values, ineffective erythropoiesis, abnormal reticulocyte (erythrocyte) count, abnormal iron load, the presence of ring sideroblasts, splenomegaly, hepatomegaly, impaired peripheral blood flow, dyspnea, increased hemolysis, jaundice, anemic pain crises, acute chest syndrome, splenic sequestration, priapism, stroke, hand-foot syndrome, and pain such as angina pectoris. [0191] The levels of BCL11A or fetal hemoglobin (HbF) can be determined by methods known in the art. For example, PCR, Western blotting, immunological methods, flow cytometric analyses, ELISA. Accordingly, the activity of BCL11A can be determined by methods known in the art, e.g., a chromatin occupancy assay, binding assays, pull-down assays, RT-PCR of fetal hemoglobin levels, animal models, etc. BCL11A activity can be assayed by measuring fetal hemoglobin expression at the mRNA or protein level following treatment with a polypeptide, nucleic acid molecule, vector, nanoparticle or other agent providing a BCL11A-specific single domain antibody or construct as described herein. [0192] In some embodiments of any of the aspects, the level or activity of BCL11A is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control. In some embodiments, a decreased level or activity of BCL11A, in a cell of the subject, increases the level and/or activity of fetal hemoglobin (HbF) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as compared to an appropriate control.   Screening for Agents that Bind BCL11A [0193] In one aspect, the discovery of single-domain antibodies that specifically bind BCL11A, but do not cross-react with BCL11B provides an approach for the identification of agents, such as small molecules, aptamers, etc., that specifically bind and inhibit the function of BCL11A. As disclosed herein, also encompassed within the objects of the present technology are methods for screening for modulators or inhibitors of BCL11A activity. [0194] In one embodiment, an approach for the screening and identification of an agent that specifically binds BCL11A comprises a competition assay that screens for agents that disrupt the interaction of a single-domain anti-BCL11A antibody as described herein with BCL11A. Such an assay uses a BCL11A-specific single-domain antibody as described herein and a polypeptide comprising the BCL11A epitope bound by that single-domain antibody as a target. A preparation including a complex of the single-domain antibody and the BCL11A polypeptide is contacted in vitro with members of a library of, e.g., small molecules or aptamers, at varying doses, and association of the members of the complex is monitored. The screen can be designed for high throughput. In one embodiment, dissociation can be monitored through fluorescence resonance energy transfer (FRET), in which, for example, one member of the binding pair is labeled with a fluorophore, and the other is labeled with a quencher of the fluorophore. Disruption of binding by a candidate agent will lead to fluorescent signal. Those of ordinary skill in the art will be aware of a number of variations on this approach or others that can be exploited to measure or detect association or dissociation as a screen. Candidate agents that disrupt the association of the single domain antibody with the BCL11A polypeptide can be evaluated in a bioassay, e.g., using cultured erythroid progenitors, to determine whether they inhibit BC11A sufficiently to induce HbF expression therein. [0195] Small molecules agents can be identified from within a small molecule library, which can be obtained from commercial sources such as Affymetrix, ArQule, Neose Technologies, Sarco, Ciddco, Oxford Asymmetry, Maybridge, Aldrich, Panlabs, Pharmacopoeia, Sigma, Tripose, AMRI (Albany, NY), AsisChem Inc. (Cambridge, MA), TimTec (Newark, DE), among others, or from libraries as known in the art. [0196] Aptamers can be identified with cell-SELEX, using 52 base-pair (bp) random region DNA aptamer library (10 ¹⁶ unique sequences) and human macrophages derived from monocytes, isolated from human leukoreduction system (LRS) chambers. See, e.g., Sefah et al, Nat Protoc, 5: 1169-1185 (2010), which is incorporated herein. [0197] Confirmatory assays can also be performed using an appropriate animal model. [0198] The technology provided herein can further be defined by the following numbered paragraphs. [0199] Paragraph 1: A single domain antibody polypeptide that specifically binds to BCL11A and does not cross-react with BCL11B. [0200] Paragraph 2: The single domain antibody polypeptide of paragraph 1, wherein the single domain antibody polypeptide is a camelid or cartilaginous fish single domain antibody polypeptide, a humanized version of a camelid or cartilaginous fish single domain antibody polypeptide, or a human single domain antibody polypeptide. [0201] Paragraph 3: The single domain antibody of any of the preceding paragraphs, that specifically binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [0202] Paragraph 4: The single domain antibody of any of the preceding paragraphs, that specifically binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide. [0203] Paragraph 5: The single domain antibody of any of the preceding paragraphs, which has an amino acid sequence at least 90% identical to SEQ ID NO: 4. [0204] Paragraph 6: The single domain antibody of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 4 occurs at one or more of amino acids according to Table 2. [0205] Paragraph 7: The single domain antibody of any of the preceding paragraphs, which has an amino acid sequence at least 90% identical to SEQ ID NO: 26. [0206] Paragraph 8: The single domain antibody of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 26 occurs at one or more of amino acids at amino acid number 102 or 108. [0207] Paragraph 9: The single domain antibody of any of the preceding paragraphs, in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 41-48, CDR2 has an amino acid sequence selected from SEQ ID NOs: 49-57; and CDR3 has an amino acid sequence selected from SEQ ID NOs 58-63. [0208] Paragraph 10: The single domain antibody of any of the preceding paragraphs, in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 29); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 30); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 31), ADQDGYPYEYW (SEQ ID NO: 32) and ADQDVYPYEYL (SEQ ID NO: 33). [0209] Paragraph 11: A single domain antibody polypeptide of any of the preceding paragraphs, comprising the amino acid sequence of any one of SEQ ID NOs: 1-28, or SEQ ID NO: 33. [0210] Paragraph 12: A fusion polypeptide comprising a single domain antibody polypeptide of any of the preceding paragraphs. [0211] Paragraph 13: A fusion polypeptide comprising a single domain antibody polypeptide of any of the preceding paragraphs and an Fc domain. [0212] Paragraph 14: The fusion polypeptide of any of the preceding paragraphs, wherein the Fc domain is a human Fc domain. [0213] Paragraph 15: The fusion polypeptide of any of the preceding paragraphs, comprising a protease polypeptide. [0214] Paragraph 16: The fusion polypeptide of any of the preceding paragraphs, comprising an E3 ubiquitin-protein ligase. [0215] Paragraph 17: The fusion polypeptide of any of the preceding paragraphs, comprising a TRIM21 polypeptide. [0216] Paragraph 18: The fusion polypeptide of any of the preceding paragraphs, comprising a cell-penetrating peptide. [0217] Paragraph 19: An isolated nucleic acid encoding a single domain antibody of any of the preceding paragraphs or a fusion polypeptide of any of the preceding paragraphs. [0218] Paragraph 20: A vector comprising a nucleic acid of any of the preceding paragraphs. [0219] Paragraph 21: The vector of any of the preceding paragraphs, wherein the vector comprises a plasmid or a viral vector. [0220] Paragraph 22: The vector of any of the preceding paragraphs, wherein the viral vector is an adeno-associated virus (AAV) vector. [0221] Paragraph 23: A nanoparticle comprising nucleic acid encoding a single domain antibody polypeptide of any of the preceding paragraphs or a fusion polypeptide of any of the preceding paragraphs. [0222] Paragraph 24: The nanoparticle of any of the preceding paragraphs, wherein the nanoparticle is a lipid nanoparticle. [0223] Paragraph 25: The nanoparticle of any of the preceding paragraphs, wherein the nucleic acid is DNA or RNA. [0224] Paragraph 26: A method of inhibiting BCL11A activity, the method comprising introducing a single domain antibody polypeptide of any of the preceding paragraphs, a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, a vector of any of the preceding paragraphs or a nanoparticle of any of the preceding paragraphs to a cell that expresses BCL11A, wherein BCL11A activity is inhibited by the introducing. [0225] Paragraph 27: The method of any of the preceding paragraphs, wherein the cell is an erythroid cell. [0226] Paragraph 28: The method of any of the preceding paragraphs, wherein the cell is a stem cell. [0227] Paragraph 29: The method of any of the preceding paragraphs, wherein the stem cell is a hematopoietic stem cell. [0228] Paragraph 30: A method of treating a hemoglobinopathy disorder, the method comprising administering a construct comprising or encoding the expression of a single domain antibody polypeptide or fusion polypeptide of any of the preceding paragraphs, or administering a nucleic acid, vector or nanoparticle of any of the preceding paragraphs to a subject in need thereof, whereby the single domain antibody polypeptide is introduced to or expressed in an erythroid cell, and whereby BCL11A expression or activity is inhibited, and expression of fetal hemoglobin (HbF) is induced, thereby treating the hemoglobinopathy disorder. [0229] Paragraph 31: A method of screening for small molecules that bind to and inhibit BCL11A, the method comprising contacting a complex comprising BCL11A polypeptide and a single domain antibody polypeptide or fusion polypeptide of any of the preceding paragraphs with members of a small molecule library and detecting disruption of the complex, wherein identification of a small molecule that disrupts the complex identifies the small molecule as a candidate BCL11A inhibitor. [0230] Paragraph 32: The fusion polypeptide of any of the preceding paragraphs, comprising a speckle type POZ (SPOP) polypeptide. [0231] Paragraph 33: The fusion polypeptide of any of the preceding paragraphs, comprising a Ring Finger Protein 4 (RNF4) polypeptide.   EXAMPLES Example 1: BCL11A-specific Nanobody Selection and Evolution [0232] Nanobodies were first selected from a synthetic yeast display nanobody library kindly provided by Andrew Kruse (doi: 10.1038/s41594-018-0028-6). Three consecutive rounds of selection were performed, including two rounds of magnetic-activated cell sorting (MACS) and one round of FACS. 72 unique clones directed against ZF456 were expressed in E. coli, and 69 were successfully purified. Four unique binders (Nb15, Nb16, Nb53, and Nb61) were identified by pulldown assay and NMR. [0233] Nb14, Nb53, and Nb61 were matured by error-prone PCR and suitable molecules were selected from one round of MACS and two rounds of FACS. After confirmation by surface plasmon resonance (SPR), seven matured Nb14, five matured Nb61, and one matured Nb53 were identified with binding affinity higher than 3 μM (Table 3). Table 3

[0234] Next, the crystal structures of ZF6 protein in complex with Nb6101 (Fig. 9C) and Nb5344-N74D (not shown) were determined. The structures showed specific recognition of nanobodies binding with BCL11A (as compared to BCL11B, which has different sequence in the binding region). Key residues of Nb6101 on the interaction interface are Ser 31, Tyr 32, Tyr 37, Glu 44, Leu 47, Ser 57, Leu 99, Asp100, Tyr101, Val 102, and ILE103 (Fig. 9C). Besides the hydrogen-bond network involving all complementarity-determining regions (CDRs) that account for the binding, it was found that residue Met45 is important for high-affinity engagement (Fig. 9C), which is consistent with the presence of residue 45 mutation in almost all of the highly matured nanobodies. The binding affinity of Nb6101 was further improved by residue substitutions to form hydrogen bonds with Lys806, including aspartic acid, glutamic acid, serine, and threonine. SPR measurement and gel-shift assay indicated the aspartic acid replacement improved the binding affinity about 6-fold (K _d =157 nM). [0235] In order to increase affinity further, the loops before and after the ZF6 domain were modified or designed with a reasonable distance from the Nb6101-M45D. Single-state design of the nanobodies was performed with Rosetta software (found on the world wide web at rosettacommons.org/software) developed by the group of David Baker at the University of Washington. Mutations were computationally introduced into the interaction interface while the ZF6 domain was fixed. ~76,000 models were generated and the top 30 models with low energy were selected for protein expression and purification (Table 4). By alpha screen, four nanobodies with higher affinity (20~ 80 nM) were confirmed (Table 3 and Figure 9B). Nb58, which does not bind, was used as a negative control. Table 4 

  Example 2: Evolution of nanobodies specific for BCL11A [0236] Transcription factors (TFs) control numerous genes that are directly relevant to many human disorders. However, developing specific reagents targeting TFs within intact cells is challenging due to the presence of highly disordered regions within these proteins. Intracellular antibodies offer opportunities to probe protein function and validate therapeutic targets. Described herein is the optimization of nanobodies specific for BCL11A, a validated target for the treatment of hemoglobin disorders. First-generation nanobodies directed to a region of BCL11A comprising zinc fingers 4 to 6 (ZF456) were obtained from a synthetic yeast surface display library, and error-prone mutagenesis, structural determination, and molecular modeling to enhance binding affinity were employed. Engineered nanobodies recognized ZF6 and mediated targeted protein degradation (TPD) of BCL11A protein in erythroid cells, leading to the anticipated reactivation of fetal hemoglobin (HbF) expression. Evolved nanobodies distinguished BCL11A from its close paralog BCL11B, which shares an identical DNA- binding specificity. The following describes the experiments, results and conclusions in further detail. [0237] Introduction: Single variable domains of heavy chain-only antibodies, known as nanobodies, are small polypeptides (~15 kDa) capable of stably binding their targets with high affinity. Human single-chain Fv antibody fragments (scFv) have been used for targeting proteins of interest (POIs) in intracellular antibody-capture technology (1). Both nanobodies and scFv have been employed to stabilize proteins for crystallization and structural determinations (2–7), in vivo live cell imaging of biological processes (8), and more recently as therapeutic single-domain antibodies (2). For instance, neutralizing nanobodies directed to the SARS-CoV-2 spike receptor-binding domain can be deployed for the treatment of patients with COVID-19 (9). Moreover, several procedures have been described to leverage nanobodies (or antibodies) for targeted protein degradation (TPD) through the recruitment of POIs to the proteasome (10). [0238] Nanobodies are produced and further engineered by several approaches, most often by immunization of camelids (llama or alpaca) with cell extracts or purified proteins. This approach requires availability of live animals and accompanying animal husbandry, which is often expensive and time-consuming. As an alternative method not requiring immunization, nanobodies can be isolated from synthetic libraries (11–13). Previous efforts have sought to retrieve nanobodies by combining phage display (13, 14), yeast display (12), or ribosome display (15) with a synthetic library. Primary nanobodies obtained in this manner generally exhibit affinities for targets that are modest and insufficient for biological studies. Higher affinity nanobodies can be isolated by random limited mutagenesis (16) and structure-directed evolution (17), which is comparable to enhanced intracellular antibody capture (18). Computational affinity maturation can also be considered, in which residues in the complementarity determining regions (CDRs) are altered based on interface analysis and energy calculations (19, 20). The Rosetta software suite addressing protein structure prediction and design (21) can be used for redesign of antigen-antibody interfaces starting from existing experimental or computational models (22, 23). Taken together, a variety of strategies are available for improving the affinity of the first-generation synthetic nanobodies for specific targets. [0239] Here, nanobodies have been explored as an aid in the characterization and targeted degradation of BCL11A, a transcriptional repressor critical in the silencing of the fetal (γ-) globin gene in the switch from fetal-to-adult hemoglobin in red cell development. To optimize BCL11A-directed nanobodies for functional studies, several available methods were combined. In the end, nanobodies were reported that permit efficient TPD of BCL11A within intact cells and can also serve as tools for discovery of small molecule ligands. Results: Identification of Synthetic Nanobodies Directed to BCL11A [0240] Full-length BCL11A protein contains a CCHC zinc finger (ZF), six regulatory C2H2 ZFs, and several disordered regions (Fig.22A). The C-terminal three ZFs (ZF456) recognize the DNA sequence TGACCA (24), which is present in the promoters of the γ-globin genes and critical for repression of γ-globin gene transcription (24, 25). The structure of this region bound to DNA reveals that ZFs four and five exhibit base contacts (26). Given the ordered nature of individual ZFs and the important functional role of ZF456 in vivo, this region of BCL11A was chosen as a target for the generation of nanobodies. [0241] To isolate candidate nanobodies, a synthetic nanobody library assembled in yeast (12) was screened against purified ZF456 protein. As shown in Fig.1A, three consecutive rounds of enrichment were performed using BCL11A ZF456 protein with different epitope tags as bait. In the first two rounds, potential binders were selected by magnetic-activated cell sorting (MACS) using protein with streptavidin-binding peptide (SBP) and Flag tags, respectively. After the initial round of MACS, the positive cell population increased from ∼0.7 to ∼3.2% as assessed by staining with Alexa Fluor 488 (Figs. 1A and B). Anti-flag antibody labeled with FITC was used in the second round of MACS enrichment, yielding ∼4.8% of the total yeast cells in the positive pool (Figs.1A and B). With a subsequent round of FACS to further enrich for binders, ∼9.9% of the total yeast cells in the pool displayed positive binding (Figs.1A and B). [0242] After three rounds of enrichment, 96 clones were subjected to DNA sequencing. The clones were diverse. However, 14 clones were identical. Seventy-two unique clones directed against ZF456 were cloned and expressed in Escherichia coli; 69 were successfully purified for characterization. [0243] Four unique clones (Nb15, Nb14, Nb53, and Nb61) scored as positive by in vitro pulldown assay with ZF456 protein (Fig.1C and Fig.22A and B). To confirm binding by an independent method, the interaction of these nano-bodies with ZF456 was examined by NMR. Peak intensity losses were observed as expected for slow exchange regime binding. Residue- specific assignments of ZF456 were determined. All tested nanobodies bound specifically to ZF6 (Fig. 2 and Fig. 23 A and B). As assessed by size exclusion chromatography, purified Nb61 and Nb53 formed stable complexes with ZF456 (Fig. 23C) and were prioritized for further characterization. Affinity Maturation of Nanobodies by Error-Prone Mutagenesis [0244] The binding affinities of the primary Nbs were insufficient for the crystallization of the ZF456-Nb complex. To identify more avid Nbs, error-prone PCR mutagenesis of Nb61 and Nb53 (12) was performed. Mutant libraries contained ~1 to 7 amino acid substitutions per clone (Figs.3A and 24A). High-affinity clones were enriched by one round of MACS and two rounds of FACS with streptavidin magnetic beads, AF647, and atto488, respectively (Fig. 3A). Following the selection, 72 clones of mutated Nb53 and 96 clones of mutated Nb61 were subjected to DNA sequencing. (Fig.17). Ultimately, 18 clones of Nb53 and 39 clones of Nb61 were successfully expressed and purified. Three affinity-matured Nb61 mutants were identified by pulldown assay. Surface plasmon resonance (SPR) confirmed that the binding affinities were improved and <700 nM (Fig.3 B and C). Arginine 45 was mutated in all clones (Fig.3C), suggesting an important contribution of this residue. The nanobodies formed a complex with ZF456 as assessed by size exclusion chromatography (Fig.38D). Specificity of binding to ZF6 was confirmed by gel shift assay (Fig.3E). Among the Nb53 mutants, Nb5344 exhibited the most favorable binding affinity, 3.42 ± 0.08 µM (Fig.25 B and C). The replacement of aspartic acid for asparagine at residue 74 further improved the binding affinity to 1.2 ± 0.05 µM (Fig. 25 B and C). Evolution of Nanobodies by Structural Protein Design [0245] To generate nanobodies with yet higher affinity, the molecular determinants of nanobody binding to ZF6 were explored through analysis of the crystal structures of Nb6101 and Nb5344N74D bound to ZF6. Nb6101-ZF6 crystals grew in space group P3121 and diffracted with a resolution of 2.2 Å. After determining phases using a selenomethionine solution, and performing iterative building and refinement, the structure reached an Rwork/ Rfree of 19.1%/23.2% with four copies of the complex in an asymmetric unit. The overall structure of the Nb6101-ZF6 complex is shown in Fig.19A. ZF6 laid in the backbone groove of the nanobody, while the zinc ion was oriented to the complementarity determining region 3 (CDR3) loop (Fig.4A and Fig.25A). The alpha helix of ZF6 contacted the CDR2 loop and the C terminus interacted with the loops of CDR2 and CDR3 (Fig.4A and Fig.25A). Ser 824, Asp 825, and Arg 826 were located in the C-terminal region after ZF6. Ser 31 and D100 in Nb6101 specifically contacted Ser 824 and Arg 826, respectively (Fig.18A). Both Tyr 32 and Tyr 101 formed hydrogen bonds with Asp 825 (Fig. 18A). The side chain of Glu 44 in Nb6101 interacted with the side chain of Lys 801 in β-strands of ZF6 (Fig.18B). There were also other interactions with the conserved residues. For instance, Tyr 37 was bound to the carbonyl group of Cys 805 in ZF6 (Fig.18B). Ser 57 formed a hydrogen bond with Lys 821 in ZF6 (Fig.18C). An hydrophobic interaction was present between Ile 103 and Ile 804. [0246] The amino acid sequence of ZF456 in BCL11A is highly conserved with its paralog BCL11B (27). Nonetheless, in the region of ZF6 bound by the nanobodies, the primary sequences of BCL11A and B diverge. Consistent with the structure of nanobodies bound to BCL11A ZF6, detectable binding to ZF456 of BCL11B was not observed, as assessed by alpha-screen (Fig.19A) with negative control Nb58 (Fig. 19B). The interaction of nanobody with the Ser 824, Asp 825, Arg 826, Lys 801, and Lys 806 via hydrogen bonds revealed the specific recognition mechanism (Fig.18 A-C). [0247] Apart from the hydrogen-bond network involving all CDRs, Met45 appeared most important for engagement. This conclusion was consistent with the presence of residue 45 mutation in nearly all of the highly evolved nanobodies (Fig.3C). Met45 was located close to Lys 806 of ZF6 but exhibited no direct interaction (Fig.4B). Methionine (or glycine) at residue 45 abolished the strong positive charge clash between the original arginine in Nb61 and lysine in ZF6. To improve the binding affinity, substitutions that could form hydrogen bonds with Lys806, including aspartic acid, glutamic acid, serine, and threonine were tested. SPR measurements and gel shift assay indicated that aspartic acid replacement improved the binding affinity ~six fold (Kd = 157 ± 9 nM) (Fig.19H and Figs.24 B and C). [0248] To enhance the affinity even further, computational modeling was employed and loops were built before and after the ZF6 domain with a reasonable distance from the Nb6101(M45D) (Fig.4D). Single-state design of the nanobody was performed with Rosetta software (available on the world wide web at rosettacommons.org/software) (28). Mutations were computationally introduced into the interaction interface, while the ZF6 domain was held constant. Approximately 76,000 models were generated and evaluated with rmsd vs. total score (Fig. 20A), dSASA_in vs. dG_separated (Fig.20B), and Lennard-Jones attractive (Fig.26A) plots. Rmsd indicated the difference between modeled structures and the structure with the lowest energy. dSASA_int is the solvent-accessible surface area buried on the interface, and dG_separated is a change in energy of the forming complex. The top 30 models with the lowest energy and biggest interface area were selected for protein expression and purification. By an alpha-screen assay, four nanobodies with substantially greater affinities were identified, Nb6101-14 (26.7 ± 0.7 nM), Nb6101-19 (21.9 ± 0.4 nM), Nb6101-20 (77.2 ± 0.5 nM), and Nb6101-22 (54.6 ± 0.9 nM) (Fig.20C and Fig. 26B) with negative control Nb58 (Fig.19G). The sequence alignment of the four variants showed common mutation sites, including S25D, I28D, S53A, S57Y, D100S, I103A, and D104E (Fig.26C), which may play a role in enhancing interactions. [0249] Despite Nb5344(N74D) having a broader binding region and variability in its own CDRs, the identified crystal structure of this nano-body with ZF6 revealed a similar recognition mode (Fig. 25D). Comparison of the two structures indicated that they recognized the same ZF6 epitope with few differences in the CDRs. Nanobody-Mediated Degradation of BCL11A and Induction of HbF Expression [0250] The next step was to assess the functional potential of the evolved nanobodies within intact cells for TPD of native BCL11A. Trim-Away employs antibodies (or nanobodies) and the RING E3 ligase TRIM21 to ubiquitinate non-canonical targets for degradation by the ubiquitin-proteasome (29). First, TRIM21-nanobody chimeras were generated and evaluated for their potential to promote the degradation of BCL11A protein in HEK293T cells transfected with DNA constructs (Fig.27A). The expression of Nb6101-14, Nb6101-19, Nb6101-20, and Nb6101-22 conjugated with TRIM21 reduced the level of BCL11A protein, as revealed by Western blotting (Fig.21A). A construct with a M10E/ M72E substitution in TRIM21, which abrogates its function, was used as a control and showed no degradation (Fig.21A). TRIM21 is expressed widely but not in all cells. Since TRIM21 is expressed in HEK293T cells and possesses high-affinity antibody-binding activity, a construct of Nb6101-19 in fusion with an Fc domain was tested. Fc-fusions directed BCL11A to the ubiquitin-proteasome system for disposal, as shown in Fig.6B (Right) which demonstrates nanobody-directed TPD of BCL11A within intact cells. Alpha-screen assay revealed that Nb6101 did not bind detectably to BCL11B (Fig. 19A). Consistent with the structure of the nanobody-ZF6 complex, BCL11B protein is neither recognized nor degraded in the cells (Fig.6B). [0251] BCL11A acts as a highly specific and potent repressor of fetal (γ-) globin gene transcription. Human umbilical cord blood-derived erythroid progenitor-2 (HUDEP2) cells, which are immortalized human erythroid progenitor cells, serve as a convenient model of red cell differentiation and model aspects of globin gene switching. HUDEP2 cells express predominantly f3-globin (and therefore, HbA, α ₂ f3 ₂ ). Upon down-regulation of BCL11A, γ- globin gene transcription is reactivated. HUDEP2 cells were trans-duced with lentivirus harboring Nb6101-19 fused to an Fc domain on day 0 and then cultured under differentiation conditions (Fig. 27B). The level of BCL11A protein was reduced, as assayed on day 0 (Fig. 21B). The expression of γ-globin transcripts was increased at day 4 and day 7 (Fig.21C). Fetal hemoglobin (HbF) protein level was markedly elevated on day 7 (Fig. 21D). In additional control experiments, BCL11A protein level was unchanged in the HUDEP2 cells in which residues 724 to 835 were deleted by CRISPR/Cas9-mediated editing (Fig.21B). [0252] To examine the functional consequence of Nb6101-19-Fc fusion protein in primary cells, adult primary human stem and progenitor CD34+ cells were used. Under appropriate culture conditions, CD34+ cells differentiate along the erythroid lineage, and express largely f3-globin (hence, HbA, α2f32). The transduction of CD34+ cells with Nb6101-19-Fc fusion protein reduced the level of BCL11A protein (Fig. 21E) on day 0 and reactivated γ-globin expression (and HbF) on day 9 and day 12 (Fig. 21F). Nb6101-19 fused with Fc domain elevated HBG RNA expression and HbF protein level dramatically on day 12 of CD34+ cells differentiation compared to green fluorescent protein (GFP) and nanobody alone (Fig. 21 F and G). The percentage of HbF was increased from 2.9 to 35.2% (Fig.21G). [0253] Taken together, these findings indicate that nanobodies directed to ZF6 are functional in erythroid precursor cells for TPD of native BCL11A and reactivation of HbF. Discussion [0254] Given the hierarchical and interconnected relationships of gene regulatory networks, establishing the roles of transcription factors (TFs) in specific cellular pathways, for instance, in developmental decisions and cancer, is often challenging. The knockout strategies test loss of function after a genetic manipulation and an inherent time lag, which allows for secondary consequences of TF loss and subsequent compensation. TPD offers a means to test direct rela- tionships between a TF and a cellular process or gene target. When a ligand has been identified for a POI, proteolysis targeting chimeras (PROTACs) represent a tractable option, although the design of an effective degrader is not straightforward and may depend on the nature and size of the linker bridging the ligand to an E3 ubiquitin ligase recruiter, as well as the specific E3 ubiquitin ligase chosen (30). A variation on the PROTAC theme, TRAFTAC, in which a DNA sequence is employed to recruit a TF, takes advantage of the high affinity of TFs for specific recognition motifs to facilitate TPD (31, 32). However, this method relies on prior knowledge of the DNA recognition sequence for a given TF and cannot distinguish between different TFs that bind a common sequence. Therefore, the need remains for improved systems for TPD of TFs, where implementation of a PROTAC approach is limited. [0255] With these considerations in mind, TPD was leveraged for studies of BCL11A, a critical effector of HbF silencing in the red cell lineage. Preclinical studies identified BCL11A as a regulator of HbF through genome-wide association studies (33, 34), knockout experiments in mice (35), and gene editing in erythroid cells (36–39). Moreover, recent clinical trials have validated BCL11A as a therapeutic target for the reactivation of HbF in sickle cell disease and β-thalassemia (37, 40–42). Consistent with these findings, the dTAG platform for TPD was recently employed to examine the immediate consequences of BCL11A depletion on transcription and identify primary gene targets (43). Here, nanobodies specific for the DNA- binding region of BCL11A were developmed and deployed for TPD. This approach allows for proteolytic degradation of BCL11A in its native form, absent any appended tags. While commonly used tags are generally well tolerated, the addition of the variant FKBP employed in the dTAG system modestly reduced the normally long half-time (~24 h) of BCL11A to ~7.5 h (43). [0256] Synthetic nanobodies that recognize one of the C-terminal ZFs of BCL11A critical for its in vivo function were identified and optimized. Using error-prone mutagenesis, structural determination and molecular modeling, nanobodies that specifically recognize ZF6 of BCL11A in a region that is divergent from its close paralog BCL11B were engineered. The evolved nanobodies mediated the degradation of BCL11A protein in cells. Following their expression in immortalized erythroid (31) HUDEP-2 cells and primary CD34-derived erythroid cells and subsequent differentiation, nanobodies elicited increased expression of HbF comparable to that observed with genetic down-regulation or TPD with CRISPR/Cas9 editing and the dTAG system, respectively. [0257] Validation of POIs as targets for biological or therapeutic manipulation has most often been assessed through genetic inactivation but TPD offers an alternative means for validation. Nanobodies have specificities and affinities comparable to those of conventional antibodies and are amenable to high-throughput engineering to target diverse proteins, including TFs containing large unstructured regions. These experiments illustrate how nano-bodies may be used to modulate nuclear regulators in an approach that distinguishes closely related TFs, such as BCL11A and BCL11B, which share identical DNA-binding specificity (24). This feature distinguishes nanobody-mediated TPD from that elicited with TRAFTAC, which is based on a DNA recognition sequence (31, 32). [0258] A potential application of intracellular antibodies in drug discovery relates to the development of small-molecule surrogates using antibody-derived (Abd) technology (44). The specific interaction interface between the antibody fragment and target protein would be mimicked by chemical compound surrogates. Chemical species identified through competition assays inside cells occupy the effector binding region and interfere with protein–protein interactions and signal transduction. The concept was initially applied in isolating drug leads directed to RAS from two commercial libraries, guided by intracellular antibodies that bind activated RAS isoforms (45, 46). Chemical surrogates that bind to LMO2 were also identified using an inhibitory intracellular antibody fragment as a competitor in a compound library screen (47). Similarly, nanobodies may be suitable for surrogate compound development in cells. Materials and Methods [0259] Cloning and Protein Purification. DNA sequences encoding ZF456 (residues 737 to 835), ZF56 (residues 768 to 835), ZF6 (residues 797 to 826), SBP-ZF456, and Flag-ZF456 were cloned into a vector PET28a containing a N-terminal His-SUMO tag for expression in E. coli. Proteins were purified on a nickel column followed by cleavage of the sumo tag with Ulp1 protease. Protein was further purified on a Heparin column (Cytiva) and concentrated for nanobody screening, crystallization, SPR, pull-down assay, and gel shift assays. [0260] Sequences encoding nanobodies for selection were cloned into vector PET26b with a C-terminal His-tag. Proteins were expressed in Escherichia coli and purified on a one-step NTA column. To remove imidazole, proteins were dialyzed to buffer (20 mM HEPES pH 7.5, 150 mM sodium chloride) for 2 h followed by concentration. [0261] 15N labeled ZF456 was expressed in Escherichia coli in M9 medium containing 0.1% ¹⁵ N-NH ₄ Cl, 0.4% glucose, 0.1 mM CaCl ₂ , 2 mM MgSO ₄ , 1 1ig/mL thiamine, 50 1ig/mL kanamycin, and trace element solution (1 uM MnCl ₂ , 3.1 mM FeCl ₃ , 0.62 mM ZnCl ₂ , 761iM CuCl ₂ , 421iM CoCl ₂ , 1621iM H ₃ BO ₃ , 8.11iM MnCl ₂ ). ¹⁵ N-ZF456 was prepared as ZF456 except for exchanging buffer to 1 × PBS on size exclusion column (SEC). [0262] Isolation of Primary Nanobodies. Nanobodies were first selected from the synthetic yeast display nanobody library (12) using MACS with streptavidin microbeads and anti-flag microbeads (Miltenyi). The enriched pool was used to select higher affinity binders by FACS sorting with anti-flag-FITC antibody and anti-his-AF647 antibody. All the MACS and FACS were performed in buffer (20 mM HEPES pH 7.5, 150 mM sodium chloride, 0.1% BSA, 1 mM DTT). After FACS selection, yeast cells were plated as single colonies which were picked and grown as clonal populations in a 96-well plate. Plasmids encoding the nanobodies were isolated with the Yeast DNA Extraction Kit (Thermo Scientific, cat# 78870) and subjected to DNA sequencing. [0263] Affinity Maturation of Nanobodies by Error-Prone PCR. The affinity maturation library was prepared by assembly PCR with oligonucleotide primers with the GeneMorph II Random Mutagenesis Kit (Cell Signaling Technologies, cat# 2350s). The PCR product was further amplified with primers. Mutagenic nanobody DNA and linearized pYDS649 plasmid (cut with NheI-HF and BamH1-HF) were co-electroporated into BJ5465 S. serevisiae cells to yield a library of transformants. Following one round of MACS and two rounds of FACS selection (Fig.23A), positive clones were enriched and sequenced. Proteins were expressed in Escherichia coli and validated by pull-down assay, gel shift assay, SPR, and an alpha-screen assay. [0264] Pull-Down Assay. GST and GST-ZF456 protein were purified with glutathione agarose beads (Thermo Scientific Pierce) and confirmed by SDS-PAGE.101iM GST or GST- ZF456 was incubated with 501iL of glutathione agarose beads in binding buffer (20 mM Tris- HCl, pH 7.5, 150 mM NaCl, and 10 1iM ZnSO ₄ , and 1 mM DTT) for 30 min at room temperature. The beads were pelleted by centrifuge at 500 × g and washed twice with the binding buffer. Hundred microliters of 501iM nanobodies were added and incubated for 30 min at 4 °C, followed by eluting with the binding buffer plus 10 1iM reduced glutathione (Sigma, cat#G4251). Eluted samples were analyzed on SDS-PAGE. [0265] NMR. NMR experiments were conducted on a Bruker Avance III spectrometer operating at 800 MHz, equipped with a triple-channel ¹ H, 13C, ¹⁵ N cryogenically cooled probe. Data were processed using TopSpin (Bruker) and analyzed using CCPNmr Analysis (51). [0266] Samples of 50 mM ¹⁵ N-labeled ZnF456 (BCL11A aa 738-835) were measured in PBS buffer pH = 7.4, 1 mM dithiothreitol (DTT) and 10% v/v ² H ₂ O at 25 °C. Interactions were tested in the presence of 1:1 molar equivalent of nanobodies (with or without 1.1:1 molar equivalent of 12-mer DNA). [0267] Combined chemical shift perturbations were calculated as [(∆o1H) ² + (0.102 ·∆o15N) ² ] ^1/2 . SD to the mean was calculated excluding outliers with values higher than 3 × SDM according to previously reported procedure (52). [0268] Crystallization of ZF6-Nanobody Complex. Sequences encoding ZF6 and nanobodies were sub-cloned into the PETDuet-1 vector (Sigma-Aldrich, cat#7116) and coexpressed in Escherichia coli Rosetta (DE3) (Novagen). The complex was purified on a nickel column in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 1iM ZnSO ₄ . Next, the His-SUMO tag at the N terminus of ZF6 was removed by Ulp1 protease and Q column (Cytiva). Additional nanobody protein was removed from the complex on Hiload 16/600 Superdex 75 g gel filtration column (Cytiva). Samples used for crystallization were measured for final absorbance of ~50 cm ⁻¹ using a Nanodrop spectrophotometer (Thermo Scientific). Crystals were obtained by mixing 11iL of complex solution and 1.21iL of reservoir solution. Crystals of ZF6-Nb6101 complex were grown from 0.1 M Bis-Tris propane, pH 6.0- 7.0, 0.2 M NaKPO ₄ and 18 to 20% PEG 3350. Crystals of ZF6-Nb5344-N74D complex were grown from 0.1 M HEPES, pH 7.5 to 8.2, 2.2M Li ₂ SO ₄ . All crystals were cryo-protected using corresponding reservoir buffers and flash-frozen in liquid nitrogen. Diffraction data sets were collected at the Stanford Synchrotron Radiation Lightsource. All diffraction data sets were processed with iMosflm (53). The phases of complexes were solved by the selenium single- wavelength anomalous diffraction (SAD) method using PHENIX (54). Iterative cycles of crystallographic refinement were performed using PHENIX. Coot was used for model building manually (55). The structure figures were prepared using PyMOL (available on the world wide wed at www.pymol.org). [0269] Maturation of Nanobodies by Protein Design. BCL11A loops (residue 793 to 800 and 825 to 831) were built around the nanobody with distance around 4 Å in the ZF6-Nb6101 complex structure using the software Coot (55). Before running a Program database (PDB) file through Rosetta (28), water molecules and all ligands that were non-essential to the protocol were removed. The residues on the nanobody–ZF6 interface were defined in the resfile file. The input complex was relaxed while restraining the atoms to their starting positions. This allowed Rosetta to relieve clashes while preventing the structure from moving too far from what was experimentally determined. Through a RosettaScripts XML file, a single round of fixed backbone design was performed. Interface residues on the nanobody were redesigned, and those on the ZF6 side were repacked. After the design step, metrics of interest were then evaluated including the score, the solvent-accessible surface buried in contact and binding energy to rank the designs and select the best models to move forward for further validation by expression of protein and affinity measurements by alpha-screen. [0270] SPR. Twin-Strep-tagged ZF456 was immobilized to a single flow cell of a sensor chip CM5 (Cytivia) coated with the Strep-Tactin®XT (Twin-Strep-tag® Capture Kit, Iba, cat# 2- 4370-000) using a Biacore T200 (GE Healthcare). The chip was regenerated using 3 M GuHCl. Three samples containing only running buffer, composed of 10 mM HEPES pH 7.5, 150 mM NaCl and 0.005% Tween 20, were injected over both ligand and reference flow cells, followed by nanobodies serially diluted from 7 nM to 1 pM, with a replicate of the 30 nM concentration. The resulting data were double-reference subtracted and fit to a 1:1 binding model using the Biacore T200 Evaluation software. [0271] Alpha-Screen Assay. The assay was performed in 384-well plates where a constant concentration of biotinylated ZF456 protein was incubated with varying concentrations of His- tag nanobodies for 30 min at room temperature. Ten microliters of streptavidin donor beads and 10 pL of nickel chelate (Ni-NTA) acceptor beads (PerkinElmer, cat# 6760619C) in assay buffer were added and incubated in the dark for 1 h. Centrifuge for 15 s at 161 × g. The fluorescent signal was measured by a plate reader at 580 nm after excitation at 680 nM. The interaction of the two proteins brings the beads in proximity, leading to energy transfer from one bead to the other, and a burst in fluorescent signal at 520 to 620 nm which correlates with the strength of the interaction. [0272] Cell Culture. Human HEK293T cells (female) were purchased from ATCC. Cells were cultured in DMEM, high glucose (Thermo Fisher Scientific, 11965) with 10% FCS and 2 mM L-Glutamine. HUDEP-2 cells (RCB4557) were obtained from Riken BioResource Research Center and cultured as reported before (56). Cells were maintained in expansion medium containing StemSpan serum-free expansion medium (SFEM, Stemcell Technologies), 2% Penicillin–Streptomycin solution (10,000 U/mL stock), 3 IU/mL Epoetin alfa (Epogen, Amgen), 0.4 ig/ mL dexamethasone, 1 ig/mL doxycycline, and 50 ng/mL recombinant human stem cell factor (SCF, Stemcell Technologies). The differentiation was achieved by switching expansion medium to EDM-2 medium, which contains Iscove’s modified Dulbecco’s medium (IMDM), 1% L-glutamine (this is in addition to the L-glutamine present in IMDM), 2% Penicillin–Streptomycin solution (10,000 U/ mL stock concentration), 330 ig/mL human holo- transferrin, 10 ig/mL recombinant human insulin solution, 2 IU/mL heparin, 5% inactivated human plasma type AB, 3 IU/mL Epoetin alfa, 100 ng/mL SCF and 1 ig/mL doxycycline. After 4d cells were transferred to EDM-3 medium (same as EDM-2 medium but without SCF) and cultured for another 3 d. Finally, cells were moved to EDM medium (no doxycycline) for 2 d. [0273] Peripheral blood-derived CD34 ⁺ cells were obtained from the NIDDK-Center of Excellence in Hematology at the Fred Hutchinson Cancer Research Center and cultured as described before (57). In brief, cells were cultured in erythroid differentiation medium (EDM) which contains IMDM supplemented with stabilized glutamine, 330 ig/mL holo-human transferrin, 10 ug/mL recombinant human insulin, 2 IU/mL heparin Choay, and 5% inactivated human plasma type AB. The expansion procedure comprised three phases. In the first phase (Days 0 to 7), 10 ⁴ /mL CD34+ cells were cultured in EDM in the presence of 10 ⁻⁶ M hydrocortisone (Stemcell Technologies), 100 ng/mL SCF, 5 ng/mL IL-3 (Stemcell Technologies), and 3 IU/mL Epoetin alfa. On Day 4, 1 volume of cell culture was diluted in four volumes of fresh medium containing SCF, IL-3, Epoetin alfa, and hydrocortisone. In the second phase (Days 7 to 11), the cells were resuspended at 10 ⁵ /mL in EDM supplemented with SCF and Epo. In the third phase (Days 11 to 18), the cells were cultured in EDM supplemented with Epo alone. Cell counts were adjusted to a range of 7.5 × 10 ⁵ to 1 × 10 ⁶ on Day 11. [0274] BCL11A Degradation in HEK293T Cells. 100K HEK293T cells were seeded in a six-well plate and transfected with 1.5 pg total amount of DNA by lipofectamine 2000 (80 ng BCL11A-HA, 800 ng Nb-Fc or Nb, and 620 ng mCherry-Trim21). Cells were sorted 24 h later followed by expanding for 48 h. 500K cells were collected for protein extraction. Forty micrograms of protein was loaded for Western blotting with anti-HA antibody (Invitrogen, cat#26183) to detect BCL11A level. The expression of fusion nanobody proteins was confirmed with anti-flag M2 antibodies (sigma, cat#F1804-50UG). [0275] BCL11A Degradation in Erythroid Progenitor Cell Lines. Lentivirus particles were collected from HEK293T supernatant 3 days after cotransfection of psPAX2, VSVG, and pLVX-EF1a-IRES-Puro (Addgene, cat# 85132) plasmid constructs containing Nb6101-19 or Nb6101-19-Fc. The supernatant was filtered at 0.45 pm before storage at −80 °C. HUDPEP2 cells, ZF456 deletion HUDEP2 cells (deletion of BCL11A 724-835), and CD34+ cells were transduced with lentivirus particles at a multiplicity of ~0.1 transducing units per cell for 24 h. GFP-positive cells were sorted by flow cytometry. Protein extracted from the cells was used for Western blot with anti-BCL11A antibody (Abcam, cat#191401). RNA from differentiated HUDEP2 cells on day 0, day 4, and day 7 was prepared for quantitative RT-PCR. RNA from differentiated CD34+ cells was extracted on day 0, day 8, and day 12. [0276] Quantitative Reverse Transcription PCR (RT-qPCR). Total RNA from cells was obtained with the RNeasy Plus Mini Kit (QIAGEN Cat# 74134), followed by reverse transcription to produce cDNA by using the iScript cDNA Synthesis Kit (BioRad, Cat# 1708890). Quantitative real-time PCR was performed with primers and iTaq universal SYBR Green supermix (BioRad, #1725120) on a Biorad CFX384 real-time system (C1000 Touch Thermal Cycler). The level of globin transcripts was quantified by Cq values and normalized to a GAPDH internal control. [0277] Data, Materials, and Software Availability. The crystallographic structure for Nb6101-ZF6 (PDB 8DTN) and Nb5344N74D-ZF6 (PDB 8DTU) has been deposited to the publicly accessible database Protein Data Bank available on the world wide web at www. rcsb.org/structure. References 1. E. Tse et al., Intracellular antibody capture technology: Application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein. J. Mol. 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The identification of such ligands for therapeutically relevant but “undruggable” proteins remains challenging. Herein, employed is a yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and /3-thalassemia. This strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins. The following describes the experiments, their results and conclusions in further detail. [0279] Introduction: Proteolysis targeting chimeric molecules (PROTACs) and molecular glue degraders, such as the immunomodulatory imide drugs, hijack the cellular protein ubiquitination machinery to specifically degrade proteins of interest (POIs). ^1,2 They offer exciting opportunities for use as therapeutics and serve as powerful research tools for biological inquiry. ^3,4 While PROTAC molecules are being used clinically with notable success and great promise, ⁵ it remains challenging to target many therapeutically relevant protein families due to challenges inherent to ligand discovery and optimization. This is especially true for transcription factors (TFs), which generally contain unstructured domains and lack obvious “ligandable” pockets. ^6,7 Additionally, the development of PROTACs requires a substantial synthetic effort to test various combinations of recruited ubiquitin E3 ligases and linkers that are optimal for forming a ternary complex of the PROTAC, POI, and ubiquitin E3 ligase. ⁸ Other degradation platforms, including the degradation tag system ⁹ and transcription factor targeting chimeras, ¹⁰ have been developed for difficult protein targets. However, the utility of these platforms is either limited to engineered proteins, or their specificity for the targeted proteins remains unexplored. [0280] Unlike the small molecule ligands that are typically used in PROTACs, antibodies exploit features of protein surfaces to recognize antigens and show exceptional specificity and remarkable affinity for their antigens. Even fragments of single variable heavy chain domains, termed nanobodies (Nbs), retain antigen specificity and can be used as the POI ligand in a PROTAC. The recent development of a yeast surface display platform to screen large libraries of synthetic nanobodies provides a straightforward and low-cost method to obtain Nb ligands for proteins. ¹¹ While protein-based degraders have been explored, ¹²⁻¹⁸ their potential is hindered either by the challenge of delivering these ligands to intracellular targets or because the ligands do not target endogenous proteins. Appending a cell-penetrating moiety to Nb degraders can overcome these limitations and provide a broad strategy to degrade endogenous proteins, including poorly structured targets for which small molecule ligands do not exist. To exemplify the approach, the transcription factor BCL11A, which is a clinically validated target for the treatment of hemoglobin disorders, including /3-thalassemia and sickle cell disease (SCD) was focused on. ^19,20 [0281] Reactivation of fetal hemoglobin (HbF, α2γ2) is a promising strategy to ameliorate clinical severity in patients with hemoglobin disorders. ²¹ Patients with SCD who produce elevated HbF exhibit significantly improved survival rates. ²² BCL11A represses HbF expression through direct binding at the γ-globin promoters, eliciting the switch from fetal to adult hemoglobin (HbA, α ₂ /3 ₂ ) expression during erythropoiesis. ^19,23,24 Genetic approaches, notably clustered regularly interspaced short palindromic repeats (CRISPR)−Cas9 ^25,26 editing and RNA interference, ²⁷ have been used to down-regulate BCL11A in patients and validated the clinical utility of disabling BCL11A. However, the resource-intensive care and high cost of ex vivo genetic manipulation of cells in clinical trials limit the application of these treatments. PROTACs can provide an alternative means to deplete BCL11A in a temporal, reversible, precise, and cost-effective manner. [0282] PROTAC development for BCL11A faces a central challenge in that there is no available small molecule ligand specific for the protein. This is largely due to the vast amount of predicted structural disorder and the protein’s high similarity to a paralog, BCL11B. To overcome this challenge, protein-based ligands were first identifiedusing a library of synthetic nanobodies displayed on the surface of yeast cells. Expression of top hits from the screen fused to the Fc domain of Immunoglobulin G1 led to Trim21-mediated loss of BCL11A but not its paralog BCL11B, indicating that the ligands are specific. Further functionalization of a top- performing candidate for cell penetration and E3 ligase recruitment created a cell-permeant, protein-based degrader for the degradation of endogenous BCL11A. Moreover, loss of BCL11A in response to treatment with the degrader resulted in a significant induction of fetal hemoglobin. Results: Target Selection and Ligand Screen. [0283] Although BCL11A is predicted to be largely unstructured, the protein contains several well-ordered regions, including a CCHC-type zinc finger domain (ZnF0) that might mediate self-association ²⁸ and six C2H2-type zinc finger domains (ZnF1, ZnF23, and ZnF456) (Figure 28A). These well-folded domains were targeted to identify Nb ligands for BCL11A. Because of the close sequence similarity between BCL11A and its paralog BCL11B in all of the zinc finger regions, ²⁹ ligands that bind to these domains might demonstrate affinity for both paralogs. With the aim of achieving specificity in ligands selected for BCL11A, ZnF23 and an “extended” zinc finger domain (exZnF23), which includes the C-terminal unstructured region that diverges in sequence between the two paralogs were produced (Figure 28A). The extended protein fragment, which is 69.3% identical to BCL11B (the ZnF23 fragment is 93.2% identical), was expressed in Escherichia coli and purified, and the recombinant protein was used in a screen of yeast surface display to identify synthetic Nbs binders (Figure 28B). An initial Nb hit (wt2D9) was produced in E. coli (Figure 33A), and its affinity for BCL11A was assessed using a pull-down assay (Figure 33B) and MicroScale Thermophoresis (MST, Figure 33C). Affinity maturation through random mutagenesis of wt2D9 was performed, after which 7 Nbs with better affinities were obtained (Figure 34). Of these, 2D9_V102G (hereafter referred to as 2D9) and 2D9_W108L were selected for additional studies because of their stability, high affinity, and specificity for exZnF23 of BCL11A (Figure 28C). Size-exclusion chromatography coupled with multiangle light scattering revealed that BCL11A exZnF23 is monomeric and formed a stable complex with 2D9 (Figure 35). A crystal of 2D9 complexed with exZnF23 of BCL11A was obtained, but insufficient electron density precluded modeling of BCL11A. Nonetheless, the high-resolution structure of 2D9 combined with maturation mutagenesis data suggested that some of the interaction with BCL11A is mediated through a loop in Complementarity Determining Region (CDR) 3 of the Nb (Figure 36). Functionalization of Ligands for Cell Penetration [0284] Ligands intended for depletion of BCL11A must be delivered to the nucleus of erythroid precursor cells. Because Nbs are too large and unfavorably charged to traverse the plasma membrane, 2D9 was functionalized for cell penetration by appending a cell permeant miniature protein called ZF5.330−32 (Figure 29A). MST measurements demonstrated that appending ZF5.3 to 2D9 did not significantly alter the affinity of 2D9 for BCL11A exZnF23 (Figure 29B). A pull-down assay using purified 2D9 or the fusion protein ZF5.3-2D9 added into the lysate of human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells ³³ confirmed their association with endogenous, full-length BCL11A (Figure 29C). Cellular entry and protein localization of ZF5.3-2D9 were first monitored by confocal imaging. Experiments in which ZF5.3-2D9 was incubated with HUDEP-2 cells that were subsequently immunostained revealed accumulation of a significant fraction of the protein localized to the nucleus (Figure 29D). Immunoblotting demonstrated a dose- (Figure 29E) and time-dependent cellular uptake (Figure 29F), and cell fractionation showed that the fusion protein was largely present in the nucleus for at least 24 h (Figure 29G). Co-immunoprecipitation of BCL11A from HUDEP-2 cells incubated with ZF5.3-2D9 further revealed its cellular entrance and binding to BCL11A (Figure 29H). Nanobody-Mediated Degradation of BCL11A [0285] Having demonstrated that ZF5.3-2D9 penetrated HUDEP-2 cells and engaged BCL11A, the cell-permeant Nb was used as a handle to mediate the proteasomal degradation of BCL11A. The rational design of PROTACs is difficult due to poor understanding of rules that govern formation of the ternary complex between the POI, ubiquitin E3 ligase, and the PROTAC. Unlike small molecule PROTACs, “all protein” degraders using reengineered E3 ligases are reported to exhibit high flexibility to various targets. ^12,15 However, these ligands are rarely cell permeant and do not degrade endogenous proteins, thereby limiting their utility. [0286] To confirm that 2D9 can mediate selective degradation of BCL11A, plasmids of the Nb fused to the Fc domain of Immunoglobulin G1 (Nb-Fc) or Trim21 were produced. With these designs, Trim21 would mediate proteasomal degradation via the Trim-Away method. ¹⁸ As expected, lentiviral trans-duction of HUDEP-2 cells with Nb 2D9 or 2D9_W108L did not affect BCL11A expression. However, transduction of 2D9-Fc or 2D9_W108L-Fc induced significant loss of BCL11A (Figure 30A). Similar experiments were performed with 2D9- wtTrim21, 2D9_W108L-wtTrim21 and their corresponding variants (2D9-mutTrim21 or 2D9_W108L-mutTrim21). As shown in Figure 30B, Nb-wtTrim21 induced BCL11A down- regulation, but no change was observed when mutTrim21 was used. To assess the specificity of the Nbs for BCL11A, BCL11A or BCL11B were overexpressed in HEK293T cells and transfected the cells with Nb-Fc and Nb-Trim21; as with HUDEP-2 cells, Nb-Fc and Nb- wtTrim21 induced loss of BCL11A. However, neither promoted loss of BCL11B (Figure 30C and 30D). Together, these data indicate that Nbs 2D9 and 2D9_W108L can distinguish BCL11A from BCL11B and target endogenous BCL11A with high specificity. [0287] Cell-permeant, nanobody-based degraders for BCL11A were designed by incorporating two ubiquitin E3 ligases: engineered SPOP (speckle type POZ protein) and RNF4. SPOP is an E3 adaptor protein that functions in complex with cullin-3 (CUL3); it is composed of a substrate binding MATH domain and a CUL3-binding BTB domain. RNF4 is an E3 ligase that contains an N-terminal SUMO substrate binding site and a C-terminal RING domain responsible for dimerization and E2 binding. By replacing the native substrate recognition domain of SPOP and RNF4 with ZF5.3-2D9, the proteins ZF5.3-2D9-tSPOP and ZF5.3-2D9-tRNF4 were created (Figure 31A). Both proteins were expressed and purified from E. coli (Figure 37) and were delivered in pure forms to HUDEP-2 cells through incubation (Figure 38). Upon their delivery, BCL11A levels were lowered steadily over time (Figure 31B, and Figure 39). With ZF5.3-2D9-tSPOP, the loss was striking, as up to 70% of BCL11A was depleted within 12 h of incubation with 10 μM of the degrader (Figure 31C). Moreover, the cell viability was minimally affected 24 h after treatment (Figure 40). Because of its far greater degradation effect than ZF5.3-2D9-tRNF4, ZF5.3-2D9-tSPOP was used for subsequent studies. With this degrader protein, loss of BCL11A in HUDEP-2 cells was sustained for at least 72 h (Figure 41). To exclude the possibility that the truncated SPOP delivered might have negative effects on the HbF repressive role of endogenous SPOP, ³⁴ the construct ZF5.3-tSPOP was created, where ZF5.3 was conjugated directly to the BTB domain of SPOP. In control experiments with ZF5.3-tSPOP, BCL11A levels were verified to remain unchanged (Figure 31D). Treatment with a proteasome inhibitor (MG-132) prevented the degradation of BCL11A, thereby confirming that the loss of the protein proceeded via proteasomal degradation in a ligand-dependent manner (Figure 31E). Fetal Hemoglobin Induction [0288] Endogenous expression of BCL11A in HUDEP-2 cells varies during differentiation (Figure 42) and represses the expression of the fetal-stage γ-globin genes. Given the sustained loss of BCL11A in response to treatment with the degrader in undifferentiated HUDEP-2 cells, its effect on differentiated HUDEP-2 cells was explored. HUDEP-2 cells were first transduced with 2D9, 2D9_W108L, and their corresponding Fc/Trim conjugates (2D9-Fc, W108L-Fc, 2D9-Trim21, and W108L-Trim21). The downregulation of BCL11A by Nb-Fc or Nb-Trim21 induced a significant increase of γ-globin transcripts in HUDEP-2 cells; no increase was observed for Nb only or Nb-mutTrim21 (Figure 43). Next, whether the cell-permeant degrader can also promote fetal hemoglobin induction was investigated. HUDEP-2 cells were treated with ZF5.3-2D9-tSPOP on Days 0 and 3 of differentiation, and samples were collected on Days 3 (prior the second treatment), 4, and 7 for analysis (Figure 32A). RT-qPCR of hemoglobin transcripts in samples from Day 7 (Figure 32B) revealed an increase of γ-globin transcripts in HUDEP-2 cells treated with ZF5.3-2D9-tSPOP. Immunoblots revealed that the degradation of BCL11A was maintained throughout and that significant fetal hemoglobin (HbF) was induced by Day 4 (Figure 32C). Fluorescence activated cell sorting of HbF-immunostained HUDEP-2 cells provided additional confirmation that treatment with ZF5.3-2D9-tSPOP resulted in a 3.5- fold increase of the HbF ⁺ population, whereas treatment with ZF5.3-tSPOP, an effect similar to that of untreated cells (Figure 32D). These results further confirmed that the HbF reactivation observed is due to 2D9-dependent proteasomal degradation of BCL11A. [0289] The effect of ZF5.3-2D9-tSPOP in human primary CD34 ⁺ progenitor cells was further investigated. The cells were cultured under differentiation conditions (Figure 44) and treated with ZF5.3-2D9-tSPOP on Day 8 of the differentiation (Figure 32E). RT-qPCR of hemoglobin transcripts in samples from Day 13 showed that upon treatment of ZF5.3-2D9-tSPOP, γ-globin increased to 30% of the total /3-like hemoglobin. As observed with the experiments in HUDEP- 2 cells, no induction of HbF was detected when CD34 ⁺ cells were treated with ZF5.3-tSPOP or with a second construct that replaced 2D9 with a Nb targeting GFP (ZF5.3-GNb-tSPOP, Figure 32F). Immunoblots of samples from Days 9−13 revealed sustained degradation of BCL11A over 5 days and marked HbF induction as of Day 11 (Figure 32G). As shown in Figure 32H, a range of a 2.4- to 3.9-fold increase in the levels of HBG transcripts was observed upon ZF5.3-2D9-tSPOP treatment in CD34 ⁺ cells from multiple donors, suggesting little donor-to-donor variability in globin induction. Additionally, treatment with the degrader did not affect the differentiation of CD34 ⁺ cells, as assessed by flow cytometric analyses of cell surface markers CD235a and CD36 (Figures 32I, and Figure 45. Although a similar percentage of viable differentiating CD34 ⁺ cells was observed, the cell proliferation rate was ∼2-fold slower in samples treated with the degrader when compared to control (Figure 46). This slower expansion effect is similar to what others have reported, ³⁵ and might be due to the loss of BCL11A as opposed to off-target toxicity. Regardless, these data together provide convincing evidence that a single treatment of the degrader ZF5.3-2D9-tSPOP is sufficient to permeate erythroid precursor cells, significantly deplete BCL11A, and elicit an increase in the expression of HbF. DISCUSSION [0290] Targeted protein degradation is attractive in that it has the potential to modulate proteins that have historically been intractable to conventional small molecule inhibitors. However, a central challenge remains: a small molecule ligand for the protein of interest must first be available to enable degrader synthesis. Because of this, a significant portion of the human proteome remains “undruggable”. Among these proteins are transcriptional regulators (activators and repressors), intrinsically disordered proteins, and scaffolding proteins. Successful modulation of these proteins with targeted protein degradation tools would invariably expand the scope of “druggable” human proteome. While difficult to be controlled with small molecules, many “undruggable” proteins contain surfaces to which proteins (or other large biomolecules) can bind, and these biomolecular ligands can be used as handles for protein modulation. In this work, a cell-permeant nanobody was used to degrade a traditionally undruggable protein. BCL11A was selected to demonstrate the utility of this approach because of its clinical significance for the /3-hemoglobin disorders and because of its high sequence similarity to a close paralog, BCL11B. 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Cell Rep.2016, 16 (12), 3181−3194. Materials and Methods [0291] Safety hazards. No unexpected or unusually high safety hazards were encountered. [0292] Cell culture. HUDEP-2 cells (RCB4557) were obtained from Riken BioResource Research Center (Japan). Cells were cultured according to the reported method (33). In brief, cells were maintained in expansion medium, which contains StemSpan serum-free expansion medium (SFEM, Stemcell Technologies), 2% Penicillin-Streptomycin solution (10,000 U/mL stock), 50 ng/mL recombinant human stem cell factor (SCF, Stemcell Technologies), 3 IU/mL Epoetin alfa (Epogen, Amgen), 0.4 tg/mL dexamethasone, and 1 tg/mL doxycycline. For differentiation, cells were transferred from expansion medium to EDM-2, which contains Iscove’s modified Dulbecco’s medium (IMDM), 1% L-glutamine (this is in addition to the L- glutamine present in IMDM), 2% Penicillin-Streptomycin solution (10,000 U/mL stock concentration), 330 tg/mL human holo-transferrin, 10 tg/mL recombinant human insulin solution, 2 IU/mL heparin, 5% inactivated human plasma type AB, 3 IU/mL Epoetin alfa, 100 ng/mL SCF and 1 tg/mL doxycycline. After culturing for 4 days, cells were transferred to EDM-3 (same as EDM-2 but without SCF) and cultured for another 3 days. After that, cells were cultured in EDM (no doxycycline) for 2 days. [0293] Human HEK293T cells (female) were purchased from ATCC. The cells were cultured in DMEM, high glucose (Thermo Fisher Scientific, 11965) with 10% FCS and 2 mM L- Glutamine. [0294] Peripheral blood-derived CD34 ⁺ cells from multiple donors were obtained from the NIDDK-Center of Excellence in Hematology at the Fred Hutchinson Cancer Research Center. The cells were cultured according to the reported method ¹ . In brief, cells were cultured in erythroid differentiation medium (EDM) which contains IMDM supplemented with stabilized glutamine, 330 ug/mL holo-human transferrin, 10 ug/mL recombinant human insulin, 2 IU/mL heparin Choay, and 5% inactivated human plasma type AB. The expansion procedure comprised 3 steps. In the first step (Day 0 to Day 7), 10 ⁴ /mL CD34 ⁺ cells were cultured in EDM in the presence of 10 ^-6 M hydrocortisone (Stemcell Technologies), 100 ng/mL SCF, 5 ng/mL IL-3 (Stemcell Technologies), and 3 IU/mL Epoetin alfa. On Day 4, 1 volume of cell culture was diluted in 4 volumes of fresh medium containing SCF, IL-3, Epoetin alfa, and hydrocortisone. In the second step (Day 7 to Day 11), the cells were resuspended at 10 ⁵ /mL in EDM supplemented with SCF and Epo. In the third step (Day 11 to Day 18), the cells were cultured in EDM supplemented with Epo alone. Cell counts were adjusted to a range of 7.5 × 10 ⁵ - 1 × 10 ⁶ on Day 11. Beyond Day 18, the culture medium containing Epo was renewed twice a week. [0295] Plasmid construction. All gene blocks were obtained from GENEWIZ while primers were obtained from Integrated DNA Technologies. KOD hot start polymerase was used for PCR reaction, NEBuilder HiFi DNA Assembly was used for plasmid fusion. NdeI, Xhol and T4 ligase were purchased from New England Biolabs.pET-20b 2D9 Plasmid Construction [0296] The sequence of 2D9 with a N-terminal Strep-tactin tag was codon-optimized for expression in E. coli and cloned into a linearized pET-20b vector at the NdeI and XhoI restriction sites. pET-20b ZF5.3-2D9 Plasmid Construction [0297] The sequence of ZF5.3 was codon optimized for expression in E. coli and cloned into a linearized pET20b_2D9 plasmid with a N-terminal Strep-tactin tag. pET-28a ZF5.3-2D9 -tSPOP Plasmid Construction: [0298] The sequence of SPOP167-374 was codon-optimized for expression in E. coli and cloned into a linearized pET-20b_2D9 plasmid with a N-terminal Strep-tactin tag. After the HiFi assembly, pET-20b_ZF5.3-2D9-tSPOP was digested with restriction enzymes NdeI and Xhol and ligated into pET-28a containing a N-terminal His6 tag to obtain pET-28a_ZF5.3- 2D9-tSPOP plasmid. pET-20b ZF5.3-2D9-tRNF4 Plasmid Construction: [0299] The sequence of RNF475-194 was codon-optimized for expression in E. coli and cloned into a linearized pET-20b_2D9 plasmid with a N-terminal Strep-tactin tag. [0300] Expression and purification of proteins ZnF23, exZnF23 of BCLJJA and exZnF23 of BCLJJB. The cDNA of ZnF23 (residues 372-430) and exZnF23 (residues 372-484) of human BCL11A, exZnF23 of human BCL11B (residues 422-528) were cloned into pET28a vector and expressed as N-terminal His6-tag fusion proteins in E. coli. Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT). After sonication and centrifugation, the supernatant was applied to the Ni ²⁺ -NTA resin (Qiagen) equilibrated with lysis buffer and incubated at 4°C for 1 hr. The resin was washed with wash buffer I (50 mM Tris-HCl pH 8.0, 1 M NaCl, 10 mM imidazole, 1 mM DTT), then wash buffer II (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT), and stepwise eluted with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 100/200/300/500 mM imidazole, 1 mM DTT). The eluate was examined by SDS-PAGE stained with Coomassie blue. Fractions containing the target proteins were combined and then dialyzed with dialysis buffer (1×PBS, 150mM NaCl, 2mM DTT) at 4 °C overnight and concentrated before loading onto the HiLoad 16/600 Superdex 75 prep-grade column (Cytiva) equilibrated with the purification buffer (1×PBS, 1 mM DTT). Purified proteins were concentrated, flash frozen in liquid nitrogen and stored at -80°C for yeast screening and binding assays. [0301] Isolation of BCL11A-exZnF23-binding nanobodies from yeast synthetic library. Purified exZnF23 of BCL11A protein was labeled with AlexaFluor647 dye (Invitrogen) or fluorescein isothiocyanate (FITC) (Sigma-Aldrich) according to the manufacturer’s protocols. [0302] For the first round of magnetic-activated cell sorting (MACS), 1x10 ¹⁰ S. cerevisiae cells expressing a surface displayed library of synthetic nanobodies (11) were centrifuged, resuspended in selection buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 5 mM maltose) and then incubated with anti-AlexaFluor647 microbeads (Miltenyi) at 4°C for 40 min. The yeast cells were then passed through an LD column (Miltenyi) to remove any yeast expressing nanobodies that non-specifically interacted with the microbeads. Yeast cells that flowed through the column were centrifuged, resuspended in selection buffer, and incubated with 1 µM of AlexaFluor647-labeled exZnF23 of BCL11A at 4°C for 1 hr. Yeast cells were then centrifuged, resuspended in selection buffer with anti-AlexaFluor647 microbeads, and incubated at 4°C for 20 min before passing through an LS column (Miltenyi). The eluted yeast cells were collected and expanded to a subsequent round of MACS to further enrich for exZnF23-binding nanobodies. The second round of MACS was performed similarly to the first round but using fluorescein isothiocyanate (FITC)-labeled exZnF23 of BCL11A and anti-FITC microbeads. After MACS selections, yeast cells were co-stained with AlexaFluor647- and FITC-labeled exZnF23 of BCL11A and sorted by flow cytometry (Sony SH800Z). Double- positive yeast cells were selected and plated as single colonies, which were randomly picked and grown as clonal populations in 96-well plates. Yeast cells in 96-well plates were induced, stained with AlexaFluor647- or FITC-labeled exZnF23 of BCL11A and analyzed by the plate reader. Yeast DNA was extracted using standard methods and sequenced from the high activity clones. [0303] Affinity maturation of nanobody wt2D9. Error-prone PCR was performed on nanobody wt2D9 DNA using the GeneMorph II Random Mutagenesis Kit (Agilent) and the resulting library was scaled up with a second PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs).100 mL of BJ5465 S.cerevisiae cells were grown to OD600nm of 1.8 and were then made into electrocompetent yeast cells with 100 mM lithium acetate ² . The electrocompetent cells were transformed with 56 jig of the error prone library and 17 jig of linearized pYDS649 plasmid (11) using an ECM 830 Electroporator (BTX-Harvard Apparatus) with 500 V and 15 ms single pulse. The resulting library of nanobody 2D9 mutants has a mean mutation rate of about 1 amino acid change per nanobody clone. [0304] 1×10 ⁶ yeast cells from the error prone library were stained with anti-HA AlexaFluor488 antibody in selection buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 5mM maltose, 1mM DTT) to assess nanobody expression levels. In order to obtain high-affinity binders to exZnF23 of BCL11A, 4 rounds of MACS selections were performed, and the yeast cells were stained with 1jiM of FITC-labeled exZnF23, 1jiM of FITC-labeled exZnF23, 1.5jiM of His6- SBP-exZnF23, and 1 jiM of FITC-labeled exZnF23 to enrich for binders with higher affinities. After MACS selections, 2 rounds of FACS were performed. In the first round of FACS, the yeast cells were co-stained with 0.5 jiM of AlexaFluro647-labeled exZnF23 and 0.75jiM of FITC-labeled exZnF23. A total of ~80,000 yeast cells from the first round of FACS were expanded and used for a second round to further enrich for high affinity nanobodies. In second round of FACS, the yeast cells were co-stained with 0.1 jiM of AlexaFluro647-labeled exZnF23 and 0.15 jiM of FITC-labeled exZnF23. After the second round of FACS, approximately 5,000 yeast cells were plated as single colonies using serial dilutions.96 yeast clones were randomly selected, mini-prepped and sequenced to reveal consensus mutations contributing to affinity. The sequence of wt2D9 is found in Table 5. Nanobody degrader production Transformation of plasmids in BL21 cells. [0305] 2 tL of plasmid (concentration ≥20 ng/tL) containing either 2D9, ZF5.3-2D9, ZF5.3- 2D9 -tSPOP, ZF5.3-2D9 -tRNF4, or ZF5.3-tSPOP was added to a thawed tube of E. coli BL21 cells and incubated on ice for 15 min. The cells were heat shocked at 42 °C for 45 s and placed on ice for 2 min.900 tL of LB media were added and the cells cultured for 1 h at 37 °C while shaking at 200 rpm. Cells were centrifuged for 3 min at 2500 × g and 900 tL LB media was removed. The recovered cells were resuspended in 100 µL remaining media and added to ampicillin-(100 µg/mL) or kanamycin-containing (50 µg/mL) agar plates and incubated at 37 °C overnight. 2D9 expression and purification [0306] The plasmid encoding Strep-2D9 was used to transform E. coli BL21 cells. Individual colonies were selected on the basis of Amp resistance and used to inoculate 50 mL of LB media supplemented with Amp (100 mg/L). The primary culture was grown overnight and then used to inoculate 6 L of ZYM-5052 autoinduction (AI) media ³ supplemented with ampicillin, which was then allowed to grow at 37 °C with shaking at 200 rpm. When the OD600nm reached 0.5, the temperature was changed to 18 °C and the cells cultured overnight. The cells were harvested by centrifugation at 6000 × g for 30 min at 4 °C, resuspended in buffer containing 25 mM Tris- HCl pH 8.0, 300 mM NaCl, and 10% glycerol, supplemented with 1 mM PMSF and lysed via microfluidization. The lysate was clarified by centrifuging at 15,000 × g for 30 min at 4 °C and the cleared lysate manually added to a column with 5 mL Strep-Tactin® Sepharose® resin (IBA Lifesciences). The column was washed with 5 column volumes of 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol. Then protein was eluted from the resin using 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol supplemented with 2 mM of desthiobiotin. Elution fractions were analyzed by SDS-PAGE, and fractions containing the desired protein combined and concentrated using spin concentrators (Millipore). Following concentration, proteins were additionally purified via size exclusion chromatography (Cytiva HiLoad 26/600 Superdex-200 pg column, #GE28-9898-36). Pure proteins were concentrated, flash frozen in liquid nitrogen, and stored at −80 °C until further use. ZF5.3-2D9 expression and purification [0307] The plasmid encoding Strep-ZF5.3-2D9 was used to transform E. coli BL21 cells. Individual colonies were selected based on ampicillin resistance and used to inoculate 150 mL of LB media supplemented with ampicillin (100 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with ampicillin, which was then allowed to grow at 37 °C with shaking at 200 rpm. At OD600nm of 0.5, protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After culturing for overnight at 18 °C, cells were harvested by centrifugation (6000 × g, 30 min at 4 °C), resuspended in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1 mM ZnSO4, and 10% glycerol, supplemented with 1 mM PMSF, and lysed by microfluidization. The purification of this protein was identical to that of 2D9, with the exception that 0.1 mM of ZnSO4 was added to all buffers. ZF5.3-2D9-tSPOP expression and purification [0308] The plasmid encoding His6-ZF5.3-2D9-tSPOP was used to transform E. coli BL21 cells. Individual colonies were selected based on kanamycin resistance and used to inoculate 150 mL of LB media supplemented with kanamycin (50 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with kanamycin, which was then allowed to grow at 37 °C with shaking at 200 rpm. At OD600nm of 0.5, protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After culturing for overnight at 18 °C, cells were harvested by centrifugation at 6000 × g for 30 min at 4 °C), resuspended in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol, supplemented with 1 mM PMSF, and lysed by microfluidization. The lysate was centrifuged at 15,000 × g for 45 min at 4 ^o C, and to the clear lysate was added 8 M urea, pH 8.0. The lysate was manually added to a column with 3 mL Ni-NTA resin and washed with 300 mL of buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, and 8M urea. After that, protein was eluted from the resin using 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 8M urea supplemented with 100 mM of imidazole. The elution fractions were analyzed by SDS page and concentrated to 1 mL. The purified proteins were refolded with 100 mL buffer containing 25mM Tris-HCl pH 8.0, 300 mM NaCl, 500 mM L-arginine, 9 mM glutathione and 1 mM glutathione disulfide. The refolded protein was cleared by centrifugation at 15,000 × g for 45 min at 4 ^o C, passed through a 0.22 μm filter, and concentrated for future use. ZF5.3-2D9-tRNF4 expression and purification [0309] The plasmid encoding Strep-ZF5.3-2D9-tRNF4 was used to transform E. coli BL21 cells. Individual colonies were selected based on ampicillin resistance and used to inoculate 150 mL of LB media supplemented with kanamycin (100 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with kanamycin, which was then allowed to grow at 37 °C with shaking at 200 rpm. At OD600nm of 0.5, protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After culturing overnight, cells were harvested via centrifugation, resuspended in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol, supplemented with 1 mM PMSF, and lysed by microfluidization. The inclusion bodies were obtained by centrifugation at 15,000 × g for 45 min at 4 ^o C, and solubilized in 300 mM NaCl, 25 mM Tris-HCl pH 8.0, and 8 M urea. After another round of centrifugation at 15,000 × g for 45 min at 4 ^o C, the solubilized inclusion bodies (~2 mL) were mixed dropwise with 200 mL of refolding buffer containing 300 mM NaCl, 25 mM Tris-HCl pH 8.0, 500 mM L-arginine, 9 mM glutathione and 1 mM glutathione disulfide. The refolded mixture was cleared by centrifugation at 15,000 × g for 45 min at 4 ^o C and passed through a 0.22 μm filter. The refolded proteins were concentrated and saved for future use. ZF5.3-tSPOP expression and purification [0310] The plasmid encoding His6-ZF5.3-tSPOP was used to transform E. coli BL21 cells. The expression and purification of this protein was identical to that of ZF5.3-2D9-tSPOP. [0311] Protein binding assays. Purified His6-ZnF23, His6-exZnF23 of BCL11A and His6- exZnF23 of BCL11B were immobilized on the Ni ²⁺ -NTA resin (Qiagen) equilibrated with the binding buffer (25mM Tris-HCl pH 8.0, 150mM NaCl, 1mM DTT). After 1h incubation at 4°C, beads were washed with the binding buffer. Purified nanobody proteins were then added to the beads and incubated at 4°C for another 1h. Beads were washed for at least three times with the binding buffer. The bound proteins were separated with SDS-PAGE and stained with Coomassie blue. [0312] Molecular mass analysis. Molecular masses were analyzed by SEC-MALS with miniDawn Multi-Angle Light Scattering (MALS) detector, Optilab refractive index detector (Wyatt Technology, Santa Barbara, CA, USA) and UV (Waters 2487, Waters Corporation, Milford, MA) detectors. Volumes of injection is 15 µl (about 200 ng for each sample). The proteins were centrifuged at 13000 rpm for 10 min and then applied to pre-equilibrated Superdex 200 Increase 3.2/300 GL SEC column (Cytiva) with buffer containing 25 mM Tris- HCl, pH 8.0, 300 mM NaCl, 100 μM ZnSO4 and 7 mM BME. Proteins were separated using a mobile phase flow rate of 0.15 mL/min at room temperature. Molecular masses were calculated using the Astra software (version 6) provided with the instrument. [0313] Microscale thermophoresis assay. Purified His6-exZnF23 of BCL11A at a concentration of 1mg/mL was labeled with Alexa Fluor 647 dye according to the manufacturer’s instruction (Invitrogen). After labeling, 100 nM BCL11A exZnF23 in buffer containing 25mM HEPES and 150 mM NaCl was used. 25 μL of 2D9 or ZF5.3-2D9 in the same buffer at the concentration of 67 and 22.6 μM, respectively, were used for 1:1 serial dilution into PCR-tubes. For each sample, 10 i.iL of labelled BCL11A exZnF23 was mixed with 10 i.iL ligand. Samples were loaded into capillaries and measured by MST with 40% LED power and medium MST power. Data was analyzed by the software MO Affinity Analysis (NanoTemper) using the “Kd” model and plotted in Prism. The equation below was used for fitting: Conc: Concentration Unbound: Response value of unbound state Bound: Response value of bound state TargConc: final concentration of fluorescent molecules [0314] Crystallization of nanobody. Sparse-matrix crystallization screens were performed with purified proteins at concentrations of about 11 mg/ml. Crystals were obtained by the sitting-drop vapor diffusion method by mixing 0.5 μL protein and 0.5 μL precipitant solution at 20°C. 2D9 nanobody crystals appeared in the SaltRX-F3 condition containing 1.5 M Ammonium sulfate, 0.1 M Tris-HCl, and pH 8.5 after 4 weeks of incubation at 20 °C. Crystals were cryoprotected with paraffin oil before being flash-cooled and stored in liquid nitrogen. [0315] Crystal data collection, processing, and structure determination. All diffraction datasets were collected at 100 K at the Stanford Synchrotron Radiation Lightsource (Menlo Park, CA, USA). 2D9 datasets were collected at a fixed wavelength at 0.97946 Å, using an Eiger 16M detector at beamline BL12-1. All diffraction datasets were indexed and processed by XDS 4, 5. The structure was phased by molecular replacement in Phenix (Phaser) using model of 1NLB-H ⁶ . Subsequent density modification gave excellent electron-density maps, which allowed the building of the model. Iterations of refinement were carried out with Phenix Refine, and model building was performed in Coot. All structural figures were prepared using PyMOL. [0316] Western blot. After treatment, cells were washed with PBS buffer and lysed using lysis buffer (1% SDS, 150 mM NaCl, 0.1 U Benzonase nuclease (Santa Cruz biotechnology, #sc- 391121B), EDTA-free protease inhibitor cocktail (Bimake, #B14002), 20 mM Tris-HCl, pH 8.0). Protein extracts were quantified by Pierce BCA Protein Assay Kit (Thermo Scientific, #PI23235) with a Nanodrop One ^C (Thermo Scientific) according to the manufacturer’s instruction. Protein lysates (~ 50 µg/lane) were resolved by SDS-PAGE and transferred onto poly (vinylidene difluoride) (PVDF) membranes. Membranes were incubated with 5% non-fat milk in 0.1% Tween 20/PBS for 1 h. The blots were then probed with the relevant primary antibodies in blocking solution at 4°C overnight with gentle agitation. Membranes were washed 5 min with 0.1% Tween 20 in PBS three times and were incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Antigens were detected by addition of ClarityTM Western ECL Substrate (Biorad, #1705060). The membranes were visualized by chemiluminescence on a GE Amersham Imager 600. Antibodies used: Anti-BCL11A (Santa Cruz biotechnologies, #sc-514842), StrepMAB-Classic HRP (IBA, #2-1509-001), Anti-His-HRP (Sigma-Aldrich, #A7058), Anti-GAPDH (Santa [0317] Cruz Biotechnology, #sc-365062), Anti-Lamin B1 (Proteintech, #66095-1), Anti-β hemoglobin (Sigma Aldrich, #WH0003043M1), Anti g-hemoglobin (Proteintech, #25728-1- AP). Secondary antibodies used: Anti-Mouse-HRP (Abcam, #ab6728), Anti-Rabbit-HRP (Promega, #W4018). [0318] BCL11A co-immunoprecipitation from HUDEP-2 cell lysate.8×10 ⁶ HUDEP-2 cells was lysed with 400 μL cell lysis buffer (containing 25 mM Tris, 150 mM NaCl, 1% Triton- X100, 1% protease inhibitor cocktail, and 0.1 % SDS) for 10 min on ice to obtain the cell lysate. To 180 μg of total protein in the cell lysate was added 20 μg pure Nb proteins or buffer (as control). The cell lysate was first incubated for 3 h at 4 ^o C with gentle agitation and then incubated with 100 μL Strep-Tactin® Sepharose® for 4h at 4 ^o C. After that, resin was centrifuged (1000 × g, 2 min), and supernatant was carefully collected as flow-through. The resin was then washed with lysis buffer, centrifuged (1000 × g for 2 min), and the supernatant removed carefully. Finally, resin was incubated with elution buffer containing 2.5 mM desthiobiotin, centrifuged, and the eluate collected in the supernatant. Protein input and the eluate were immunoblotted with StrepMAB-Classic HRP and anti-BCL11A antibodies. [0319] Protein delivery. 4 × 10 ⁵ cells were washed with PBS twice and seeded into 24-well plate in 500 μL serum free medium or medium containing proteins (as indicated in figures) for 45 min at 37 ^o C. After this, the cells were collected via by centrifugation (300 × g for 5 min) and incubated in full expansion medium at 37 ^o C for 45 min (or as indicated in the figures). Finally, the cells were harvested by centrifuge at 300 × g for 5 min and lysed for western blot. [0320] Nucleus isolation. To isolate the nuclei, HUDEP-2 cells were first lysed with lysis buffer containing 25 mM Tris, 150 mM NaCl, 1% Triton-X100, 1% protease inhibitor cocktail, and 0.1% SDS for 10 min on ice. After this, cells were spun down at the maximum speed to get the nuclei at the bottom as white pellets. The supernatant was collected as cytosolic fractions. The nucleus fraction was then washed three times with PBS buffer and lysed with buffer containing 25 mM Tris, 1% SDS, 150 mM NaCl, 0.1% benzonase, and 2 mM EDTA to obtain the nuclear proteins. [0321] Confocal imaging. ZF5.3-2D9V102G at the concentration of 10 μM was delivered into 4 × 10 ⁵ HUDEP-2 cells, and cells were cultured for another 24 h in full expansion medium at 37 ^o C. After that, cells were washed with PBS for 3 times, and seeded into confocal plates coated with poly-lysine (Fisher Scientific, #80824). Cells were cultured at 37 ^o C for 1h to allow settling down after seeding. Cells were then and fixed by addition of 4% paraformaldehyde for 20 min at 4 ^o C and permeabilized by adding 0.1% Triton-X100 in PBS for 5 min. After staining with Anti-Strep, and Anti-Rab7 (Cell Signaling, #9367) at 4 ^o C overnight, fixed cells were washed with 0.1% Tween 20 in PBS for 3 times, cells were then incubated with 1 μg/mL DAPI, secondary antibodies Goat anti-rabbit IgG (H+L) -488 (Thermo Scientific, #A32371) and Goat anti-Mouse-IgG (H+L) -Alexa Fluor 647 (Thermo Scientific, #A21235) for 1h at room temperature. Cells were washed 3 times with Tween 20 in PBS and imaged with a Zeiss LSM980 with Airyscan 2 Confocal Microscope with 60 × oil immersion objective at excitations of 653, 493 and 353 nm. [0322] AlphaScreen assay. The assay was performed in a light grey 384-well AlphaPlate (PerkinElmer) containing 10 nM biotinylated Avi-tagged nanobody protein and a serial dilution of His6-tagged exZnF23 of BCL11A in a total volume of 20 μL in AlphaScreen buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, mM DTT, 0.1% Tween, 0.1% BSA). Reaction mixtures were incubated at RT for 30 min. Streptavidin Donor beads and nickel chelate (Ni- NTA) AlphaScreen Acceptor beads PerkinElmer) each at a concentration of 10 μg/mL were added to the mixture. The plate was incubated at RT for 1 hr in the dark and then analyzed by a plate reader. [0323] Transduction or transfection of Nb-Fc, Nb-Trim21 and Nb-mutTrim21. To generate nanobody-Fc, nanobody-wtTrim21 and nanobody-mutTrim21 fusions, the nanobody coding sequence followed by the hIgG1-Fc coding sequence (pFuse-hIgG1-Fc1; Invivogen), or full- length wild-type Trim21 or mutant Trim21 (M10E/M72E) were subcloned into the lentiviral vector pLVX-EF1a-IRES-ZsGreen (Clontech #631982). Lentiviruses were packaged in HEK293T cells as described previously ^7. [0324] For expression in HEK293T cells, plasmids were transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Cells were harvested 24 hours after transfection, subjected to SDS-PAGE and analyzed by Western Blotting. [0325] HUDEP-2 cells were transduced with lentiviruses at a confluency of 2×10 ⁵ cells/mL. GFP positive cells were FACS sorted 72hr post-transduction. After sorting, cells were collected at Day 7 after differentiation for Western Blotting and RT-qPCR. [0326] RT-qPCR. Quantitative real-time PCR Quantitative real-time PCR was performed to quantify RNA abundance. For each sample, total RNA was isolated by using the RNeasy Plus Mini Kit (QIAGEN Cat# 74134), followed by cDNA synthesis using the iScript cDNA Synthesis Kit (BioRad, Cat# 1708890). qRT-PCR primers were ordered from Integrated DNA Technologies. Quantitative real-time PCR was performed using the PrimePCR assay with the iTaq universal SYBR Green supermix (BioRad, #1725120) and run on a Biorad CFX384 real- time system (C1000 Touch Thermal Cycler) according to the manufacturer instructions. Cq values were used to quantify RNA abundance. The relative abundance of the hemoglobin was normalized to a GAPDH internal control by using this equation: ∆Cq = Cq (gene of interest) – Cq (GAPDH) R = 2-∆Cq [0327] Analytical flow cytometry of HbF ⁺ cells. Cells were harvested and washed twice with PBS buffer and centrifuged at 350-500 × g for 5 min. Then, cells were fixed with 4% paraformaldehyde for 20 min at 4 ^o C and permeabilized with 0.2 % Triton-X100 in PBS for 5 min. Following that, 100 μL of 0.1% BSA in 0.1% Tween 20/PBS buffer was added for blocking, and cells were incubated with anti g-hemoglobin (Proteintech, #25728-1-AP) for 2 h, washed 3 times with 0.1% Tween 20 in PBS buffer and stained with Goat anti-rabbit IgG (H+L) -488 (Thermo Scientific, #A32371) for 1 h at room temperature. After washing 3 times with 0.1% Tween 20 in PBS buffer, cells were analyzed on a BD Accuri ^TM C6 Plus cell sorter. [0328] Analytical flow cytometry of differentiated CD34 ⁺ cells. 10 ⁶ CD34 ⁺ cells were collected on different days and centrifuged at 500 × g for 5 min. Following that, cells were washed with PBS buffer and fixed with 4% paraformaldehyde for 10 min at room temperature. Following that, cells were washed with PBS buffer twice and stored in 4 ^o C in PBS buffer. After all samples were collected, cells were stained with 100 μL of 0.1% CD235a-APC (BD Bioscience, #551336) and 0.1% CD36-FITC (Biolegend, #336232) in PBST containing 0.1% BSA for 1h at room temperature. Cells were washed 3 times with PBST and analyzed on a BD Accuri ^TM C6 Plus cell analyzer and sorter. [0329] HUDEP-2 cells viability. HUDEP-2 cells at the density of 2 × 10 ⁵ cells/ml with or without ZF5.3-2D9-tSPOP delivered were seeded into 96-well plates for 24 h. Cell viability was measured by CellTiter-Glo® Luminescent Cell Viability reagent (CellTiter reagent, G7570, Promega) according to the manufacturer’s instruction. The luminescence was measured using a microplate reader (Infinite 200 pro, TECAN). [0330] Cell counting and viability measurement.10 μL cells in medium was mixed with 10 μL 0.4% Trypan Blue solution and counted by Countess 3 cell counter. [0331] Statistics. All data reported were mean ± SD. Statistical analysis represents p values obtained from one-way ANOVA, two-way ANOVA or unpaired Student’s t-test where necessary; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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