WO2023215877A1 - Compositions and methods for targeted protein degradation - Google Patents

Compositions and methods for targeted protein degradation Download PDF

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WO2023215877A1
WO2023215877A1 PCT/US2023/066674 US2023066674W WO2023215877A1 WO 2023215877 A1 WO2023215877 A1 WO 2023215877A1 US 2023066674 W US2023066674 W US 2023066674W WO 2023215877 A1 WO2023215877 A1 WO 2023215877A1
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polypeptide
bcl11a
fusion polypeptide
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Stuart H. Orkin
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Boston Childrens Hospital
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    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • TPD Targeted protein degradation
  • PROTACs proteolysis-targeting chimeras
  • E3 ubiquitin ligases or their associated subunits, e.g. cereblon or von Hippel-Lindau (VHL) proteins, to recruit a protein of interest (POI) to the proteasome for degradation.
  • a bispecific construct including binding domains for a target polypeptide and an erythroid-enriched E3 ubiquitin ligase can act as a tether, bringing the target polypeptide into close proximity with the erythroid-enriched E3 ubiquitin ligase.
  • the E3 ubiquitin ligase involved is expressed in an erythroid-enriched manner, such a construct will function to selectively channel the target polypeptide into the degradation pathway in erythroid cells.
  • the E3 ubiquitin ligase itself is fused to a molecule that specifically binds a desired target polypeptide.
  • compositions for targeting desired polypeptides for degradation and methods of using them are described herein.
  • the constructs described herein can be used therapeutically to target desired polypeptides expressed in erythroid cells for degradation.
  • Non-limiting examples include the transcription factor BCL11A, which is involved in repression of fetal hemoglobin (HbF) expression in adult erythroid lineage cells.
  • Targeting compositions and methods described herein can promote the degradation of BCL11A in erythroid cells, thereby promoting re-expression of HbF in those cells, which can provide therapeutic benefit for subjects suffering from ⁇ - hemolglobinopathies.
  • a fusion polypeptide comprising a binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and a binding domain that specifically binds an erythroid-enriched polypeptide of interest.
  • the fusion polypeptide mediates degradation of the polypeptide of interest in erythroid cells.
  • the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase specifically binds TRIM10 or TRIM58.
  • the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase comprises an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
  • the binding domain that specifically binds an erythroid-enriched polypeptide of interest comprises an antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
  • the polypeptide of interest is selected from BCL11A, LRF and ZNF410.
  • the E3 ubiquitin ligase is TRIM10 or TRIM58.
  • the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
  • ZNF6 zinc-finger 6
  • the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
  • ZNF 23 is shorthand for zinc fingers 2 and 3, or a polypeptide fragment of BCL11A including those zinc fingers
  • ZNF456 is shorthand for zinc fingers 4, 5 and 6, or a polypeptide fragment of BCL11A including those zinc fingers.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
  • amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2.
  • variation relative to SEQ ID NO: 12 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.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
  • amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids at amino acid number 102 or 108.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50; CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40) and ADQDVYPYEYL (SEQ ID NO: 41).
  • the fusion polypeptide further comprises a cell-penetrating peptide.
  • the fusion polypeptide further comprises a linker peptide between the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and the binding domain that specifically binds an erythroid-enriched polypeptide of interest.
  • described herein is a nucleic acid comprising sequence encoding a fusion polypeptide as described herein.
  • the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
  • described herein is a vector comprising a nucleic acid comprising sequence encoding a fusion polypeptide as described herein.
  • the vector is a viral vector.
  • the viral vector is an AAV vector.
  • described herein is a method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to an erythroid cell.
  • a method of targeted degradation of BCL11A comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a cell expressing BCL11A.
  • the targeted degradation of BCL11A is targeted to erythroid cells.
  • a method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
  • a method of treating a hemoglobinopathy disorder comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
  • a fusion polypeptide comprising a TRIM10 or TRIM58 polypeptide fused to a binding domain that specifically binds a target polypeptide.
  • the binding domain that specifically binds a target polypeptide comprises an antigen-binding domain of an antibody.
  • the binding domain that specifically binds a target polypeptide comprises an scFv, a single domain antibody or a nanobody.
  • the target polypeptide is expressed in an erythroid cell.
  • the target polypeptide is selected from BCL11A, LRF and ZNF410.
  • the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
  • ZNF6 zinc-finger 6
  • the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
  • amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2. In some embodiments, variation relative to SEQ ID NO: 12 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.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
  • amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids number 102 or 108.
  • amino acid sequence variation relative to SEQ ID NO: 34 occurs only at amino acids number 102 or 108.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50; CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
  • the polypeptide of interest is BCL11A
  • the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40) and ADQDVYPYEYL (SEQ ID NO: 41).
  • the fusion polypeptide further comprises a cell-penetrating peptide.
  • the fusion polypeptide further comprises a linker peptide between the TRIM10 or TRIM58 polypeptide and the binding domain that specifically binds a target polypeptide.
  • described herein is a nucleic acid comprising sequence encoding a fusion polypeptide as described herein.
  • the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
  • a vector comprising a nucleic acid comprising sequence encoding a fusion polypeptide as described herein.
  • the vector is a viral vector.
  • the viral vector is an AAV vector.
  • described herein is a method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to an erythroid cell.
  • a method of targeted degradation of BCL11A comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a cell expressing BCL11A.
  • the targeted degradation of BCL11A is targeted to erythroid cells.
  • a method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
  • HbF fetal hemoglobin
  • a method of treating a hemoglobinopathy disorder comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
  • the terms “increased”, “increase”, “enhance”, “activate” are all used herein to refer to an increase by a statistically significant amount.
  • 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.
  • the term “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • “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.
  • “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.
  • 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.
  • 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).
  • 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.
  • the term “consisting essentially of” refers to those elements required for a given embodiment.
  • Figs 1A-1C show erythroid cell enriched expression of TRIM10 and TRIM48
  • Fig. 1A The modular structures of TRIM10 and TRIM58.
  • Fig.1B RNA expression of E3 ligases during cell maturation.
  • Fig.1C Heat-map depicting the relative expression of UBE20, TRIM10, and TRIM58 across cell types. The dots indicate erythroid cells.
  • Fig 2 shows fusion of TRIM10/58 to the recognition domain of cereblon (CRBN). Schematic showing the fusion proteins of TRIM10/58 with the CRBN recognition domain along with control contructs.
  • Fig 3 shows degradation of BCL11A protein by TRIM10/58. Western blot for BCL11A depicting fusions of TRIM10/58 with the recognition domain of CRBN were capable of degrading tagged BCL11A upon addition of dTAG47 PROTAC. WT-CRBN served as a positive control and inactive form of dTAG47 served as a negative control.
  • Fig 4 shows degradation of BCL11A protein by TRIM10/58 in HUDEP-2 erythroid cells.
  • HUDEP-2 cells with CRBN or lacking CRBN were infected with lentiviruses expressing CRBN cDNA or TRIM10 fused with the recognition domain of CRBN and then dTAG47 PROTAC was added.
  • BCL11A protein was degraded in the presence of dTAG47 in CRBN- cells rescued with CRBN cDNA and the fusion of TRIM10 with the recognition domain of CRBN leads to a reduction in BCL11A protein.
  • Fig.5 shows targeted protein degradation of BCL11A by TRIM10/58 in 293T cells.
  • E3 ubiquitin ligases target proteins in the cells in which they are expressed for degradation via the ubiquitin-mediated proteasome pathway
  • the identification of erythroid-specific or erythroid enriched E3 ubiquitin ligases provides avenues for erythroid specific, targeted degradation of a protein of interest.
  • the ability to target a protein of interest for degradation in an erythroid-enriched manner has therapeutic implications, in that, as but one example, hemoglobinopathies generally stem from defects in cells of the erythroid lineage, and targeted degradation of, e.g., BCL11A, which represses expression of fetal hemoglobin (HbF), can de-repress and promote the expression of HbF, providing a new source of active hemoglobin.
  • BCL11A which represses expression of fetal hemoglobin (HbF)
  • HbF fetal hemoglobin
  • Ubiquitin-mediated protein degradation [00065] The degradation of proteins occurs mainly through two major pathways: autophagy and the ubiquitin proteasome system (UPS), both of which are critical for maintaining cellular homeostasis (see, e.g., Dikic, I., Ann. Rev. Biochem.86: 193-224 (2017)).
  • UPS ubiquitin proteasome system
  • the UPS is a cascade reaction, and an important mechanism for the degradation of misfolded and damaged proteins, as well as a regulated mechanism that ensures short lives of certain regulatory proteins, among others.
  • Degradation of a protein via the ubiquitin pathway generally involves two discrete and successive steps: (a) covalent attachment of multiple ubiquitin molecules to the protein substrate – so called “ubiquitination” or “ubiquitylation;” and (b) degradation of the ubiquitin-targeted protein by the 26S proteasome complex, with the release of free and reusable ubiquitin.
  • ubiquitination or “ubiquitylation” refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers.
  • the ubiquitin-proteasome system consists of several components that act in concert.
  • ubiquitin an evolutionarily conserved protein of 76 residues (human ubiquitin has the amino acid sequence: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDY NIQKESTLHLVLRLRGG (SEQ ID NO: 1), is activated in its C-terminal Gly to a high- energy thiol-ester intermediate. This reaction is catalyzed by the ubiquitin-activating enzyme, E1.
  • E2s After ubiquitin activation, one of several ubiquitin-carrier proteins or ubiquitin- conjugating enzymes, referred to as E2s, transfers the activated ubiquitin moiety from E1 to a member of the ubiquitin-protein ligase family, E3, to which the substrate protein is bound. E3 catalyzes the last step in the conjugation process, covalent attachment of ubiquitin to the substrate protein.
  • the first ubiquitin moiety is transferred to the ⁇ -NH2 group of a lysine residue of the protein substrate to generate an isopeptide bond.
  • a polyubiquitin chain is generated by processive transfer of additional activated ubiquitin moieties to a lysine residue (one of the most prominently used is Lys48, or K48, but each of K6, K11, K27, K29, K33, K63 and the N-terminal methionine 1 (Met1) are also functional sites in various ubiquitination reactions) of the previously conjugated ubiquitin molecule.
  • the polyubiquitin chain serves as a recognition marker for the 26S proteasome.
  • the binding of the substrate to E3 is generally specific and indicates that E3 ubiquitin ligases play a major role in recognition and selection of proteins for conjugation and subsequent degradation.
  • E3 Ubiquitin Ligases [00067] Hundreds of different E3 ubiquitin ligases have been identified. These are reviewed and classified in, e.g., Yang et al., Molecular Biomedicine 2: 23 (2021). Different types of E3 ubiquitin ligases have low sequence homology and large differences in composition.
  • the tripartite motif (TRIM) protein family is a subset of the so-called RING-type E3 ligase subfamily.
  • TRIM10 and TRIM58 are expressed in an erythroid-enriched manner.
  • erythroid enriched refers to the preferential expression and activity of the TRIM10 and TRIM58 E3 ubiquitin ligases in cells of the erythroid lineage.
  • preferential in this context is meant that TRIM10 and TRIM58 are most highly expressed in cells of this lineage, relative to their expression in other cell types or tissues.
  • TRIM10 and TRIM58 renders these E3 ubiquitin ligase polypeptides useful for the targeted degradation of a protein of interest in an erythroid-enriched manner.
  • 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.
  • TRIM10 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: 2) and polypeptide (e.g., Isoform 1: NCBI Ref Seq: NP_006769.2, SEQ ID NO: 3).
  • TRIM10 can refer to human TRIM10, including naturally occurring variants, molecules, and alleles thereof.
  • SEQ ID NO: 2 is a mRNA sequence for TRIM10. Note: U’s replaced with T’s in this representation.
  • 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: 4) and polypeptide (e.g., NCBI Ref Seq: NP_056246.3, SEQ ID NO: 5). TRIM58 can refer to human TRIM58, including naturally occurring variants and alleles thereof.
  • Antibodies that specifically bind TRIM58 are known; see, e.g., Novusbio Trim58 Antibody (2H3) H00025893-M02.
  • Single domain antibody fragments or nanobodies can be screened using a fully in vitro platform based on yeast surface display as in McMahon et al Nature 25: 289-296 (2016).
  • SEQ ID NO: 4 is a mRNA sequence for TRIM58. Note: U’s replaced with T’s in this representation. 1 gcagaccgcg aggggagacg gtgcgggcgg ccgggagcgc agccctccgg gaggcgggtc 61 atggcctggg cgccgccgg ggagcggctg cgaggatg cgcggtgccc ggtgtgcctg 121 gatttcctgc aggagccggt cagcgtggac tgcggccaca gcttctgcct caggtgcatc 181 tccgagttct gcgagaagtc ggacggcgcg cagggcggcg tactacgctg tccgcagtgcatc 181
  • a binding agent that specifically binds TRIM10 or TRIM58 can be joined to a binding agent that specifically binds a protein of interest.
  • a bispecific construct can bring the protein of interest into close proximity with the TRIM E3 ubiquitin ligase, resulting in ubiquitination of the protein of interest, thereby promoting the targeted degradation of the protein of interest in an erythroid-enriched manner.
  • the binding agent that binds either or both of the TRIM polypeptide and the protein of interest can be, for example, antibodies or antigen-binding fragments thereof.
  • the binding agents can be joined, for example, by a chemical or peptide linker, e.g., a flexible peptide linker, including, but not limited to a serine/glycine-rich peptide linker.
  • a TRIM10 or TRIM58 polypeptide can be directly fused, e.g., as a fusion polypeptide, to a binding agent that specifically binds a protein of interest.
  • the polypeptide of interest upon introduction to a cell expressing the polypeptide of interest, the polypeptide of interest is brought into close proximity with the TRIM E3 ubiquitin ligase, resulting in ubiquitination of the protein of interest, thereby promoting the targeted degradation of the protein of interest.
  • the binding agent that binds the protein of interest can be, for example, an antibody or antigen-binding fragment thereof.
  • the binding agents can be joined, for example, by a chemical or peptide linker, e.g., a flexible peptide linker, including, but not limited to a serine/glycine-rich peptide linker.
  • Erythroid targets for degradation [00074] Any polypeptide expressed in an erythroid cell, whether erythroid specific, erythroid enriched, or simply expressed in an erythroid lineage cell, can be target for proteasome- mediated degradation using constructs as described herein.
  • Non-limiting examples of erythroid-enriched factors include LRF, ZNF410, and BLC11A.
  • LRF Leukemia/Lymphoma-Related Factor
  • ZBTB7A Zinc Finger and BTB Domain Containing 7A
  • FBI-1 Factor binding IST protein 1
  • Pokemon is a member of the POK family of transcription factors that possesses a POZ-domain at the N- terminus and Krüppel-like zinc finger at the c-terminus. See, e.g., Gupta et al., Cancer Lett. 483, 22-34 (2020), which is incorporated herein by reference in its entirety. LRF is involved in several fundamental biological processes including cell proliferation, differentiation, and development.
  • LRF also negatively regulates SMAD4-DNA complex.
  • LRF LRF/ZBTB7A
  • mRNA e.g., LRF variant 2 NCBI Ref Seq NM_001317990.2, SEQ ID NO: 6
  • polypeptide e.g., LRF: NCBI Ref Seq: NP_001304919.1, SEQ ID NO: 7
  • Antibodies for specific detection or binding to LRF/ZBTB7A are available, for example, from R&D Systems, see Human/Mouse ZBTB7A/Pokemon Antibody (clone 466407) MAB3496; from Origen, see Pokemon (ZBTB7A) Mouse Monoclonal Antibody (clone BC17-2.9F), AM32711PU-N; from Novusbio, see ZBTB7A/Pokemon Antibody (clone 3T2J1) NBP3-15914; from Abcam, see Anti-ZBTB7A antibody (EPR13178B) ab175918; and from Invitrogen, see Pokemon (LRF) monoclonal antibody (13E9) 14-3309-82.
  • LRF can refer to human LRF, including naturally occurring variants and alleles thereof.
  • SEQ ID NO: 6 is a transcript variant 2, mRNA of human LRF.
  • U’s replaced with T’s in this representation.
  • 1 actgccgcct cccggccct cggagggagc cagcccagcc gcagccgccg ccaccgccgc 61 cgccggggcc gggcccctc gcgccccgggaaggag gtctcggcgc ggaagatggc 121 cggcggcgtg gacggcccca tcgggatccccgttcccgac cacagcagcg acatcctgag 181 tgggctgaac gagcagcgga cgcagggcct gtgtgtg
  • a binding agent e.g., an antibody or antigen-binding fragment thereof specific for TRIM10 or TRIM58 and a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for LRF can be fused, optionally via a peptide linker.
  • TRIM10 or TRIM58 polypeptide itself can be fused to a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for LRF.
  • Introduction of either or both of these constructs to a cell e.g., an erythroid cell, for example as discussed herein for BCL11A-targeting constructs, can target LRF for degradation in those cells.
  • ZNF410 Zinc Finger Protein 410
  • APA-1 Zinc Finger Protein 410
  • ZNF410 binds to the sequence motif 5’-CATCCCATAATA-3’ (SEQ ID NO: 66) in regulatory elements and modulates transcription of target genes.
  • SEQ ID NO: 66 sequence motif 5’-CATCCCATAATA-3’
  • CHD4 a component of the NuRD complex, which leads to repression of fetal globin genes HBG1 and HBG2.
  • ZNF410 Sequences of ZNF410 are known for a number of species, e.g., human ZNF410 (NCBI GeneID: 57862), mRNA (e.g., ZNF410 Isoform a: NCBI Ref Seq NM_001242924.2, SEQ ID NO: 8) and polypeptide (e.g., ZNF410 Isoform a: NCBI Ref Seq: NP_001229853.1, SEQ ID NO: 9).
  • NCBI GeneID 57862
  • mRNA e.g., ZNF410 Isoform a: NCBI Ref Seq NM_001242924.2, SEQ ID NO: 8
  • polypeptide e.g., ZNF410 Isoform a: NCBI Ref Seq: NP_001229853.1, SEQ ID NO: 9).
  • ZNF410 can refer to human ZNF410, including naturally occurring variants and alleles thereof.
  • SEQ ID NO: 8 is an mRNA sequence of human ZNF410. Note: U’s replaced with T’s in this representation.
  • a binding agent e.g., an antibody or antigen-binding fragment thereof specific for TRIM10 or TRIM58 and a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for ZNF410 can be fused, optionally via a peptide linker.
  • TRIM10 or TRIM58 polypeptide itself can be fused to a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for ZNF410.
  • BCL11A B-cell lymphoma/leukemia 11A
  • Evi9 also known as Evi9, CTIP1, or ZNF856
  • 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 (2016) 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.
  • BCL11A 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: 10) and polypeptide (e.g., BCL11A isoform 1: NCBI Ref Seq: NP_075044.2, SEQ ID NO: 11).
  • BCL11A can refer to human BCL11A, including naturally occurring variants and alleles thereof.
  • SEQ ID NO: 10 is a mRNA sequence for Isoform 1 of human BCL11A. Note: U’s replaced with T’s in this representation.
  • 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: 67: ytcttckqpftsawfllqhaqnth); Zinc finger domain 2 amino acids: 377-399 (SEQ ID NO: 68: kscefcgktfkfqsnlvvhrrsh); Zinc finger domain 3 amino acids: 405-429 (SEQ ID NO: 69: ykcnlcdhactqasklkrhmkthmh); Zinc finger domain 4 amino acids: 742-764 (SEQ ID NO: 70: dtceycgkvfkncsnltvhrrsh); Zinc finger domain 5 amino acids: 770-792 (SEQ ID NO:
  • BCL11A can be targeted for degradation in erythroid cells, for example, through introduction of a bispecific construct as described herein that includes an antibody or antigen- binding fragment thereof that specifically binds BCL11A and a binding element that specifically binds TRIM10 or TRIM58.
  • the binding element that specifically binds TRIM10 or TRIM58 can be an antibody or antigen-binding fragment thereof.
  • BCL11A can also be targeted for degradation, for example, through introduction of a fusion construct as described herein that includes an antibody or antigen-binding fragment thereof that specifically binds BCL11A fused to a TRIM10 or TRIM58 polypeptide.
  • the fusion construct can be introduced to erythroid cells in any of a number of ways, including, but not limited to introduction of the construct or a nucleic acid encoding it via lipid nanoparticles, introduction (e.g., as a fusion) via cell-penetrating peptides, or introduction of a vector, e.g., a viral vector, among others, encoding the construct.
  • ANTIBODIES As discussed above, in various embodiments, described herein are antibodies or antigen-binding fragments thereof that specifically bind to BCL11A or to a TRIM10 or TRIM58 polypeptide.
  • 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.
  • CDRs complementarity determining regions
  • 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.
  • 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.
  • 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.
  • 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.
  • an "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.
  • an "antigen” is a molecule that is bound by a binding site on an antibody agent.
  • 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.
  • an 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.
  • an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen- binding domain of a monoclonal antibody.
  • 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).
  • an antibody in another example, includes two heavy (H) chain variable regions and two light (L) chain variable regions, as occurs, for example, in an IgG immunoglobulin.
  • 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.
  • 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.
  • CDR complementarity determining region
  • 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.
  • 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.
  • 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.
  • CDR complementarity determining region
  • a target ligand-binding recognition domain is a single-domain antibody.
  • 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.
  • 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).
  • 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).
  • libraries include immune libraries derived from llama, shark or human immunized with the target antigen; non- immune/na ⁇ ve libraries derived from non-immunized llama, camel, shark or human; or synthetic or semi-synthetic libraries such as VH, VL, VHH or VNAR libraries.
  • the sdAb can be a heavy variable domain (VH).
  • 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.
  • Nbs 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.
  • 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.
  • 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.
  • 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.
  • the affinity can also be expressed as the affinity constant (K A ), which is 1/ K D ).
  • 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 KD 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.
  • a molecule that “specifically binds,” “selectively binds” or “is specific for” a given target will bind with a KD 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.
  • 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.
  • 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.
  • antigen-binding proteins such as an antibody or portion of an antibody as described herein
  • KD dissociation constant
  • KA association constant
  • K D value greater than 10 -4 mol/liter is generally considered to indicate non-specific binding.
  • the KD for biological interactions which are considered meaningful 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 .
  • 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.
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • sandwich competition assays sandwich competition assays
  • 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.
  • 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.
  • 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.
  • 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.
  • a “target” can be one which it is wished to specifically degrade or inhibit, which can be the case when BCL11A is a target.
  • a “target” can be one which it is wished to bind and tether to another moiety, which can be the case when the target is, e.g., a TRIM10 or TRIM58 polypeptide.
  • 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.
  • K D dissociation constant
  • 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.
  • an antibody reagent that specifically binds to TRIM10 or to TRIM58 binds to TRIM10 or TRIM58 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.
  • K D dissociation constant
  • 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.
  • 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.
  • 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.
  • 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.
  • Nanobodies that target BCL11A ZNF6 [000103] The following provides amino acid sequence for nanobodies that specifically bind human BCL11A.
  • SEQ ID NO: 13 ⁇ NB15 QVQLVESGGG LVQAGGSLRL SCAASGSTFS DYAMGWYRQA PGKERELVAV ITASDDITYY 60
  • ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAAAV TPG ⁇ QDLDYW GQGTQVTVSS (119)
  • Nanobodies directed to ZNF23 “extended region” SEQ ID NO: 34 ⁇ 2D9 / Consensus QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTYY (60) ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADQ DVYPYEYWGQ GTQVTVSS (118) SEQ ID NO: 35 ⁇ V102G QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTYY (60) ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADQ DGYPYEYWGQ GTQVTVSS (118) SEQ ID NO: 36 ⁇ W108L QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTY
  • 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: 12 occurs only at the sites listed in Table 2. Table 2: BCL11A ZNF6 nanobody amino acid variations. Compared to Nb14 (SEQ ID NO: 12).
  • Fetal hemoglobin is a tetramer of two adult ⁇ -globin polypeptides and two fetal ⁇ -like ⁇ -globin polypeptides. During gestation, the duplicated ⁇ -globin genes constitute the predominant genes transcribed from the ⁇ -globin locus.
  • ⁇ -globin becomes progressively replaced by adult ⁇ -globin, a process referred to as the "fetal switch.”
  • the developmental switch from production of predominantly fetal hemoglobin or HbF ( ⁇ 2 ⁇ 2) to production of adult hemoglobin or HbA ( ⁇ 2 ⁇ 2) 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.
  • 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)).
  • 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 ⁇ -globin in sufficient amounts, while others involve the failure to produce normal ⁇ -globin entirely. Those disorders specifically associated with the ⁇ -globin protein are referred to generally as ⁇ -hemoglobinopathies. For example, ⁇ -thalassemias result from a partial or complete defect in the expression of the ⁇ - globin gene, leading to deficient or absent HbA.
  • Sickle cell anemia or sickle cell disease results from a point mutation in the ⁇ -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)).
  • the search for treatment aimed at reduction of globin chain imbalance in patients with ⁇ -hemoglobinopathies has focused on the manipulation of fetal hemoglobin ( ⁇ 2 ⁇ 2; HbF).
  • treating or reducing a risk of developing a hemoglobinopathy in a subject means to ameliorate at least one symptom of hemoglobinopathy.
  • the methods described herein feature methods of treating, e.g., reducing severity or progression of, a hemoglobinopathy in a subject.
  • 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.
  • 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.
  • a subject with a diagnosis of SCD with genotype HbSS, HbS/ ⁇ 0 thalassemia, HbSD, or HbSO, and/or HbF ⁇ 10% by electrophoresis is indicated for treatment using compositions and methods as described herein.
  • 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).
  • a control subject or population e.g., a healthy subject or population.
  • 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.
  • 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.
  • SCD single cell disease
  • 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.
  • SCD 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.
  • SCD 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.
  • THAL thalassemia
  • thalassemia refers to a hereditary disorder characterized by defective production of hemoglobin.
  • the term encompasses hereditary anemias that occur due to mutations affecting the synthesis of hemoglobins.
  • the term includes any symptomatic anemia resulting from thalassemic conditions such as severe or ⁇ -thalassemia, thalassemia major, thalassemia intermedia, ⁇ -thalassemias such as hemoglobin H disease.
  • ⁇ -thalassemias are caused by a mutation in the ⁇ ⁇ ⁇ globin chain, and can occur in a major or minor form. In the major form of ⁇ -thalassemia, children are normal at birth, but develop anemia during the first year of life. The mild form of ⁇ -thalassemia produces small red blood cells.
  • Alpha-thalassemias are caused by deletion of a gene or genes from the globin chain.
  • HbF is functional for carrying and delivering oxygen to tissues
  • the re- induction or de-repression of HbF expression provides an avenue for treating ⁇ - hemoglobinopathies.
  • One approach as described herein targets BCL11A, which is responsible for repression of HbF expression.
  • BCL11A 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 [000113]
  • the activity of BCL11A is inhibited or decreased, e.g., by targeted protein degradation.
  • 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 compositions and methods described herein, than a comparable, control cell or population, wherein BCL11A is not targeted for degradation.
  • 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 BCL11A is not targeted for degradation.
  • BCL11A activity can be assayed by determining the amount of BCL11A at the protein level, using techniques standard in the art.
  • BCL11A activity can be determined using a reporter construct, wherein the reporter construct is sensitive to BCL11A activity.
  • BCL11A activity can be assayed by measuring fetal hemoglobin expression at the mRNA or protein level following treatment targeting BCL11A for degradation. Increased expression of endogenous HbF in adult cells treated or contacted with a construct targeting BCL11A for degradation is indicative of de-repression, and therefore reduced activity of BCL11A.
  • Treatment of hemoglobinopathies by targeting BCL11A [000115] BCL11A activity and/or expression has been shown to repress expression of fetal hemoglobin isoforms.
  • inhibition or degradation of BCL11A removes or reduces this repression and permits fetal hemoglobin isoforms to be re-induced, for example, in an adult.
  • targeted degradation of BCL11A via introduction of a targeted protein degradation construct as described herein can treat a hemoglobinopathy.
  • “increasing the fetal hemoglobin levels” in a cell indicates that HbF is at least 5% higher in populations treated with the construct than in a comparable, control population, wherein no BCL11A targeting construct is present.
  • the percentage of HbF expression in a BCL11A targeting construct-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.
  • 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 targeting construct.
  • any method known in the art can be used to measure an increase in fetal hemoglobin expression, e. g. Western Blot analysis of fetal ⁇ -globin protein and PCR quantification of mRNA encoding fetal hemoglobin (e.g., HBG1 or HBG2 mRNAs).
  • HBG1 or HBG2 mRNAs mRNA encoding fetal hemoglobin
  • 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.
  • 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 [000118]
  • 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 to the antibody or antigen-binding fragment thereof.
  • fusion of an antibody or antigen-binding fragment thereof that binds TRIM10 or TRIM58 to an antibody or antigen-binding fragment thereof that binds an erythroid-specific or erythroid- enriched polypeptide can provide an avenue for targeted protein degradation of the erythroid- specific or erythroid enriched polypeptide in erythroid cells.
  • a TRIM10 or TRIM58 polypeptide can be fused to an antibody or antigen-binding fragment thereof that specifically binds an erythroid-specific or erythroid-enriched polypeptide to provide a targeted protein degradation construct for that erythroid-specific or erythroid-enriched polypeptide.
  • TRIM10, TRIM58, or encoding an antibody or antigen-binding fragment thereof that specifically binds TRIM10 or TRIM58 can generate a fusion construct that fuses the TRIM10 or TRIM58 polypeptide or TRIM10 or TRIM58-specific antibody sequence to a given fusion partner, e.g., antibody that binds a target for targeted protein degradation where the sequence for the fusion partner is known. Fusion can optionally include a peptide linker between the fusion partners. In one embodiment, the linker can be a serine/glycine rich linker.
  • sequences for TRIM10, TRIM58, and nanobodies that specifically bind BCL11A are provided herein.
  • Expression and Delivery of Targeted Protein Degradation Constructs [000120]
  • fusion proteins as described herein that target a protein of interest, e.g., an erythroid protein of interest, for degradation can be expressed from a vector as a recombinant polypeptide.
  • Sequences encoding a fusion protein as described herein can be contained in or expressed by a desired vector. Many such vectors useful for transferring exogenous genes into target mammalian cells are available.
  • a vector can be viral or non-viral.
  • 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.
  • An expression vector can direct expression of a polypeptide (e.g., a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein) 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.
  • a vector comprising a nucleic acid encoding a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein as described herein.
  • 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.
  • AAV adeno-associated virus
  • the vector is an AAV vector.
  • 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.
  • 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.
  • 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.
  • a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components.
  • 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.
  • 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.
  • 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.
  • 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.
  • expression control sequences 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.
  • 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.
  • the promoter can be a promoter for an erythroid-enriched or erythroid-specific gene, e.g., a hemoglobin gene.
  • a host cell transformed with a nucleic acid molecule encoding a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein as described herein.
  • 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.
  • 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.
  • 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 lymphatic system, e.g., an hematopoietic cell, an erythroid cell or erythroid precursor, from muscles, any organ, gland, the skin, brain, lung, liver, kidney, etc..
  • 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 ⁇ cells, among others.
  • introduction of nucleic acid encoding the fusion proteins 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.
  • expression from a vector e.g., a viral vector, e.g., an AAV vector as discussed above, can be used to introduce sequence encoding the fusion protein into a cell expressing the target protein (including, but not limited to BCL11A, ZNF410 LRF) to thereby promote degradation of the target protein.
  • the host cell is a stem cell.
  • 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.
  • progenitor cell 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.
  • ES human embryonic stem
  • ICM inner cell mass
  • EG human primordial germ
  • 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.
  • 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.
  • the stem cell is a partially differentiated or differentiating cell.
  • the stem cell is an induced pluripotent stem cell (iPSC), which has been reprogrammed or de-differentiated.
  • iPSC induced pluripotent stem cell
  • Stem cells can be obtained from embryonic, fetal or adult tissues.
  • 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.
  • 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.
  • lineage-restricted precursor cells such as a hematopoietic progenitor cell
  • 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.
  • 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.
  • Hematopoietic stem or progenitor cell 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.
  • 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.
  • cells of the “erythroid lineage”, as the term is used herein, comprise hematopoietic progenitor cells, rubriblasts, prorubricytes, erythroblasts, metarubricytes, reticulocytes, and erythrocytes.
  • 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+.
  • the hematopoietic progenitor cells have several of these markers.
  • the hematopoietic progenitor cells of the erythroid lineage have the cell surface marker characteristic of the erythroid lineage: CD71 and Ter119.
  • a construct or vector encoding a fusion protein as described herein 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.
  • the cells can be autologous to the subject.
  • the cells can be allogeneic to the subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • a viral vector e.g., an AAV vector, among others
  • Such delivery can inhibit the function of the protein targeted for degradation in the target hematopoietic progenitor, erythroid progenitor or erythroid cells.
  • the fusion protein introduced targets BCL11A, such expression can thereby increase HbF expression in the cells and/or their progeny.
  • the degradation-targeting fusion protein can be produced, e.g., as a further fusion with a cell-penetrating peptide to thereby facilitate introduction of the degradation-targeting fusion protein to a target cell.
  • 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.
  • 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).
  • 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.
  • the hematopoietic progenitors are derived from non-autologous sources.
  • the use of iPSCs negates the need for cells obtained from an embryonic source.
  • the stem cells used in the disclosed methods are not embryonic stem cells.
  • 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.
  • differentiated cells e.g., undifferentiated cells
  • 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.
  • the cell to be reprogrammed can be either partially or terminally differentiated prior to reprogramming.
  • 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.
  • reprogramming of a differentiated 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).”
  • 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. [000143] 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Somatic cells 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.
  • 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.
  • the somatic cell is a primary cell line or is the progeny of a primary or secondary cell line.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • markers can include Dnmt3L; Sox15; Stat3; Grb2; ⁇ -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.
  • 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.
  • 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.
  • 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.
  • cell-penetration peptides or cell-penetrating peptides can be used as a transmembrane drug delivery agent for delivery of degradation-targeting fusion polypeptide 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.
  • Delivery of a fusion protein as described herein, or nucleic acids encoding them can include the use of lipid complexes or lipid nanoparticles complexed or loaded with the fusion protein or nucleic acid encoding same.
  • the term “nanoparticle” refers to particles that are on the order of about 10 -9 or one to several billionths of a meter.
  • nanoparticle includes nanospheres; nanorods; nanoshells; and nanoprisms; these nanoparticles may be part of a nanonetwork.
  • 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
  • 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.
  • 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.
  • administering introducing
  • transplanting are used interchangeably in the context of the placement of an agent, e.g. a targeted protein degradation fusion protein or composition 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., a fusion protein as 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.
  • 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.
  • 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.
  • the agent e.g., fusion protein or composition described herein
  • systemically administration refers to the administration of a formulation (whether including cells expressing a fusion protein or composition described herein, or including nucleic acid or a vector encoding a fusion protein or composition described herein, or including an isolated 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.
  • an agent e.g., a fusion protein or composition described herein
  • 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 ⁇ -globin to predominantly ⁇ -globin.
  • the prophylactic administration of, e.g., a modified hematopoietic progenitor cell population can serve to prevent a hemoglobinopathy, as described herein.
  • the agent 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.
  • the term “effective amount” as used herein refers to the amount of an agent (e.g., a fusion protein or composition as 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.
  • terapéuticaally 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.
  • a treatment comprising an agent (e.g., a fusion protein or composition described herein) as described herein for the treatment of a hemoglobinopathy can be determined by the skilled clinician.
  • 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.
  • 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. [000159] 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.
  • 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.
  • 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. [000161]
  • 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.
  • 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 ⁇ g/kg body weight to 100 ⁇ g/kg body weight.
  • 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.
  • the treatment is sustained until, for example, the hemoglobinopathy is treated, as measured by the methods described above or known in the art.
  • 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.
  • 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.
  • 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.
  • the treatment as described herein ameliorates one or more symptoms associated with, e.g., a ⁇ -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.
  • 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.
  • 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.
  • 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.
  • HbF fetal hemoglobin
  • the technology provided herein can further be defined by the following numbered paragraphs.
  • Paragraph 1 A fusion polypeptide comprising a binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and a binding domain that specifically binds an erythroid-enriched polypeptide of interest.
  • Paragraph 2 The fusion polypeptide of paragraph 1 which mediates degradation of the polypeptide of interest in erythroid cells.
  • Paragraph 3 The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase specifically binds TRIM10 or TRIM58.
  • Paragraph 4 The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase comprises an antibody or antigen-binding fragment thereof.
  • Paragraph 5 The fusion polypeptide of any of the preceding paragraphs, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
  • Paragraph 6 The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched polypeptide of interest comprises an antibody or antigen-binding fragment thereof.
  • Paragraph 7 The fusion polypeptide of any of the preceding paragraphs, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
  • Paragraph 8 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is selected from BCL11A, LRF and ZNF410.
  • Paragraph 9 The fusion polypeptide of any of the preceding paragraphs, wherein the E3 ubiquitin ligase is TRIM10 or TRIM58.
  • Paragraph 10 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
  • ZNF6 zinc-finger 6
  • Paragraph 11 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
  • Paragraph 12 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
  • Paragraph 13 The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2.
  • Paragraph 14 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
  • Paragraph 15 The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids at amino acid number 102 or 108.
  • Paragraph 16 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
  • Paragraph 17 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO: 41).
  • Paragraph 18 The fusion polypeptide of any of the preceding paragraphs which further comprises a cell-penetrating peptide.
  • Paragraph 19 The fusion polypeptide of any of the preceding paragraphs, further comprising a linker peptide between the binding domain that specifically binds an erythroid- enriched E3 ubiquitin ligase and the binding domain that specifically binds an erythroid- enriched polypeptide of interest.
  • Paragraph 20 A nucleic acid comprising sequence encoding a fusion polypeptide of any of the preceding paragraphs.
  • Paragraph 21 A nucleic acid of any of the preceding paragraphs, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
  • Paragraph 22 A vector comprising a nucleic acid of any of the preceding paragraphs.
  • Paragraph 23 The vector of any of the preceding paragraphs, which is a viral vector.
  • Paragraph 24 The viral vector of any of the preceding paragraphs, which is an AAV vector.
  • Paragraph 25 A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to an erythroid cell.
  • Paragraph 26 A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell expressing BCL11A.
  • Paragraph 27 The method of any of the preceding paragraphs, wherein the targeted degradation of BCL11A is targeted to erythroid cells.
  • Paragraph 28 A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
  • HbF fetal hemoglobin
  • Paragraph 29 A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
  • Paragraph 30 A fusion polypeptide comprising a TRIM10 or TRIM58 polypeptide fused to a binding domain that specifically binds a target polypeptide.
  • Paragraph 31 The fusion polypeptide of claim 30, wherein the binding domain that specifically binds a target polypeptide comprises an antigen-binding domain of an antibody.
  • Paragraph 32 The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds a target polypeptide comprises an scFv, a single domain antibody or a nanobody.
  • Paragraph 33 The fusion polypeptide of any of the preceding paragraphs, wherein the target polypeptide is expressed in an erythroid cell.
  • Paragraph 34 The fusion polypeptide of any of the preceding paragraphs, wherein the target polypeptide is selected from BCL11A, LRF and ZNF410.
  • Paragraph 35 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
  • Paragraph 36 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
  • Paragraph 37 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
  • Paragraph 38 The fusion polypeptide of claim 37, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2.
  • Paragraph 39 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
  • Paragraph 40 The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids number 102 or 108.
  • Paragraph 41 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
  • Paragraph 42 The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO: 41).
  • Paragraph 43 The fusion polypeptide of any of the preceding paragraphs which further comprises a cell-penetrating peptide.
  • Paragraph 44 The fusion polypeptide of any of the preceding paragraphs, further comprising a linker peptide between the TRIM10 or TRIM58 polypeptide and the binding domain that specifically binds a target polypeptide.
  • Paragraph 45 A nucleic acid comprising sequence encoding a fusion polypeptide of any one of any of the preceding paragraphs.
  • Paragraph 46 The nucleic acid of any of the preceding paragraphs, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
  • Paragraph 47 A vector comprising a nucleic acid of any of the preceding paragraphs.
  • Paragraph 48 The vector of any of the preceding paragraphs, which is a viral vector.
  • Paragraph 49 The viral vector of claim 48, which is an AAV vector.
  • Paragraph 50 A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to an erythroid cell.
  • Paragraph 51 A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell expressing BCL11A.
  • Paragraph 52 The method of any of the preceding paragraphs, wherein the targeted degradation of BCL11A is targeted to erythroid cells.
  • Paragraph 53 A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
  • HbF fetal hemoglobin
  • Paragraph 54 A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of claim 45 or 46, or a vector of any of the preceding paragraphs to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
  • the invention comes in part from investigation into erythroid specific E3 ubiquitin ligases.
  • TRIM10 and TRIM58 are members of the tripartite motif family that includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled- coil region (FIG 1A). Furthermore, TRIM10 and TRIM58 were found to be enriched in erythroid cells as they are highly expressed in that cell type but expressed at much lower levels across other cell types.
  • Known erythroid cell specific E3 ligase UBE2O was used as a positive control (FIG. 1C).
  • TRIM10 or TRIM58 were fused to the recognition domain of cereblon (CRBN), then CRBN -/- HEK293T cells were co-transfected with the E3 ligase construct and BCL11A tagged with FKBP, which binds to dTAG47 PROTAC (a heterobifuncational molecule including a thalidomide derivative and an FKBP12 F36V -selective ligand) (Fig.
  • dTAG47 was added to cells for 48 hrs and cells were collected via fluorescent activated cell sorting (FACS).
  • FACS fluorescent activated cell sorting
  • Western blot analysis with BCL11A antibody was used. Results indicate that fusions of TRIM10/58 with the recognition domain of CRBN are capable of degrading tagged BCL11A upon addition of dTAG47 PROTAC.
  • dTAG47i An inactive form of dTAG47 (dTAG47i) is inactive for degradation and showed no effects, indicating that degradation is dependent on recruitment through the CRBN recognition domain (Fig.3).
  • HUDEP-2 erythroid cells
  • HUDEP-2 cells were tagged with FKBP, which permits the use of dTAG47 PROTAC for recruitment to CRBN for degradation.
  • HUDEP-2 cells lacking CRBN were generated by CRISPR/Cas9 editing.
  • CRBN+ and CRBN- cells were infected with lentiviruses expressing CRBN cDNA or TRIM10 fused with the recognition domain of CRBN and cells were cultured with and without dTAG47 PROTAC.
  • Degradation of BCL11A was analyzed by Western blot using BCL11A antibody.
  • the fusion of TRIM10 with the recognition domain of CRBN led to a reduction in BCL11A.
  • TRIM10 In CRBN- cells, degradation of BCL11A does not occur (Fig. 4).
  • BCL11A could be targeted for degradation by TRIM10/58 using BCL11A nanobodies.
  • 293T cells were transfected with constructs expressing either TRIM10 or TRIM58 fused to a BCL11A-specific nanobody. Analysis by Western blot with BCL11A allowed for detection of endogenous BCL11A.
  • mutant versions of TRIM10 or TRIM58 C16A in which a critical, conserved residue of TRIM proteins is mutated were used. Cells were transfected and assayed 2 days later. Results show marked reduction in BCL11A in cells treated with TRIM10-Nb19 (BCL11A) fusion.
  • BCL11A TRIM10 and to a lesser extent TRIM58 are able to direct degradation of wild-type, untagged BCL11A protein.

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Abstract

Described herein are compositions and methods for targeted erythroid-specific protein degradation. The compositions described comprise a binding domain for a erythroid protein of interest, e.g., BCL11A, and a binding domain for a erythroid enriched E3 ubiquitin ligase. Compositions and methods described can be used to promote fetal hemoglobin and treat hemoglobinopathy disorders.

Description

x COMPOSITIONS AND METHODS FOR TARGETED PROTEIN DEGRADATION   CROSS-REFERENCE TO RELATED APPLICATIONS  [0001] This application includes a claim of priority under 35 U.S.C. 119(e) to U.S. provisional patent application No. 63/339,208, filed May 6, 2022, the entirety of which is hereby incorporated by reference. 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 4, 2023, is named “701039-191700WOPT_SL2.xml” and is 93,396 bytes in size. GOVERNMENT SUPPORT [0003] This invention was made with government support under Grant Number HL032259, awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND [0004] Targeted protein degradation (TPD) in cells is most commonly achieved with molecular glues or proteolysis-targeting chimeras (PROTACs). In the majority of instances, PROTACs leverage widely expressed E3 ubiquitin ligases (or their associated subunits), e.g. cereblon or von Hippel-Lindau (VHL) proteins, to recruit a protein of interest (POI) to the proteasome for degradation. In order to restrict TPD to specific cells, rather than risk TPD in many different cell types, it would be advantageous to leverage cell-restricted E3 ubiquitin ligases. SUMMARY [0005] The technology described herein relates, in part, to the discovery that certain ubiquitin ligases are enriched for expression in cells of the erythroid lineage. Where ubiquitination marks proteins for ubiquitin-mediated degradation, this discovery provides approaches for the targeted degradation of a desired target polypeptide in an erythroid-preferring manner. In one approach, a bispecific construct including binding domains for a target polypeptide and an erythroid-enriched E3 ubiquitin ligase can act as a tether, bringing the target polypeptide into close proximity with the erythroid-enriched E3 ubiquitin ligase. Where the E3 ubiquitin ligase involved is expressed in an erythroid-enriched manner, such a construct will function to selectively channel the target polypeptide into the degradation pathway in erythroid cells. In another approach, the E3 ubiquitin ligase itself is fused to a molecule that specifically binds a desired target polypeptide. Similar to the first approach, tethering of the E3 ubiquitin ligase to a degradation target in this manner results in the ubiquitination and subsequent degradation of the tethered target. Compositions for targeting desired polypeptides for degradation and methods of using them are described herein. In various embodiments, the constructs described herein can be used therapeutically to target desired polypeptides expressed in erythroid cells for degradation. Non-limiting examples include the transcription factor BCL11A, which is involved in repression of fetal hemoglobin (HbF) expression in adult erythroid lineage cells. Targeting compositions and methods described herein can promote the degradation of BCL11A in erythroid cells, thereby promoting re-expression of HbF in those cells, which can provide therapeutic benefit for subjects suffering from ^- hemolglobinopathies. [0006] In one aspect, described herein is a fusion polypeptide comprising a binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and a binding domain that specifically binds an erythroid-enriched polypeptide of interest. [0007] In one embodiment of this and any other aspect described herein, the fusion polypeptide mediates degradation of the polypeptide of interest in erythroid cells. [0008] In another embodiment of this and any other aspect described herein, the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase specifically binds TRIM10 or TRIM58. [0009] In another embodiment of this and any other aspect described herein, the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase comprises an antibody or antigen-binding fragment thereof. In another embodiment of this and any other aspect described herein, the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody. [00010] In another embodiment of this and any other aspect described herein, the binding domain that specifically binds an erythroid-enriched polypeptide of interest comprises an antibody or antigen-binding fragment thereof. In another embodiment of this and any other aspect described herein, the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody. [00011] In another embodiment of this and any other aspect described herein, the polypeptide of interest is selected from BCL11A, LRF and ZNF410. [00012] In another embodiment of this and any other aspect described herein, the E3 ubiquitin ligase is TRIM10 or TRIM58. [00013] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [00014] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised 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. [00015] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12. In another embodiment of this and any other aspect described herein, amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2. In some embodiments, variation relative to SEQ ID NO: 12 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. [00016] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34. In another embodiment of this and any other aspect described herein, amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids at amino acid number 102 or 108. In another embodiment of this and any other aspect described herein, variation occurs only at amino acids 102 or 108 relative to SEQ ID NO: 34. [00017] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50; CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65. [00018] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40) and ADQDVYPYEYL (SEQ ID NO: 41). [00019] In another embodiment of this and any other aspect described herein, the fusion polypeptide further comprises a cell-penetrating peptide. [00020] In another embodiment of this and any other aspect described herein, the fusion polypeptide further comprises a linker peptide between the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and the binding domain that specifically binds an erythroid-enriched polypeptide of interest. [00021] In another aspect, described herein is a nucleic acid comprising sequence encoding a fusion polypeptide as described herein. [00022] In one embodiment of this and any other aspect described herein, the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells. [00023] In another aspect, described herein is a vector comprising a nucleic acid comprising sequence encoding a fusion polypeptide as described herein. In one embodiment of this and any other aspect described herein, the vector is a viral vector. In another embodiment of this and any other aspect described herein, the viral vector is an AAV vector. [00024] In another aspect, described herein is a method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to an erythroid cell. [00025] In another aspect, described herein is a method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a cell expressing BCL11A. [00026] In one embodiment of this and any other aspect described herein, the targeted degradation of BCL11A is targeted to erythroid cells. [00027] In another aspect, described herein is a method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression. [00028] In another aspect, described herein is a method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid comprising sequence encoding a fusion polypeptide as described herein or a vector comprising sequence encoding a fusion polypeptide as described herein to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder. [00029] In another aspect, described herein is a fusion polypeptide comprising a TRIM10 or TRIM58 polypeptide fused to a binding domain that specifically binds a target polypeptide. [00030] In one embodiment of this and any other aspect described herein, the binding domain that specifically binds a target polypeptide comprises an antigen-binding domain of an antibody. In another embodiment of this and any other aspect described herein, the binding domain that specifically binds a target polypeptide comprises an scFv, a single domain antibody or a nanobody. [00031] In another embodiment of this and any other aspect described herein, the target polypeptide is expressed in an erythroid cell. [00032] In another embodiment of this and any other aspect described herein, the target polypeptide is selected from BCL11A, LRF and ZNF410. [00033] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [00034] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide. [00035] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12. In another embodiment of this and any other aspect described herein, amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2. In some embodiments, variation relative to SEQ ID NO: 12 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. [00036] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34. In another embodiment of this and any other aspect described herein, amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids number 102 or 108. In another embodiment of this and any other aspect described herein, amino acid sequence variation relative to SEQ ID NO: 34 occurs only at amino acids number 102 or 108. [00037] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50; CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65. [00038] In another embodiment of this and any other aspect described herein, the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40) and ADQDVYPYEYL (SEQ ID NO: 41). [00039] In another embodiment of this and any other aspect described herein, the fusion polypeptide further comprises a cell-penetrating peptide. [00040] In another embodiment of this and any other aspect described herein, the fusion polypeptide further comprises a linker peptide between the TRIM10 or TRIM58 polypeptide and the binding domain that specifically binds a target polypeptide. [00041] In another aspect, described herein is a nucleic acid comprising sequence encoding a fusion polypeptide as described herein. [00042] In one embodiment of this and any other aspect described herein, the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells. [00043] In another aspect, described herein is a vector comprising a nucleic acid comprising sequence encoding a fusion polypeptide as described herein. In one embodiment of this and any other aspect described herein, the vector is a viral vector. In another embodiment of this and any other aspect described herein, the viral vector is an AAV vector. [00044] In another aspect, described herein is a method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to an erythroid cell. [00045] In another aspect, described herein is a method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a cell expressing BCL11A. [00046] In one embodiment of this and any other aspect described herein, the targeted degradation of BCL11A is targeted to erythroid cells. [00047] In another aspect, described herein is a method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression. [00048] In another aspect, described herein is a method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide as described herein, a nucleic acid encoding a fusion polypeptide as described herein, or a vector comprising a nucleic acid encoding a fusion polypeptide as described herein to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder. [00049] 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, 4th 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. [00050] 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. [00051] 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. [00052] 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. [00053] 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. [00054] 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). [00055] 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. [00056] 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. [00057] 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 FIGURES [00058] Figs 1A-1C show erythroid cell enriched expression of TRIM10 and TRIM48 (Fig. 1A) The modular structures of TRIM10 and TRIM58. (Fig.1B) RNA expression of E3 ligases during cell maturation. (Fig.1C) Heat-map depicting the relative expression of UBE20, TRIM10, and TRIM58 across cell types. The dots indicate erythroid cells. [00059] Fig 2 shows fusion of TRIM10/58 to the recognition domain of cereblon (CRBN). Schematic showing the fusion proteins of TRIM10/58 with the CRBN recognition domain along with control contructs. [00060] Fig 3 shows degradation of BCL11A protein by TRIM10/58. Western blot for BCL11A depicting fusions of TRIM10/58 with the recognition domain of CRBN were capable of degrading tagged BCL11A upon addition of dTAG47 PROTAC. WT-CRBN served as a positive control and inactive form of dTAG47 served as a negative control. [00061] Fig 4 shows degradation of BCL11A protein by TRIM10/58 in HUDEP-2 erythroid cells. HUDEP-2 cells with CRBN or lacking CRBN were infected with lentiviruses expressing CRBN cDNA or TRIM10 fused with the recognition domain of CRBN and then dTAG47 PROTAC was added. BCL11A protein was degraded in the presence of dTAG47 in CRBN- cells rescued with CRBN cDNA and the fusion of TRIM10 with the recognition domain of CRBN leads to a reduction in BCL11A protein. [00062] Fig.5 shows targeted protein degradation of BCL11A by TRIM10/58 in 293T cells. Western blot for BCL11A of 293T cells transfected with constructs expressing either TRIM10 or TRIM58 fused to a BCL11A-specific nanobody (Nb19). The nanobody provides the recognition domain to direct the TRIM to BCL11A. DETAILED DESCRIPTION [00063] The technology described herein relates, in part, to the discovery that certain ubiquitin ligases are enriched for expression in cells of the erythroid lineage. In particular, it was discovered that the E3 ubiquitin ligases TRIM10 and TRIM58 are expressed in an erythroid- enriched or erythroid-specific manner. Where E3 ubiquitin ligases target proteins in the cells in which they are expressed for degradation via the ubiquitin-mediated proteasome pathway, the identification of erythroid-specific or erythroid enriched E3 ubiquitin ligases provides avenues for erythroid specific, targeted degradation of a protein of interest. The ability to target a protein of interest for degradation in an erythroid-enriched manner has therapeutic implications, in that, as but one example, hemoglobinopathies generally stem from defects in cells of the erythroid lineage, and targeted degradation of, e.g., BCL11A, which represses expression of fetal hemoglobin (HbF), can de-repress and promote the expression of HbF, providing a new source of active hemoglobin. [00064] Described herein below are constructs for targeting proteins of interest for degradation in cells of the erythroid lineage, as well as formulations and uses of such constructs for the treatment of diseases or disorders including, but not limited to hemoglobinopathies. The following describes the various considerations for one of skill in the art to practice the described technology. Ubiquitin-mediated protein degradation [00065] The degradation of proteins occurs mainly through two major pathways: autophagy and the ubiquitin proteasome system (UPS), both of which are critical for maintaining cellular homeostasis (see, e.g., Dikic, I., Ann. Rev. Biochem.86: 193-224 (2017)). The UPS is a cascade reaction, and an important mechanism for the degradation of misfolded and damaged proteins, as well as a regulated mechanism that ensures short lives of certain regulatory proteins, among others. Degradation of a protein via the ubiquitin pathway generally involves two discrete and successive steps: (a) covalent attachment of multiple ubiquitin molecules to the protein substrate – so called “ubiquitination” or “ubiquitylation;” and (b) degradation of the ubiquitin-targeted protein by the 26S proteasome complex, with the release of free and reusable ubiquitin. As used herein, “ubiquitination” or “ubiquitylation” refers to the post-translational modification of a protein by the covalent attachment (via an isopeptide bond) of one or more ubiquitin monomers. [00066] The ubiquitin-proteasome system consists of several components that act in concert. One of these, ubiquitin, an evolutionarily conserved protein of 76 residues (human ubiquitin has the amino acid sequence: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDY NIQKESTLHLVLRLRGG (SEQ ID NO: 1), is activated in its C-terminal Gly to a high- energy thiol-ester intermediate. This reaction is catalyzed by the ubiquitin-activating enzyme, E1. After ubiquitin activation, one of several ubiquitin-carrier proteins or ubiquitin- conjugating enzymes, referred to as E2s, transfers the activated ubiquitin moiety from E1 to a member of the ubiquitin-protein ligase family, E3, to which the substrate protein is bound. E3 catalyzes the last step in the conjugation process, covalent attachment of ubiquitin to the substrate protein. The first ubiquitin moiety is transferred to the ɛ-NH2 group of a lysine residue of the protein substrate to generate an isopeptide bond. In successive reactions, a polyubiquitin chain is generated by processive transfer of additional activated ubiquitin moieties to a lysine residue (one of the most prominently used is Lys48, or K48, but each of K6, K11, K27, K29, K33, K63 and the N-terminal methionine 1 (Met1) are also functional sites in various ubiquitination reactions) of the previously conjugated ubiquitin molecule. The polyubiquitin chain serves as a recognition marker for the 26S proteasome. The binding of the substrate to E3 is generally specific and indicates that E3 ubiquitin ligases play a major role in recognition and selection of proteins for conjugation and subsequent degradation. The structure of the system appears to be hierarchical: a single E1 appears to carry out activation of ubiquitin required for all modifications. Several major species of E2 enzymes have been characterized in mammalian cells, each of which can act with one or more E3 enzymes. E3 Ubiquitin Ligases [00067] Hundreds of different E3 ubiquitin ligases have been identified. These are reviewed and classified in, e.g., Yang et al., Molecular Biomedicine 2: 23 (2021). Different types of E3 ubiquitin ligases have low sequence homology and large differences in composition. The tripartite motif (TRIM) protein family is a subset of the so-called RING-type E3 ligase subfamily. The TRIM family of E3 ubiquitin ligases is reviewed, e.g., by Esposito et al., Biochem. Soc. Trans.45: 183-191 (2017). As discussed in the Examples herein, it was discovered that two E3 ubiquitin ligases, TRIM10 and TRIM58, are expressed in an erythroid-enriched manner. As used herein, the term “erythroid enriched” refers to the preferential expression and activity of the TRIM10 and TRIM58 E3 ubiquitin ligases in cells of the erythroid lineage. By preferential in this context is meant that TRIM10 and TRIM58 are most highly expressed in cells of this lineage, relative to their expression in other cell types or tissues. The erythroid-enriched expression of TRIM10 and TRIM58 renders these E3 ubiquitin ligase polypeptides useful for the targeted degradation of a protein of interest in an erythroid-enriched manner. [00068] 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: 2) and polypeptide (e.g., Isoform 1: NCBI Ref Seq: NP_006769.2, SEQ ID NO: 3). TRIM10 can refer to human TRIM10, including naturally occurring variants, molecules, and alleles thereof. For example, SEQ ID NO: 2 is a mRNA sequence for TRIM10. Note: U’s replaced with T’s in this representation. 1 cttctgacag tggacagctg acatatcacc actttccttc tactgtgagt gtctctggat 61 gggcagaaag gaatggccag cccctggtta tggtcatcta aggtcacctc tgaaatgctg 121 tgagcccctc ttccttcctc tcctctgcta tttcccatct ctgctgttgg caggagaata 181 gaaccctggc tgccagagat gcaagtgtgt gacgatatgg gtgctggtgc atatttagta 241 tgtgcctgtg tccagccatg tgcatgtgtg ggtgtgtgag tgtgtgaccc agcccttccc 301 ccgtggccaa gcagagagag tggccttgag gaagccatag cagcaggacc agcatggcct 361 ctgctgcctc tgtgaccagc ctggcagatg aagtcaactg ccccatctgt cagggtaccc 421 tgagggagcc ggtcactatc gactgcggcc acaacttctg ccgggcctgc cttacccgct 481 actgtgagat accaggccca gacctggagg agtcccctac ttgcccactc tgcaaagaac 541 ccttccgtcc tgggagcttc cggcccaact ggcagctggc taacgtggtg gagaacattg 601 agcgcctcca gctggtgtcc acactgggtt tgggagagga ggatgtctgc caagagcacg 661 gagagaagat ctacttcttc tgtgaggatg atgagatgca gttgtgcgtg gtgtgccggg 721 aggctgggga gcacgctacc cacaccatgc gcttcctgga ggatgcagcg gctccctata 781 gggaacaaat ccataagtgt cttaaatgtc taagaaaaga gagagaggag attcaagaaa 841 tccagtcaag agaaaataaa aggatgcaag tcctcctgac tcaggtgtcc accaagagac 901 aacaggtgat ttctgagttc gcacacctga ggaagtttct agaggaacag cagagcatcc 961 tcttagcaca attggagagc caggatgggg acatcttgag gcaacgggat gaatttgatt 1021 tgctggttgc tggggagatc tgccggttta gtgctcttat tgaagaactg gaggagaaga 1081 atgagaggcc agcaagggag ctcctgacgg acatcagaag cactctaata agatgtgaaa 1141 ccagaaagtg ccggaaaccg gtggctgtgt cgccagagct gggccagagg attcgggact 1201 ttccccagca ggccctcccg ctgcagaggg agatgaagat gtttctggaa aaactatgct 1261 ttgagttgga ctatgagcca gctcacattt ctctagaccc tcagacttcc caccccaagc 1321 tcctcttgtc cgaggaccac cagcgagctc agttctccta caaatggcag aactcaccag 1381 acaaccccca acgttttgac cgggccacct gtgttctggc ccacactggc atcacagggg 1441 ggagacacac gtgggtggtg agtatagacc tggcccatgg gggcagctgc accgtgggcg 1501 tggtgagcga ggatgtgcag cggaaggggg agcttcggct gcggccagag gagggggtgt 1561 gggctgtgag gctggcttgg ggcttcgtct cggctctggg ctccttcccc acacggctga 1621 ccctgaagga gcagccccgg caggtgaggg tgtctcttga ctatgaggtg ggctgggtga 1681 ccttcaccaa cgctgtcacc cgagagccca tctacacctt cactgcctcc ttcactagga 1741 aggtcattcc cttctttggg ctctggggcc gagggtccag tttctccctg agctcctgag 1801 aaggagcagt tacctactct cctctaagta caggactcat atcaacccaa gtaccatgtg 1861 gacttgatcc ctggctgaat cacctggatg acttggaata gaaatgactg ctttagaaga 1921 tgggatgggg ccgggtggta agggatagaa gagaggactc tcaatctact gatcaagtcc 1981 tttccccaat gcccagtgga tggccagggt acctggggac tcaggctgct gccagttctg 2041 ctcaccacca tccgtgcttg gcacagaagt agctgcatag aaagggcact ggatttgaag 2101 tcagaagacc tgggttcttg aaccagcctg tcaaccagtt gtatgacttt aaacaaggca 2161 tctcacctct tttcatcttg ttttcttcca ataatgttag agttcatgta atcacattct 2221 ctagaaccat ttagtttgtg ttaactatga accaagcagt gtggtggcca ctggtgactt 2281 gaaaatatag agaaaaaaaa aacctgctct atatctgaaa gagctcttgg gaagacagag 2341 aaacataaag aggaaattac agcacagtgt ggtgggtgtt acgggaagtc caaccccagc 2401 attatgggag ttcaggggaa ggggcatagc ccagcctgga aggagagggt gagtggggga 2461 tggctttctg aaaagggtgg tctaaaggat gcctatggtc aatagggaaa agaggggaag 2521 aagcatctta agaagaggaa acagcagaga actggctgga tgacctgtga ctctggagca 2581 ctgggttgtc tccattgtca ttatgggcag atgtgtgcca tccccagccg acgccactca 2641 ctgcctcctt cctcctggtg ttgccacttc tgggtgagat taaggtgcag ggcctggggg 2701 caggaggaca taaggtatag ccataaatca taacccaggg accacactca accctaggga 2761 aattgtcttc ctgatcagtt gattaccatc tgaggtcaag aaatgagata gtgggagcaa 2821 atgggtcaca aatagctcag ctgtgggctc agaaactgct aggtaaaaga attccagaag 2881 gaggccaggg cataagttgg atgacctatg aactttagtc taaagaattg agactaccgt 2941 aattgagact actgtagtga catctgagaa atgggatgga agagtgacca tgttttattt 3001 tcttgttctt gtcactattg tattttattt tgcataatca tgcccttcac tgacagtctc 3061 cttaacatca tctgtttact ctgctcagtg taaactacaa tgctctgtca tctccctact 3121 gggtctcctg ggaggagggg agccatccag ggtgcaaact caaaggcaga gggcacagcg 3181 tgcttaggcc caagcttaga attcaattga gaagttctgt tgttcatcct tacctcagca 3241 ggtagaaaag aggtggagat cagaagccag ggattagaga ttgaattgct ttccctggga 3301 gtgtgcagta tctcattaaa atgttgtata ttcaaaaaaa tacagacaca cacaagtgcc 3361 tatataatga taaaacatat ccaaagcacc caaacctgtg attccagaaa agtagatcct 3421 atttttgttt atttttattg gctttagctg tgtcttttga aaggactatt atcctctaaa 3481 atgtatgtgt atgaagatcc tggcttgcag ctcgggagct ttacacacct tgtgtcactt 3541 tatcttggta acgagccaag atcatgcaac cagttcatcc atgtctgacc caagagctct 3601 taactgctat gctgcactgc ctcattcaga gatagatgcc tgcatgggtc ctggcgatta 3661 ttttaatgct ggctacaccc ccacaggtga cttggattca gaaatacaat ttatatttct 3721 tctttttaaa ttgttttatt ttatttttct tttaatagtt atatgtcagt gagaacaatt 3781 tatatgtctt acactgagaa ataaaactgc tcataagtga aaaca [00069] SEQ ID NO: 3 is an amino acid sequence of TRIM10. 1 masaasvtsl adevncpicq gtlrepvtid cghnfcracl tryceipgpd leesptcplc 61 kepfrpgsfr pnwqlanvve nierlqlvst lglgeedvcq ehgekiyffc eddemqlcvv 121 creagehath tmrfledaaa pyreqihkcl kclrkereei qeiqsrenkr mqvlltqvst 181 krqqvisefa hlrkfleeqq sillaqlesq dgdilrqrde fdllvageic rfsalieele 241 eknerparel ltdirstlir cetrkcrkpv avspelgqri rdfpqqalpl qremkmflek 301 lcfeldyepa hisldpqtsh pklllsedhq raqfsykwqn spdnpqrfdr atcvlahtgi 361 tggrhtwvvs idlahggsct vgvvsedvqr kgelrlrpee gvwavrlawg fvsalgsfpt 421 rltlkeqprq vrvsldyevg wvtftnavtr epiytftasf trkvipffgl wgrgssfsls 481 s 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: 4) and polypeptide (e.g., NCBI Ref Seq: NP_056246.3, SEQ ID NO: 5). TRIM58 can refer to human TRIM58, including naturally occurring variants and alleles thereof. Antibodies that specifically bind TRIM58 are known; see, e.g., Novusbio Trim58 Antibody (2H3) H00025893-M02. One can generate monoclonal antibodies using standard procedures e.g., Corti et al, Microbiol Spectr 2 (2014), Huang et al. Nat Protoc 10, 1907-1915 (2013), and Pedrioli et al., Trends Immunol.42: 1143-1158 (2021), which are incorporated herein by reference. Single domain antibody fragments or nanobodies can be screened using a fully in vitro platform based on yeast surface display as in McMahon et al Nature 25: 289-296 (2018). [00070] SEQ ID NO: 4 is a mRNA sequence for TRIM58. Note: U’s replaced with T’s in this representation. 1 gcagaccgcg aggggagacg gtgcgggcgg ccgggagcgc agccctccgg gaggcgggtc 61 atggcctggg cgccgcccgg ggagcggctg cgcgaggatg cgcggtgccc ggtgtgcctg 121 gatttcctgc aggagccggt cagcgtggac tgcggccaca gcttctgcct caggtgcatc 181 tccgagttct gcgagaagtc ggacggcgcg cagggcggcg tctacgcctg tccgcagtgc 241 cggggcccct tccggccctc gggctttcgc cccaaccggc agctggcggg cctggtggag 301 agcgtgcggc ggctggggtt gggcgcgggg cccggggcgc ggcgatgcgc gcggcacggc 361 gaggacctga gccgcttctg cgaggaggac gaggcggcgc tgtgctgggt gtgcgacgcc 421 ggccccgagc acaggacgca ccgcacggcg ccgctgcagg aggccgccgg cagctaccag 481 gtaaagctcc agatggctct ggaacttatg aggaaagagt tggaggacgc cttgactcag 541 gaggccaacg tggggaaaaa gactgtcatt tggaaggaga aagtggaaat gcagaggcag 601 cgcttcagat tggagtttga gaagcatcgt ggctttctgg cccaggagga gcaacggcag 661 ctgaggcggc tggaggcgga ggagcgagcg acgctgcaga gactgcggga gagcaagagc 721 cggctggtcc agcagagcaa ggccctgaag gagctggcgg atgagctgca ggagaggtgc 781 cagcgcccgg ccctgggtct gctggagggt gtgagaggag tcctgagcag aagtaaggct 841 gtcacaaggc tggaagcaga gaacatcccc atggaactga agacagcatg ctgcatccct 901 gggaggaggg agctcttaag gaagttccaa gtggatgtaa agctggatcc cgccacggcg 961 cacccgagtc tgctcttgac cgccgacctg cgcagtgtgc aggatggaga accatggagg 1021 gatgtcccca acaaccctga gcgatttgac acatggccct gcatcctggg tttgcagagc 1081 ttctcatcag ggaggcatta ctgggaggtt ctggtgggag aaggagcaga gtggggttta 1141 ggggtctgtc aagacacact gccaagaaag ggggaaacca cgccatctcc tgagaatggg 1201 gtctgggccc tgtggctgct gaaagggaat gagtacatgg tccttgcctc cccatcagtg 1261 cctcttctcc aactggaaag tcctcgctgc attgggattt tcttggacta tgaagccggt 1321 gaaatttcat tctacaatgt cacagatgga tcttatatct acacattcaa ccaactcttc 1381 tctggtcttc ttcggcctta ctttttcatc tgtgatgcaa ctcctcttat cttgccaccc 1441 acaacaatag cagggtcagg aaattgggca tccagggatc atttagatcc tgcttctgat 1501 gtaagagatg atcatctcta aaattctgtt cccaagatgc agtcctagcg tagcgaacgt 1561 tcctggagtg gggtgaagga tatcaatata ctaagtttta acagataccc catttaggtc 1621 agcacttgat tcgttgttgc tgtgaaatat gtccatggga caaaagaggg aatatgaaat 1681 atttgcatat gggaagatta tagagcataa taattttgta aatggagcaa tctcaacctc 1741 tatttctaga tcacattttc ttgatgtctt ccttcaaatt aatgaccttg gattacataa 1801 ggatttctat gcattcatta taatttgtta ttcctttcaa tatccttgta tttcaaatct 1861 tccatataag aattagacat ggcaattctt aaattgattc agaatggtct gatactattc 1921 cagtatcacc tccttaattc tgtttctcct cgttttcctg attttccttc tcattctctc 1981 cttccccgct ctgtctctct ctccctgtca ctctctctct cttgttcctt attttttgtt 2041 tcttacctct tactgtttaa cctgttgctt ccttctggat taatacattt agagccattc 2101 ctttatatgg tcacatttcc tatgacttta ctcaattact tttaaaatcc tttctattct 2161 gagactaatt tttaagaatt acaaagctca ttcttctgaa tctaatatca ctaactccta 2221 gactttttcc gttttctttg gatacacttt aagtaggaat ttatcagaat tttcattcaa 2281 ctcgttcttt aatgcagata tttactagtt ataagacctt aaggctgggt gcagtggctc 2341 acgcctgtaa tcccagcact ttgggaggct gaggcgggtg gatcacaagc tcaggagttc 2401 aagaccagcc tggccaacat ggtgaaaccc tgtctctact aaaaaaaaaa aaaaaataga 2461 aaaattagct gggcatggtg gcaggagcct gtaatcccag ctattctgga ggtggagaca 2521 ggagaattgc ttgaaccctg gaggcggagg ttgcagtgag ccaatatctc accactgtac 2581 tccagcccag tgcgagactc catctcaaaa aagaaaaaag acctcaaaca acacttctct 2641 ctctctttta gctgcttgtt atggttccta tacatggaac aattatactg gcctcactgt 2701 gttatggtaa atatttaagg tcatatttga tattgctggt ttgaattcag cttttccatt 2761 taaatacatt ataatgatga tgatgaaatc atgataatat ttaacttatt tttaaagtat 2821 attctgtacc tttccaacaa aaaggttaaa agtcattgaa ggctaacctt actgccttct 2881 ttgtatcact gtcttctaaa taattattat gtctgggtac agtggctcac gcctgtaatc 2941 ccagcacttt gggaggccga ggtgggcaga tcacgaggtc aggagattga gaccatcctg 3001 gctaacacag tgaaaccccg tctctactaa aaatacaaaa agaaattagc tgggcgtggt 3061 ggtgggtgcc tgttgtccca gctacttggg aggctgaggc aggagaatgg catgaaccca 3121 ggaggcagag cttgtagtga gccgagatcg cgccactgca ctccagccgg ggcaacagag 3181 caagactcca tctcaaaaat aaataaataa ataaataaat aaataaataa ataaataaat 3241 attacacaaa tgctaaaatg tttaaatggt aaatgcttca atgctaacca aatattaatt 3301 aatggcaaat tatttaacat tatctgataa taatctgcag aaggtttaat tttcctcctc 3361 aatttgaagt tcaagatgtt tttctcttcc agggagattt tttcgactga catctttaac 3421 ttaccttcca atcatattac taacgtagcc ttcttcctag attttttaat tgtttgatca 3481 tgagcgaaca cttctactct ctgtgataga tttgcaaaca gaggaaataa cgcatcctcg 3541 tgtccctctt cttggtgttc cacaggccat gtgtgcccta gccctcgttc atgcaaggtc 3601 tgtgtaggga aggtggactt cagctcagca acagcatccc ttcccacagg gatcaggtgg 3661 gtggcttgag ataccccttc catggggcac cacccattca gtgagacggg gaagccctgg 3721 gtgggaggga gaacacctcc acatgtcttc tactctctcc ataggatgga atgagtgtcc 3781 cagtcccagg agtatccatt tcccactgtg tagcccagta ctctggtctc actgtctctg 3841 ctgaatcctg tctcactgtg catattattg tggtttatat cagtcagtaa accaatgtga 3901 gtcttcatct cttgcattct taggttcata gttttgtgtg tctcctgtaa tgactcttct 3961 ctttcccttt ccaactcctg aaagattgcc actatttcct ctggaacttt gtttcgttac 4021 cagcaaaatc ctcgacatcc atacccgttt cctggctttc cctctccttt cctctgaatg 4081 gtagtctttt atattcagct gtccacttga catcaaaata gacattttga actcaatttg 4141 cctaaaactt acccacaaat ttctccccaa gtctctccct aactgcaaca acaaaaacca 4201 caggcttctc cctgtcactg gatggcaact ccattctttt gattgcttaa gccaggcatc 4261 cgattgagta ctttcttgat ttctccagcc cacatccagt ccatcggcaa gccctgttgg 4321 tcctaccttc agaatatgtc cggggttcag ttgtcctggc caccctgctg ctgtaaccat 4381 ggtcagaact ccatcctgcc cctctggatt atgactttcg tttcctcaca gtggtcctgc 4441 ttgggctcta ggcccttcca ctcccattct ctctacagca gctgggctga ttcctttagc 4501 acccaaggat atgttggcat cacagtgact tagataccat cacaaagacc tcccattcaa 4561 cttagagtga aagtcagaat cctcacagtg aatccccagg ccctagagga tgtgaacccc 4621 caggccctag aggatctgaa cccccatccc tcctctgatt atctctccca cccccacttc 4681 cctttgcatt ctgctccagc tgccctggcc tcatggctgg gtttccacca aagcaggcac 4741 ttcccatcac agggccattt ccccgcctgt ggcttctgct tgacattccc ttttccctga 4801 tatccccttg actcattatt ccctttcttc cttaactctt ctgagatcca gcttctcagt 4861 gataccacac agccctactc cccccagagc ccatctagag ctcacctttc cagtcgccct 4921 tgccaggctc agtggaggct ctttgttccc catacagtac gtgtcgtcgt actatattgt 4981 taggcttatt taatttatgt atgttttgcc tttttgtgct aaatgtaaac accacaaggg 5041 gaggtatctt tgtctgttga caatgataca ttcaatgttt ctcaagcacc cccaatgctg 5101 gtttgtatgt ggttatcatt caatctgtat ttgttgaatg aataaatgat tgactatgtg 5161 gagagcaaaa [00071] SEQ ID NO: 5 is an amino acid sequence of TRIM58. 1 mawappgerl redarcpvcl dflqepvsvd cghsfclrci sefceksdga qggvyacpqc 61 rgpfrpsgfr pnrqlaglve svrrlglgag pgarrcarhg edlsrfceed eaalcwvcda 121 gpehrthrta plqeaagsyq vklqmalelm rkeledaltq eanvgkktvi wkekvemqrq 181 rfrlefekhr gflaqeeqrq lrrleaeera tlqrlresks rlvqqskalk eladelqerc 241 qrpalglleg vrgvlsrska vtrleaenip melktaccip grrellrkfq vdvkldpata 301 hpsllltadl rsvqdgepwr dvpnnperfd twpcilglqs fssgrhywev lvgegaewgl 361 gvcqdtlprk gettpspeng vwalwllkgn eymvlaspsv pllqlesprc igifldyeag 421 eisfynvtdg syiytfnqlf sgllrpyffi cdatplilpp ttiagsgnwa srdhldpasd 481 vrddhl [00072] As noted above, the erythroid-enriched nature of TRIM10 and TRIM58 establishes them as candidates for the targeting of a protein of interest for degradation in erythroid cells. In one embodiment, a binding agent that specifically binds TRIM10 or TRIM58 can be joined to a binding agent that specifically binds a protein of interest. Upon introduction to an erythroid cell, whether by expression therein or by more direct introduction, such a bispecific construct can bring the protein of interest into close proximity with the TRIM E3 ubiquitin ligase, resulting in ubiquitination of the protein of interest, thereby promoting the targeted degradation of the protein of interest in an erythroid-enriched manner. In such an embodiment, the binding agent that binds either or both of the TRIM polypeptide and the protein of interest can be, for example, antibodies or antigen-binding fragments thereof. The binding agents can be joined, for example, by a chemical or peptide linker, e.g., a flexible peptide linker, including, but not limited to a serine/glycine-rich peptide linker. [00073] In another embodiment, a TRIM10 or TRIM58 polypeptide can be directly fused, e.g., as a fusion polypeptide, to a binding agent that specifically binds a protein of interest. In such an embodiment, upon introduction to a cell expressing the polypeptide of interest, the polypeptide of interest is brought into close proximity with the TRIM E3 ubiquitin ligase, resulting in ubiquitination of the protein of interest, thereby promoting the targeted degradation of the protein of interest. In such an embodiment, the binding agent that binds the protein of interest can be, for example, an antibody or antigen-binding fragment thereof. The binding agents can be joined, for example, by a chemical or peptide linker, e.g., a flexible peptide linker, including, but not limited to a serine/glycine-rich peptide linker. Erythroid targets for degradation [00074] Any polypeptide expressed in an erythroid cell, whether erythroid specific, erythroid enriched, or simply expressed in an erythroid lineage cell, can be target for proteasome- mediated degradation using constructs as described herein. Non-limiting examples of erythroid-enriched factors include LRF, ZNF410, and BLC11A. LRF [00075] Leukemia/Lymphoma-Related Factor (LRF), also known as Zinc Finger and BTB Domain Containing 7A (ZBTB7A), Factor binding IST protein 1 (FBI-1), or Pokemon is a member of the POK family of transcription factors that possesses a POZ-domain at the N- terminus and Krüppel-like zinc finger at the c-terminus. See, e.g., Gupta et al., Cancer Lett. 483, 22-34 (2020), which is incorporated herein by reference in its entirety. LRF is involved in several fundamental biological processes including cell proliferation, differentiation, and development. It also participates in hematopoiesis and erythrocyte maturation, adipogenesis, chondrogenesis, cellular metabolism and alternative splicing of BCLXL, DNA repair, development of oligodendrocytes, osteoclast, and the unfolded protein response. LRF also negatively regulates SMAD4-DNA complex. Sequences of LRF are known for a number of species, e.g., human LRF/ZBTB7A (NCBI GeneID: 51341), mRNA (e.g., LRF variant 2 NCBI Ref Seq NM_001317990.2, SEQ ID NO: 6) and polypeptide (e.g., LRF: NCBI Ref Seq: NP_001304919.1, SEQ ID NO: 7). Antibodies for specific detection or binding to LRF/ZBTB7A are available, for example, from R&D Systems, see Human/Mouse ZBTB7A/Pokemon Antibody (clone 466407) MAB3496; from Origen, see Pokemon (ZBTB7A) Mouse Monoclonal Antibody (clone BC17-2.9F), AM32711PU-N; from Novusbio, see ZBTB7A/Pokemon Antibody (clone 3T2J1) NBP3-15914; from Abcam, see Anti-ZBTB7A antibody (EPR13178B) ab175918; and from Invitrogen, see Pokemon (LRF) monoclonal antibody (13E9) 14-3309-82. LRF can refer to human LRF, including naturally occurring variants and alleles thereof. For example, SEQ ID NO: 6 is a transcript variant 2, mRNA of human LRF. Note: U’s replaced with T’s in this representation. 1 actgccgcct cccggcccct cggagggagc cagcccagcc gcagccgccg ccaccgccgc 61 cgccggggcc gggccccctc gccgctgccc cgggaaggag gtctcggcgc ggaagatggc 121 cggcggcgtg gacggcccca tcgggatccc gttccccgac cacagcagcg acatcctgag 181 tgggctgaac gagcagcgga cgcagggcct gctgtgcgac gtggtgatcc tggtggaggg 241 ccgcgagttc cccacgcacc gctcggtgct ggccgcctgc agccagtact tcaagaagct 301 gttcacgtcg ggcgccgtgg tggaccagca gaacgtgtac gagatcgact tcgtcagcgc 361 cgaggcgctc accgcgctca tggacttcgc ctacacggcc acgctcaccg tcagcacagc 421 caacgtgggt gacatcctca gcgccgcccg cctgctggag atccccgccg tgagccacgt 481 gtgcgccgac ctcctggacc ggcagatcct ggcggccgac gcgggcgccg acgccgggca 541 gctggacctt gtagatcaaa ttgatcagcg caacctcctc cgcgccaagg agtacctcga 601 gttcttccag agcaacccca tgaacagcct gccccccgcg gccgccgccg ccgctgccag 661 cttcccgtgg tccgcctttg gggcgtccga tgatgacctg gatgccacca aggaggccgt 721 ggccgccgct gtggccgccg tggccgcggg cgactgcaac ggcttagact tctatgggcc 781 gggccccccg gccgagcggc ccccgacggg ggacggggac gagggcgaca gcaacccggg 841 tctgtggcca gagcgggatg aggacgcccc caccgggggt ctctttccgc cgccggtggc 901 cccgccggcc gccacgcaga acggccacta cggccgcggc ggagaggagg aggccgcctc 961 gctgtcggag gcggcccccg agccgggcga ctctccgggc ttcctgtcgg gagcggccga 1021 gggcgaggac ggggacgggc ccgacgtgga cgggctggcg gccagcacgc tgctgcagca 1081 gatgatgtca tcggtgggcc gggcgggggc cgcggcgggg gacagcgacg aggagtcgcg 1141 ggccgacgac aagggcgtca tggactacta cctgaagtac ttcagcggcg cccacgacgg 1201 cgacgtctac ccggcctggt cgcagaaggt ggagaagaag atccgagcca aggccttcca 1261 gaagtgcccc atctgcgaga aggtcatcca gggcgccggc aagctgccgc gacacatccg 1321 cacccacacg ggcgagaagc cctacgagtg caacatctgc aaggtccgct tcaccaggca 1381 ggacaagctg aaggtgcaca tgcggaagca cacgggcgag aagccgtacc tgtgccagca 1441 gtgcggcgcc gcctttgccc acaactacga cctgaagaac cacatgcgcg tgcacacggg 1501 cctgcgcccc taccagtgcg acagctgctg caagaccttc gtccgctccg accacctgca 1561 cagacacctc aagaaagacg gctgcaacgg cgtcccctcg cgccgcggcc gcaagccccg 1621 cgtccggggc ggggcgcccg accccagccc gggggccacc gcgacccccg gcgcccccgc 1681 ccagcccagc tcccccgacg cccggcgcaa cggccaggag aagcacttta aggacgagga 1741 cgaggacgag gacgtggcca gccccgacgg cttgggccgg ttgaatgtag cgggcgccgg 1801 tggaggaggt gacagcggag gtggccccgg ggccgccacc gacggtaact tcacagccgg 1861 actcgcctaa aaaccaaaaa gagaaaacag aaacccgaga aagagagaga cagagagaga 1921 gaaaaaaaat cacccaccac ccccccaaaa acacaaaaaa agaaaatcta tctatataca 1981 gatatctata tctatatata tatatacaga tatatatata tgacgcgtca cagaatctag 2041 ggtagcgctt tctcagattt ccctcctttc tgacgttttt ctccctccgc aggggccccg 2101 gccctccctg gctccccttc cccccaccac cccatcgctg ggtttcgggg cttggtttgg 2161 ggttttttgt aggacacaag gaatccgaga ccccgcacag ccccctgggc acccggcatg 2221 gggcctgggg cccgatccga ggccctgggc tggggggagg gtagacgtgg gggcgctggg 2281 gggggactgg ggtgggcttt taatttcctc ccctcgctgg tttctatgag tctttcagac 2341 aagaccttaa atgatttctg tctgctctga gcggacgtta aaatgggccc ccgtcccccg 2401 acccgcaccc tccttcctca gggcacttac taagggaggg gtctccctct ccatctcccc 2461 agtggcctcc ccgcctccaa ccctgcctgc ggcctccccc cgtcgcccac cccacgtctc 2521 ctggccactg agacacaaac ctatttattt ctaggcctgg agaaaggaga tcggactggg 2581 gttcccggtg gggcgccagg atggctcctg ggggtgctcc tgccgccttc cttcacggca 2641 cttacaaccg gcgggacccc cagggaccac ccctcagggc gcccccccac ccccgcccgg 2701 tccacctaga cccccacgtt tggagattca aaacttctgt cttcgtcctc tcccccgagc 2761 cccctctccc aaatttttaa agcacttttt agattttttt ttctctttcc tccttaaaaa 2821 caaaatttat atatagatat atatatatat ataaataata tacttttcct cagaggagca 2881 ggcaacagtg tgggataaac agagtcacga tcagaggaac cccagggtct ggtgatggca 2941 gggatggggg gagagagaga aaatccacaa attccaatgt cacaaaagca ataaaacaaa 3001 ctagaaaaaa aaaaggtttt acaaaatgaa aggaaggaaa aaaaaaaagg caaccaacca 3061 cattagaagt cttggcactt tgtaacggaa cgggtactac actttatctt aattcttaat 3121 ttaaaaacat gtttacaagt tacaaccaac ttctatgaaa agttgaaaag acaaaaaaaa 3181 aaaaaaaaag cgagcgagag agagagcgag agagagagcg agagcagaag aaattcctaa 3241 aagtcgattt atttttgtac aaaataataa aaaaaaaaac ccaccacaaa cgtagaatcc 3301 acttctgttc cccaaaaagc gagaaggggg gttcaggagg aagccatcgc aggggacctg 3361 ggagacgccc cgaggtgttt gtgcttcacc cccagacgtc agcctcgaag gcaggactgt 3421 ggggtgttcg tgctgtgttc cccccgctcc ccctttctgt cccctttttt ggttctgacg 3481 tgaagaggtc ttagcgcccg cttctgtcca cggggtctct ccttcctcct ccctagctca 3541 gggatgggcc ttccagccgg agcaccccga tccccatccg gcacccccca atcccccaac 3601 acgcctgtcc ctcccgcatg gccaccaagg agctggacct tggatgcgcc taccctgctg 3661 aggtgggtga caggggcccc ccacctccag ggccttagaa ccaccgcccc tctccccacc 3721 ccaggcaccc ctctttttac tcaaaggcac tgactgtaat ccagggggac tgggacctgc 3781 ctccccccaa cctctggctc ccacaaggcc cggtgttgac cgagccacag gccacggaca 3841 ggggccgggg ttggggagac tatgtcgcca gatgccagga cgccctcacc ccgtttgcat 3901 atgcaatgct agcatgggac cccgaaaata gacgctctgc tgcactgaga cttcttgtca 3961 atgcccaacc ggcggggggg tgtctccctg cccccgaccc ccccataccc ccttctctgt 4021 gacacacaca tcttctcgtc tctttttctt tcattgttaa agggaagctt tttaagaagg 4081 caattttcat attgtttcta caggatggtt ttggttccct tcccttccca ccccccctta 4141 agcctgtcag ccccctccaa atgtctcagg atcccccctc tcccctgggg ctgggtgaca 4201 gcaccccggc tgcgttcaca ccccagtgtc acagggcgag ctgttctgga gagaaaacca 4261 tctgtcgtgg ctgagcgggg agcttgaaca cccaggccag ggacacccct ccccagctcc 4321 cagagaggcc ccctgagggg tgagccctct ttccaccttc ccctatccat gcaccccctc 4381 gcaataaaac caactctaaa atcacagctg tcgtcctagc cagtgggggc gaccggactt 4441 ggggggtgga gccctctggg acttccgtag gaacaagggc tgcggcccac cgcgacactt 4501 acacagacct cggggattgc actaaaccct cgttcctagc tccgcactca gcttcgcctg 4561 tcctgcccgc ccactttgcc ttaactaccc gcccgtcctg ggggccacag cctctgcatg 4621 ggcccagagc cgggaccccc ccagcccagc cccgccctcc ccagactccg cgcaatcaca 4681 tactgtatat agacgtgaat cgattttatt tttattcttt aaattaaggt cgtgataaag 4741 tgttgccaaa gatacctgct gaattctcgc gtttcaggaa acaaacaaac aaaaaaaaat 4801 gatatttgag gagggtcgtg ttgactccat atgaaaggac acagctcaaa gcttttttgt 4861 ttggttgttt ggggtttttt gtgttttctt tttttggggt gttttttttt taactgcctg 4921 gtacaaaaaa aaaaagagaa aaaaaaaaaa gaaaaacaat gcgaaattgt tatttccatt 4981 ctcatggtga agttgcgtgg acgcgtgtgt gcgtgtgtgc aagagagcgg gagtgaggtc 5041 caggctgggg ttggggggct tcaggcgggg gcgcccgggg gccggggagg tggccgggcc 5101 ggagcccccg tctgcagtgc cccccagcct gccgggccca ggagagagag agaagcatct 5161 ttgctactag ctgttgctgc tacctgcctc tgccccccga cgccccccgc cttttgagat 5221 taaggaaaaa aaaaaaaagt caaaaaagtt tttaaaaatg aaaaaaaaaa attataaacc 5281 agtgaatgta aaatgccgga gcaggcccgg cctggcatgg gtgtggacct gcagccaggc 5341 aggctcgagc gggcgatacc aaagtctgcc cccccaccat tgtggccatg cagtcctgtc 5401 actgtctttt tgcttccttc cgaggggggt cccccagcct cttccagggt cttcccctgg 5461 aagtgggcgg ctgcagggaa ggtgggggac aggggtcttt gcacgattca gaccccgggg 5521 ccgtggcagg agcggtcacc tcacaggtgg tgacactgag gcaggggcct cggggtgccc 5581 cctcccgccc ggcaaccaga atggttggag gcaagacaga gagaatgaaa ggaaaaacag 5641 aagaaaaaaa aatattaaaa accaacaaaa aaagcaaaaa tcctattttt tgagaaagaa 5701 agatatttat atttgcagtt ttattttaaa aagttattta agttgaagca gccttcctgg 5761 aggtgggggg gggggggtgg tgggtggctg gcgcaggacg ggtcaggggc ctggaggctg 5821 ggggtgcccc aggagctaca acctcagagt taagactagc tcgcattaaa tacatagatt 5881 tacggggggg gggggggggg gccgggccca gggggtggag ggggccaggg agacccccat 5941 ccctcgccgg ggctgcctgg aggctgtgga ccaggatccg atgcccaggt cccgcccccc 6001 accccacccc aggcccagaa tcgaggtgcc ttggactttg gaggggccag gcctggtgaa 6061 tggggggcgg ggcggcgccc tcagggtaca gagcacagac agatagacat tccagagact 6121 gtattgagag tctttataaa gtgtgggaga tttaaaaaaa aaaaaaactg ataaaaatgc 6181 actttttggg agtggggagg gagaagcttt aaaagtaata aaaaacaaac aaaaacacaa 6241 aagatgaaaa aacaaaaaaa ttcatttttc ttgtacataa aaaaaaaaaa agaaccacta 6301 aacgcagcct gttacgacc [00076] SEQ ID NO: 7 is an amino acid sequence of human LRF. 1 maggvdgpig ipfpdhssdi lsglneqrtq gllcdvvilv egrefpthrs vlaacsqyfk 61 klftsgavvd qqnvyeidfv saealtalmd faytatltvs tanvgdilsa arlleipavs 121 hvcadlldrq ilaadagada gqldlvdqid qrnllrakey leffqsnpmn slppaaaaaa 181 asfpwsafga sdddldatke avaaavaava agdcngldfy gpgppaerpp tgdgdegdsn 241 pglwperded aptgglfppp vappaatqng hygrggeeea aslseaapep gdspgflsga 301 aegedgdgpd vdglaastll qqmmssvgra gaaagdsdee sraddkgvmd yylkyfsgah 361 dgdvypawsq kvekkiraka fqkcpicekv iqgagklprh irthtgekpy ecnickvrft 421 rqdklkvhmr khtgekpylc qqcgaafahn ydlknhmrvh tglrpyqcds ccktfvrsdh 481 lhrhlkkdgc ngvpsrrgrk prvrggapdp spgatatpga paqpsspdar rngqekhfkd 541 edededvasp dglgrlnvag aggggdsggg pgaatdgnft agla [00077] In some embodiments, LRF can be targeted for degradation in erythroid cells via TRIM10 or TRIM58 in a manner directly analogous to that described herein for targeting of BCL11A in erythroid cells. Briefly, in one embodiment, a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for TRIM10 or TRIM58 and a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for LRF can be fused, optionally via a peptide linker. In another embodiment, TRIM10 or TRIM58 polypeptide itself can be fused to a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for LRF. Introduction of either or both of these constructs to a cell, e.g., an erythroid cell, for example as discussed herein for BCL11A-targeting constructs, can target LRF for degradation in those cells. ZNF410 [00078] Zinc Finger Protein 410 (ZNF410), also known as APA-1 is a transcription factor that contains five tandem canonical C2H2 zinc fingers. See, e.g., Lan et al., Mol Cell.21: 239-254, (2021), which is incorporated herein by reference in its entirety. ZNF410 binds to the sequence motif 5’-CATCCCATAATA-3’ (SEQ ID NO: 66) in regulatory elements and modulates transcription of target genes. In erythroid cells, ZNF410 directly binds and transcriptionally activates CHD4, a component of the NuRD complex, which leads to repression of fetal globin genes HBG1 and HBG2. Sequences of ZNF410 are known for a number of species, e.g., human ZNF410 (NCBI GeneID: 57862), mRNA (e.g., ZNF410 Isoform a: NCBI Ref Seq NM_001242924.2, SEQ ID NO: 8) and polypeptide (e.g., ZNF410 Isoform a: NCBI Ref Seq: NP_001229853.1, SEQ ID NO: 9). Antibodies for specific detection of or binding to ZNF410 are available, for example, from Creative Biolabs, see Mouse Anti-ZNF410 Recombinant Antibody (clone 1C7) CBMAB-0827-LY; Mouse Anti- ZNF410 Recombinant Antibody (clone 1A12) CBMAB-0826-LY; and Anti-ZNF410 monoclonal antibody (clone DCABH-17601. ZNF410 can refer to human ZNF410, including naturally occurring variants and alleles thereof. For example, SEQ ID NO: 8 is an mRNA sequence of human ZNF410. Note: U’s replaced with T’s in this representation. 1 gtgtgtggac ggaattcggg accgactgac ggccggccgg cttcccggaa ctggaaggtt 61 acattgatta cccacctagt acaacatctt acgggaagag catagtattt cctagaggaa 121 tatgaacata acaggaaggt atcattggct ctgaattaaa tttgaacttg tcccctgaat 181 agctacaggt tttggaagct gaatcaatgt tatcagatga gttagaatcc aaaccagagc 241 tcctggtaca gtttgttcag aatacgtcca tcccattggg acaggggctt gtagaatcag 301 aagctaaaga tattacttgc ttgtccctcc ttcccgtgac tgaagcctca gaatgcagtc 361 ggctaatgtt accagaacct gttccatgga gagaagagga tggaaagtca ggctgcagtg 421 acttgaatga tactacaaat cattctaact cctccaagga ggtcccttcc tcagctgttt 481 tgagaagcct tcgggtgaat gtgggtccag acggagagga gacgagagct cagactgtac 541 agaaatcccc ggagtttttg tccacttcag agtcttctag cttgttgcaa gatctacagc 601 caagtgatag cacttctttt attcttctta acctaacaag agcaggtctg ggctcttcag 661 ctgagcactt agtgtttgta caggatgagg cagaagattc agggaatgat ttcctctcca 721 gtgagagcac agacagtagc attccatggt tcctccgggt tcaggagttg gcccatgaca 781 gtttgattgc tgctactcgt gcacaactgg caaagaatgc aaaaaccagc agcaatggag 841 aaaatgtcca ccttggttct ggtgatgggc agtcaaaaga ttctgggccc cttcctcaag 901 tggaaaagaa gctcaagtgt acagttgaag gttgtgaccg gacatttgta tggccagctc 961 actttaaata ccacctcaag actcatcgaa atgaccgctc cttcatctgt cctgcagaag 1021 gttgtgggaa aagcttctat gtgctgcaga ggctgaaggt gcacatgagg acccacaatg 1081 gagagaagcc ctttatgtgc catgagtctg gctgtggtaa gcagtttact acagctggaa 1141 acctgaagaa ccaccggcgc atccacacag gagagaaacc tttcctttgt gaagcccaag 1201 gatgtggccg ttcctttgct gagtattcta gcctccgaaa acatctggtg gttcactcag 1261 gagagaagcc tcatcagtgc caagtctgtg ggaagacctt ctctcagagt ggaagcagga 1321 atgtgcatat gagaaagcat cacctgcagc tgggagcagc tgggagtcaa gagcaggagc 1381 aaactgctga gccactaatg ggcagtagtt tgcttgaaga ggcttcagta cccagtaaaa 1441 acctggtgtc tatgaattcc cagcccagcc ttggtggaga gtccttgaac ctaccaaata 1501 ccaattctat cctgggagtt gatgatgagg tgcttgctga aggatcccca cgttccctgt 1561 cttcagtgcc tgatgtgaca catcacctgg tgaccatgca gtcagggagg caatcatatg 1621 aagtttctgt cttaactgca gtaaatccac aagagagtct cgctccgttg cccaggctgg 1681 agtgcagtgg tgcgttctcg gctcactgca acctctgcct cccaggttca agcgattctc 1741 ctgcctcagc ctcctgagta gctgctacta cagttactaa accaaggaga tttaactgaa 1801 agacggacat gagcgtgggt gctgactcct ggaagagcaa ctctatctga tctcaaaatg 1861 cgtatactgg gaacaggatg ccttagccca caacagaacc agaatgaatc tttgaaggca 1921 caagactctg cttttgccac tcttcctctt tcctggtata gaagatggat gtaggagagc 1981 ttcttttcta actaccatct gatcagacaa ggaatgaagc aatgactgtg ggctgggaaa 2041 ctgtacctac ctctcttccc actgcaaatt tctgggatag accaaaagtg aatttgatta 2101 tgtgttggct gaagttcttc attctgactg ttgaggggag gttttccttt gaagagtttt 2161 catcccagac tcagctgtct tttcacatgg atgaaataat tcctgctacc aacaacagag 2221 cttcaccagg aagttgagtt ttcaagatgc cttgttgctt tgaagaaggg agtgatgtca 2281 attctcttgt tacattctcc ctttagcaac ctgagtaaga gactctctgc cactgggctg 2341 caaaaaaata aattacttga atctcccctt ggcccaggct gaggtactat cttgtcctat 2401 acacttctac ttggtcactt tgctttcttc gttaatggaa catacagtag catgttaaga 2461 gggatttcat gtttttgttt ttttaaagtt aaaaattaca tgatgcagag attcaggttt 2521 tcctttaaat aaacaaaaca gcccagtcag ttctttggct cttgttttat acaaaatttg 2581 ttttcttaga gattaagaat ttaatctttc cctttaaaat agtgtattga tttaccctta 2641 ggattccata ccagtaaaac tgaaccgagg agtcttagga aacaacaaga acatcttcat 2701 ttcttaagcc caggtgatag ttactctgtc accaccaaaa aagacgcatc tgagaaaatg 2761 gcaataaaaa cagatacttc tgaatttttc ca [00079] SEQ ID NO: 9 is an amino acid sequence of human ZNF410. 1 mlsdeleskp ellvqfvqnt siplgqglve seakditcls llpvteasec srlmlpepvp 61 wreedgksgc sdlndttnhs nsskevpssa vlrslrvnvg pdgeetraqt vqkspeflst 121 sesssllqdl qpsdstsfil lnltraglgs saehlvfvqd eaedsgndfl ssestdssip 181 wflrvqelah dsliaatraq laknaktssn genvhlgsgd gqskdsgplp qvekklkctv 241 egcdrtfvwp ahfkyhlkth rndrsficpa egcgksfyvl qrlkvhmrth ngekpfmche 301 sgcgkqftta gnlknhrrih tgekpflcea qgcgrsfaey sslrkhlvvh sgekphqcqv 361 cgktfsqsgs rnvhmrkhhl qlgaagsqeq eqtaeplmgs slleeasvps knlvsmnsqp 421 slggeslnlp ntnsilgvdd evlaegsprs lssvpdvthh lvtmqsgrqs yevsvltavn 481 pqeslaplpr lecsgafsah cnlclpgssd spasas [00080] In some embodiments, ZNF410 can be targeted for degradation in erythroid cells via TRIM10 or TRIM58 in a manner directly analogous to that described herein for targeting of BCL11A in erythroid cells. Briefly, in one embodiment, a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for TRIM10 or TRIM58 and a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for ZNF410 can be fused, optionally via a peptide linker. In another embodiment, TRIM10 or TRIM58 polypeptide itself can be fused to a binding agent, e.g., an antibody or antigen-binding fragment thereof specific for ZNF410. Introduction of either or both of these constructs to a cell, e.g., an erythroid cell, for example as discussed herein for BCL11A-targeting constructs, can target ZNF410 for degradation in those cells. BCL11A [00081] BCL11A (B-cell lymphoma/leukemia 11A), 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: 10) and polypeptide (e.g., BCL11A isoform 1: NCBI Ref Seq: NP_075044.2, SEQ ID NO: 11). BCL11A can refer to human BCL11A, including naturally occurring variants and alleles thereof. For example, SEQ ID NO: 10 is a mRNA sequence for Isoform 1 of human BCL11A. Note: U’s replaced with T’s in this representation. 1 gtctctgtcc atccagactc ctgacgttca agttcgcagg gacgtcacgt ccgcacttga 61 acttgcagct caggggggct tttgccattt ttttcatctc tctctctctc tctccctcta 121 tctctcttct ctctctctcc ctcttttttt tttttttttt tttttttttt ttgcttaaaa 181 aaaagccatg acggctctcc cacaattcat cttccctgcg ccatctttgt attatttcta 241 atttattttg gatgtcaaaa ggcactgatg aagatatttt ctctggagtc tccttctttc 301 taacccggct ctcccgatgt gaaccgagcc gtcgtccgcc cgccgccgcc gccgccgccg 361 ccgccgcccg ccccgcagcc caccatgtct cgccgcaagc aaggcaaacc ccagcactta 421 agcaaacggg aattctcgcc cgagcctctt gaagccattc ttacagatga tgaaccagac 481 cacggcccgt tgggagctcc agaaggggat catgacctcc tcacctgtgg gcagtgccag 541 atgaacttcc cattggggga cattcttatt tttatcgagc acaaacggaa acaatgcaat 601 ggcagcctct gcttagaaaa agctgtggat aagccacctt ccccttcacc aatcgagatg 661 aaaaaagcat ccaatcccgt ggaggttggc atccaggtca cgccagagga tgacgattgt 721 ttatcaacgt catctagagg aatttgcccc aaacaggaac acatagcaga taaacttctg 781 cactggaggg gcctctcctc ccctcgttct gcacatggag ctctaatccc cacgcctggg 841 atgagtgcag aatatgcccc gcagggtatt tgtaaagatg agcccagcag ctacacatgt 901 acaacttgca aacagccatt caccagtgca tggtttctct tgcaacacgc acagaacact 961 catggattaa gaatctactt agaaagcgaa cacggaagtc ccctgacccc gcgggttggt 1021 atcccttcag gactaggtgc agaatgtcct tcccagccac ctctccatgg gattcatatt 1081 gcagacaata acccctttaa cctgctaaga ataccaggat cagtatcgag agaggcttcc 1141 ggcctggcag aagggcgctt tccacccact ccccccctgt ttagtccacc accgagacat 1201 cacttggacc cccaccgcat agagcgcctg ggggcggaag agatggccct ggccacccat 1261 cacccgagtg cctttgacag ggtgctgcgg ttgaatccaa tggctatgga gcctcccgcc 1321 atggatttct ctaggagact tagagagctg gcagggaaca cgtctagccc accgctgtcc 1381 ccaggccggc ccagccctat gcaaaggtta ctgcaaccat tccagccagg tagcaagccg 1441 cccttcctgg cgacgccccc cctccctcct ctgcaatccg cccctcctcc ctcccagccc 1501 ccggtcaagt ccaagtcatg cgagttctgc ggcaagacgt tcaaatttca gagcaacctg 1561 gtggtgcacc ggcgcagcca cacgggcgag aagccctaca agtgcaacct gtgcgaccac 1621 gcgtgcaccc aggccagcaa gctgaagcgc cacatgaaga cgcacatgca caaatcgtcc 1681 cccatgacgg tcaagtccga cgacggtctc tccaccgcca gctccccgga acccggcacc 1741 agcgacttgg tgggcagcgc cagcagcgcg ctcaagtccg tggtggccaa gttcaagagc 1801 gagaacgacc ccaacctgat cccggagaac ggggacgagg aggaagagga ggacgacgag 1861 gaagaggaag aagaggagga agaggaggag gaggagctga cggagagcga gagggtggac 1921 tacggcttcg ggctgagcct ggaggcggcg cgccaccacg agaacagctc gcggggcgcg 1981 gtcgtgggcg tgggcgacga gagccgcgcc ctgcccgacg tcatgcaggg catggtgctc 2041 agctccatgc agcacttcag cgaggccttc caccaggtcc tgggcgagaa gcataagcgc 2101 ggccacctgg ccgaggccga gggccacagg gacacttgcg acgaagactc ggtggccggc 2161 gagtcggacc gcatagacga tggcactgtt aatggccgcg gctgctcccc gggcgagtcg 2221 gcctcggggg gcctgtccaa aaagctgctg ctgggcagcc ccagctcgct gagccccttc 2281 tctaagcgca tcaagctcga gaaggagttc gacctgcccc cggccgcgat gcccaacacg 2341 gagaacgtgt actcgcagtg gctcgccggc tacgcggcct ccaggcagct caaagatccc 2401 ttccttagct tcggagactc cagacaatcg ccttttgcct cctcgtcgga gcactcctcg 2461 gagaacggga gtttgcgctt ctccacaccg cccggggagc tggacggagg gatctcgggg 2521 cgcagcggca cgggaagtgg agggagcacg ccccatatta gtggtccggg cccgggcagg 2581 cccagctcaa aagagggcag acgcagcgac acttgtgagt actgtgggaa agtcttcaag 2641 aactgtagca atctcactgt ccacaggaga agccacacgg gcgaaaggcc ttataaatgc 2701 gagctgtgca actatgcctg tgcccagagt agcaagctca ccaggcacat gaaaacgcat 2761 ggccaggtgg ggaaggacgt ttacaaatgt gaaatttgta agatgccttt tagcgtgtac 2821 agtaccctgg agaaacacat gaaaaaatgg cacagtgatc gagtgttgaa taatgatata 2881 aaaactgaat agaggtatat taatacccct ccctcactcc cacctgacac cccctttttc 2941 accactcccc ttccccatcg ccctccagcc ccactccctg taggattttt ttctagtccc 3001 atgtgattta aacaaacaaa caaacaaaca gaagtaacga agctaagaat atgagagtgc 3061 ttgtcaccag cacacctgtt ttttttcttt ttctttttct tttttctttt tccttttttt 3121 tttttttcct ttatgttctc accgtttgaa tgcatgatct gtatggggca atactattgc 3181 attttacgca aactttgagc ctttctcttg tgcaataatt tacatgttgt gtatgttttt 3241 ttttaaactt agacagcatg tatggtatgt tatggctatt ttaaattgtc cctaattcgt 3301 tgctgagcaa acatgttgct gtttccagtt ccgttctgag agaaaaagag agagagagag 3361 aaaaagacca tgctgcatac attctgtaat acatatcatg tacagtttta ttttataacg 3421 tgaggaggaa aaacagtctt tggattaacc ctctatagac agaatagata gcactgaaaa 3481 aaaatctcta tgagctaaat gtctgtctct aaagggttaa atgtatcaat tggaaaggaa 3541 gaaaaaaggc cttgaattga caaattaaca gaaaaacaga acaagtttat tctatcattt 3601 ggttttaaaa tatgagtgcc ttggatctat taaaaccaca tcgatggttc tttctacttg 3661 ttataaactt gtagcttaat tcagcattgg gtgaggtaat aaaccttagg aactagcata 3721 taattctata ttgtatttct cacaacaatg gctacctaaa aagatgaccc attatgtcct 3781 agttaatcat catttttcct ttagtttaat tttataaaca aaactgatta taccagtata 3841 aaagctactt tgctcctggt gagagcttaa aagaaatggg ctgttttgcc caaagtttta 3901 ttttttttaa acaatgatta aattgaatgt gtaatgtgca aaagccctgg aacgcaatta 3961 aatacactag taaggagttc attttatgaa gatatttgct ttaataatgt ctttttaaaa 4021 atactggcac caaaagaaat agatccagat ctacttggtt gtcaagtgga caatcaaatg 4081 ataaacttta agaccttgta taccatattg aaaggaagag gctgacaata aggtttgaca 4141 gaggggaaca gaagaaaata atatgattta ttagcacaac gtggtactat ttgccattta 4201 aaactagaac aggtatataa gctaatattg atacaatgat gattaactat gaattcttaa 4261 gacttgcatt taaatgtgac attcttaaaa aaagaagaga aagaatttta agagtagcag 4321 tatatatgtc tgtgctccct aaaagttgta cttcatttct tttccataca ctgtgtgcta 4381 tttgtgttaa catggaagag gattcattgt ttttattttt atttttttaa ttttttcttt 4441 tttattaagc tagcatctgc cccagttggt gttcaaatag cacttgactc tgcctgtgat 4501 atctgtatct tttctctaat cagagataca gaggttgagt ataaaataaa cctgctcaga 4561 taggacaatt aagtgcactg tacaattttc ccagtttaca ggtctatact taagggaaaa 4621 gttgcaagaa tgctgaaaaa aaattgaaca caatctcatt gaggagcatt ttttaaaaac 4681 taaaaaaaaa aaaactttgc cagccattta cttgactatt gagcttactt acttggacgc 4741 aacattgcaa gcgctgtgaa tggaaacaga atacacttaa catagaaatg aatgattgct 4801 ttcgcttcta cagtgcaagg atttttttgt acaaaacttt tttaaatata aatgttaaga 4861 aaaatttttt ttaaaaaaca cttcattatg tttagggggg aactgcattt tagggttcca 4921 ttgtcttggt ggtgttacaa gacttgttat ccatttaaaa atggtagtgg aaattctatg 4981 ccttggatac acaccgctct tcaggttgta aaaaaaaaaa acatacattg gggaaaggtt 5041 taagattata tagtacttaa atataggaaa atgcacactc atgttgattc ctatgctaaa 5101 atacatttat ggtctttttt ctgtatttct agaatggtat ttgaattaaa tgttcatcta 5161 gtgttaggca ctatagtatt tatattgaag cttgtatttt taactgttgc ttgttctctt 5221 aaaaggtatc aatgtacctt ttttggtagt ggaaaaaaaa aagacaggct gccacagtat 5281 atttttttaa tttggcagga taatatagtg caaattattt gtatgcttca aaaaaaaaaa 5341 aaagagagaa acaaaaaagt gtgacattac agatgagaag ccatataatg gcggtttggg 5401 ggagcctgct agaatgtcac atggatggct gtcatagggg ttgtacatat ccttttttgt 5461 tcctttttcc tgctgccata ctgtatgcag tactgcaagc taataacgtt ggtttgttat 5521 gtagtgtgct ttttgtccct ttccttctat caccctacat tccagcatct taccttcata 5581 tgcagtaaaa gaaagaaaga aaaaaaaagg aaaaaaaaaa aaaaaccaat gttttgcagt 5641 ttttttcatt gccaaaaact aaatggtgct ttatatttag attggaaaga atttcatatg 5701 caaagcatat taaagagaaa gcccgcttta gtcaatactt ttttgtaaat ggcaatgcag 5761 aatattttgt tattggcctt ttctattcct gtaatgaaag ctgtttgtcg taacttgaaa 5821 ttttatcttt tactatggga gtcactattt attattgctt atgtgccctg ttcaaaacag 5881 aggcacttaa tttgatcttt tatttttctt tgtttttatt ttttttttta tttagatgac 5941 caaaggtcat tacaacctgg ctttttattg tatttgtttc tggtctttgt taagttctat 6001 tggaaaaacc actgtctgtg tttttttggc agttgtctgc attaacctgt tcatacaccc 6061 attttgtccc tttattgaaa aaataaaaaa aattaaagta ca [00082] SEQ ID NO: 11 is an amino acid sequence of Isoform 1 of human BCL11A. 1 msrrkqgkpq hlskrefspe pleailtdde pdhgplgape gdhdlltcgq cqmnfplgdi 61 lifiehkrkq cngslcleka vdkppspspi emkkasnpve vgiqvtpedd dclstssrgi 121 cpkqehiadk llhwrglssp rsahgalipt pgmsaeyapq gickdepssy tcttckqpft 181 sawfllqhaq nthglriyle sehgspltpr vgipsglgae cpsqpplhgi hiadnnpfnl 241 lripgsvsre asglaegrfp ptpplfsppp rhhldphrie rlgaeemala thhpsafdrv 301 lrlnpmamep pamdfsrrlr elagntsspp lspgrpspmq rllqpfqpgs kppflatppl 361 pplqsappps qppvksksce fcgktfkfqs nlvvhrrsht gekpykcnlc dhactqaskl 421 krhmkthmhk sspmtvksdd glstasspep gtsdlvgsas salksvvakf ksendpnlip 481 engdeeeeed deeeeeeeee eeeelteser vdygfglsle aarhhenssr gavvgvgdes 541 ralpdvmqgm vlssmqhfse afhqvlgekh krghlaeaeg hrdtcdedsv agesdriddg 601 tvngrgcspg esasgglskk lllgspssls pfskriklek efdlppaamp ntenvysqwl 661 agyaasrqlk dpflsfgdsr qspfassseh ssengslrfs tppgeldggi sgrsgtgsgg 721 stphisgpgp grpsskegrr sdtceycgkv fkncsnltvh rrshtgerpy kcelcnyaca 781 qsskltrhmk thgqvgkdvy kceickmpfs vystlekhmk kwhsdrvlnn dikte [00083] 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: 67: ytcttckqpftsawfllqhaqnth); Zinc finger domain 2 amino acids: 377-399 (SEQ ID NO: 68: kscefcgktfkfqsnlvvhrrsh); Zinc finger domain 3 amino acids: 405-429 (SEQ ID NO: 69: ykcnlcdhactqasklkrhmkthmh); Zinc finger domain 4 amino acids: 742-764 (SEQ ID NO: 70: dtceycgkvfkncsnltvhrrsh); Zinc finger domain 5 amino acids: 770-792 (SEQ ID NO: 71: ykcelcnyacaqsskltrhmkth); Zinc finger domain 6 amino acids: 800-823 (SEQ ID NO: 72: ykceickmpfsvystlekhmkkwh). [00084] BCL11A can be targeted for degradation in erythroid cells, for example, through introduction of a bispecific construct as described herein that includes an antibody or antigen- binding fragment thereof that specifically binds BCL11A and a binding element that specifically binds TRIM10 or TRIM58. In one embodiment, the binding element that specifically binds TRIM10 or TRIM58 can be an antibody or antigen-binding fragment thereof. Any of a number of ways to introduce the construct to erythroid cells can be used, including, but not limited to introduction of the construct or a nucleic acid encoding it via lipid nanoparticles, introduction (e.g., as a fusion) via cell-penetrating peptides, or introduction of a vector, e.g., a viral vector, among others, encoding the construct. [00085] BCL11A can also be targeted for degradation, for example, through introduction of a fusion construct as described herein that includes an antibody or antigen-binding fragment thereof that specifically binds BCL11A fused to a TRIM10 or TRIM58 polypeptide. The fusion construct can be introduced to erythroid cells in any of a number of ways, including, but not limited to introduction of the construct or a nucleic acid encoding it via lipid nanoparticles, introduction (e.g., as a fusion) via cell-penetrating peptides, or introduction of a vector, e.g., a viral vector, among others, encoding the construct. ANTIBODIES [00086] As discussed above, in various embodiments, described herein are antibodies or antigen-binding fragments thereof that specifically bind to BCL11A or to a TRIM10 or TRIM58 polypeptide. 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, VH, 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 VH/VL, 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. [00087] 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. [00088] 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. [00089] 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. [00090] As noted above, the VH and VL 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. [00091] 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 VH 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 VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. [00092] 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. [00093] In some embodiments, the target ligand-binding recognition domain is a nanobody. A “nanobody” (Nb) is a single variable domain (VHH) 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. [00094] 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 (KA), which is 1/ KD). 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 KD 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 KD 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. [00095] 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 OctetTM analysis, enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA’s), for example. [00096] 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 105 to 1012 liter/moles or more, such as 107 to 1012 liter/moles or 108 to 1012 liter/moles). Any KD value greater than 10-4 mol/liter (or any KA value lower than 104 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 KD. 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. [00097] 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. [00098] 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. In some embodiments, a “target” can be one which it is wished to specifically degrade or inhibit, which can be the case when BCL11A is a target. In other instances, a “target” can be one which it is wished to bind and tether to another moiety, which can be the case when the target is, e.g., a TRIM10 or TRIM58 polypeptide. [00099] 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 (KD) 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 KD at least 100X, or at least 1000X greater than the KD with which it binds to BCL11A. [000100] Similarly, an antibody reagent that specifically binds to TRIM10 or to TRIM58 binds to TRIM10 or TRIM58 with a dissociation constant (KD) 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. [000101] 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. [000102] 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 [000103] The following provides amino acid sequence for nanobodies that specifically bind human BCL11A. SEQ ID NO: 12‐ NB14  QVQLVESGGG LVQAGGSLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAS ITWRDDSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 13‐ NB15  QVQLVESGGG LVQAGGSLRL SCAASGSTFS DYAMGWYRQA PGKERELVAV ITASDDITYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAAAV TPG‐QDLDYW GQGTQVTVSS   (119)    SEQ ID NO: 14‐ NB53  QVQLVESGGG LVQAGGSLRL SCAASGSTFS GYAMGWYRQA PGKERELVAA ITSSGASTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD EGY‐LDYDSW GQGTQVTVSS    (119)    SEQ ID NO: 15‐ NB61  QVQLVESGGG LVQAGGSLRL SCAASGFIFD SYAMGWYRQA PGKERELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)      SEQ ID NO: 16‐ NB19 (Nb6101‐M45D‐19)  RVQLVESGGG LVQAGGSLRL SCAADGFDFK SYAMGWYRQA PGREDELVAA ITASGDYTYY  (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPDD TAVYYCAALD ‐‐Y‐VAEGYW GQGTQVTVSS    (117)    SEQ ID NO: 17‐ NB5344‐N74D  QVQLVKSGGG LVQAGDSLRL SCAASGSTFS GYAMGWYRQA PGKERELVAA ITSSGASTYY    (60)  ADSVRGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD EGY‐LDYDSW GQGTQVTVSS    (119)    SEQ ID NO: 18‐ NB6101  RVQLVESGGG LVQAGGSLRL SCAASGFIFD SYAMGWYRQA PGKEMELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)    SEQ ID NO: 19‐ NB6101‐M45D  RVQLVESGGG LVQAGGSLRL SCAASGFIFD SYAMGWYRQA PGKEDELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)    SEQ ID NO: 20‐ NB1453  QVQLVESGGG LVQAGGSLHL SCAVSGSIFS FYAMGWYRQA PGKEREFVAS ITRRDDSTYY    (60)  ADSVEGRFAI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 21‐ NB1459  QVQLVESGGG LVQAEGSLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAS ITWRDDSTYY    (60)  ADSVTGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYSVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 22‐ NB1431  QVQLVESGGG LVQAGGCLRL SCAASGSIFS FYAMGWYRQA PGNEREFVAS ITWRDDSTYY   (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYTVEMDYW GQGTHVTVSS   (120)    SEQ ID NO: 23‐ NB1407  QVQLVESGGD LVQAGASLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAS ITWRDDSTYY    (60)  ADSVKGRFTI SRDNARNTVY LQMNNLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 24‐ NB1481  QVQLVESGGG LVQAGGSLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAF ITWRDDSTYY    (60)  ADSVKGRFRI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 25‐ NB1419  QVQLVESGGD LVQAGGSLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAS ITWRDDSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNRLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 26‐ NB1487  QVQLVESGGG LVQAGGSLRL SCAASGSIFS FYAMGWYRQA PGKEREFVAS ITWRDDSTYY    (60)  ADSVMGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADT DDYTVEMDYW GQGTQVTVSS   (120)    SEQ ID NO: 27‐ NB6101‐M45D‐14  QVQLVKSGGG LVQAGDSLRL SCAASGSTFS GYAMGWYRQA PGKERELVAA ITSSGASTYY    (60)  ADSVRGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD EGY‐LDYDSW GQGTQVTVSS    (119)    SEQ ID NO: 28‐ NB6130  QVQFVESGGG FVQAGGSLRL SCAASGFIFD SYAMGWYSQA PGKERELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)    SEQ ID NO: 29‐ NB6127  QVQLVESGGG LVQAGGSLRL SCAASGFIFD SYSMGWYRQA PGKEGELVAA ITSSGSSTYY    (60)  ADSVNGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)    SEQ ID NO: 30‐ NB6160  QVQLVESGGG LVQAGGSLRL SCAASGFIFD SYAMGWYRQA PGKEGELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVSVSS    (117)    SEQ ID NO: 31‐ NB6102  QVQLVESGGG LVQAGGSLRL SCAASGFIFD SYAMGWYRQA PGKEGELVAA ITSSGSSTYY    (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALD ‐‐Y‐VIDGYW GQGTQVTVSS    (117)    SEQ ID NO: 32‐ NB6101‐M45D‐26  RVQLVESGGG LVQAGGSLRL SCAADGFDFS SYAMGWYRQA PGTQDELVAA ITASGSYTYY   (60)  SDSVKGRFTI SRDNAKNTVY LQMNSLKPDD TAVYYCAALS ‐‐Y‐VAEGYW GQGTQVTVSS    (117)    SEQ ID NO: 33‐ NB6101‐M45D‐20  RVQLVESGGG LVQAGGSLRL SCAADGFDFK SYAMGWYRQA PGYEDELVAA ITASGSYTYY   (60)  SDSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAALS ‐‐Y‐VAEGYW GQGTQVTVSS    (117)    Table 1: CDR’s for Nanobodies directed to ZNF6
Figure imgf000037_0001
Figure imgf000038_0001
  Nanobodies directed to ZNF23 “extended region” SEQ ID NO: 34‐ 2D9 / Consensus   QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTYY  (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADQ DVYPYEYWGQ GTQVTVSS  (118)    SEQ ID NO: 35‐ V102G  QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTYY  (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADQ DGYPYEYWGQ GTQVTVSS  (118)    SEQ ID NO: 36‐ W108L  QVQLVESGGG LVQAGGSLRL SCAASGSIFV NNAMGWYRQA PGKERELVAA ISASGGSTYY  (60)  ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAVYYCAADQ DVYPYEYLGQ GTQVTVSS  (118)    CDR’s for Nanobodies directed to ZNF23 extended region SEQ ID NO: 37‐ 2D9 / V102G / W108L CDR 1  SIFVNNAM    SEQ ID NO: 38‐ 2D9 / V102G / W108L CDR 2  ELVAAISASGGSTYY    SEQ ID NO: 39‐ 2D9 CDR 3  ADQDVYPYEYW    SEQ ID NO: 40‐ 2D9 CDR 3  ADQDGYPYEYW    SEQ ID NO: 41‐ 2D9 CDR 3  ADQDVYPYEYL  Nanobodies directed to BCL11A ZNF4 SEQ ID NO: 42‐ Nb12  QVQLVESGGG SVQPGGSLTL SCTASGFPLS MWGMTWLRQA PGKGLERVSG IASDSTNTYY  (60)   ADSVKGRFAI SRDNAKNKLF LQMNSLKPED TAVYFCARKW TNWDLKGQGT QVTVSS    (116)    [000104] As a means of describing structure involved in or necessary for binding to BCL11A, the nanobody NB14 of SEQ ID NO: 12 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: 12 occurs only at the sites listed in Table 2. Table 2: BCL11A ZNF6 nanobody amino acid variations. Compared to Nb14 (SEQ ID NO: 12).
Figure imgf000039_0001
Figure imgf000040_0001
  HEMOGLOBINOPATHIES [000105] Fetal hemoglobin (HbF) is a tetramer of two adult ^-globin polypeptides and two fetal ^-like ^-globin polypeptides. During gestation, the duplicated ^-globin genes constitute the predominant genes transcribed from the ^-globin locus. Following birth, ^-globin becomes progressively replaced by adult ^-globin, a process referred to as the "fetal switch.” In humans, the developmental switch from production of predominantly fetal hemoglobin or HbF ( ^2 ^2) to production of adult hemoglobin or HbA ( ^2 ^2) 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)). [000106] 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 ^-globin in sufficient amounts, while others involve the failure to produce normal ^-globin entirely. Those disorders specifically associated with the ^-globin protein are referred to generally as ^-hemoglobinopathies. For example, ^-thalassemias result from a partial or complete defect in the expression of the ^- globin gene, leading to deficient or absent HbA. Sickle cell anemia or sickle cell disease (SCD) results from a point mutation in the ^-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)). [000107] The search for treatment aimed at reduction of globin chain imbalance in patients with ^-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 ^-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)). [000108] 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 ^-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. [000109] 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. [000110] 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). [000111] 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 ^-thalassemia, thalassemia major, thalassemia intermedia, ^-thalassemias such as hemoglobin H disease. ^-thalassemias are caused by a mutation in the ^ ^ ^globin chain, and can occur in a major or minor form. In the major form of ^-thalassemia, children are normal at birth, but develop anemia during the first year of life. The mild form of ^ -thalassemia produces small red blood cells. Alpha-thalassemias are caused by deletion of a gene or genes from the globin chain. [000112] 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 ^- 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 [000113] In various embodiments, the activity of BCL11A is inhibited or decreased, e.g., by targeted protein degradation. 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 compositions and methods described herein, than a comparable, control cell or population, wherein BCL11A is not targeted for degradation. 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 BCL11A is not targeted for degradation. 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. [000114] Alternatively, or in addition, BCL11A activity can be assayed by measuring fetal hemoglobin expression at the mRNA or protein level following treatment targeting BCL11A for degradation. Increased expression of endogenous HbF in adult cells treated or contacted with a construct targeting BCL11A for degradation is indicative of de-repression, and therefore reduced activity of BCL11A. Treatment of hemoglobinopathies by targeting BCL11A [000115] 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, targeted degradation of BCL11A via introduction of a targeted protein degradation construct as described herein can treat a hemoglobinopathy. [000116] In connection with contacting a cell with a targeted protein degradation construct aimed at BCL11A, “increasing the fetal hemoglobin levels” in a cell indicates that HbF is at least 5% higher in populations treated with the construct than in a comparable, control population, wherein no BCL11A targeting construct is present. It is preferred that the percentage of HbF expression in a BCL11A targeting construct-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 targeting construct. 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 ^-globin protein and PCR quantification of mRNA encoding fetal hemoglobin (e.g., HBG1 or HBG2 mRNAs). [000117] 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 [000118] 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 to the antibody or antigen-binding fragment thereof. As discussed herein above, fusion of an antibody or antigen-binding fragment thereof that binds TRIM10 or TRIM58 to an antibody or antigen-binding fragment thereof that binds an erythroid-specific or erythroid- enriched polypeptide can provide an avenue for targeted protein degradation of the erythroid- specific or erythroid enriched polypeptide in erythroid cells. Alternatively, a TRIM10 or TRIM58 polypeptide can be fused to an antibody or antigen-binding fragment thereof that specifically binds an erythroid-specific or erythroid-enriched polypeptide to provide a targeted protein degradation construct for that erythroid-specific or erythroid-enriched polypeptide. [000119] One of ordinary skill in the art, given sequences encoding TRIM10, TRIM58, or encoding an antibody or antigen-binding fragment thereof that specifically binds TRIM10 or TRIM58, can generate a fusion construct that fuses the TRIM10 or TRIM58 polypeptide or TRIM10 or TRIM58-specific antibody sequence to a given fusion partner, e.g., antibody that binds a target for targeted protein degradation where the sequence for the fusion partner is known. Fusion can optionally include a peptide linker between the fusion partners. In one embodiment, the linker can be a serine/glycine rich linker. By way of example, sequences for TRIM10, TRIM58, and nanobodies that specifically bind BCL11A are provided herein. Expression and Delivery of Targeted Protein Degradation Constructs [000120] In various embodiments, fusion proteins as described herein that target a protein of interest, e.g., an erythroid protein of interest, for degradation can be expressed from a vector as a recombinant polypeptide. [000121]   Sequences encoding a fusion protein as described herein 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. [000122] An expression vector can direct expression of a polypeptide (e.g., a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein) 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. [000123] Thus, in some embodiments, provided herein is a vector comprising a nucleic acid encoding a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein as described herein. Typically, where introduction of the sequence encoding the fusion protein 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. [000124] 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. [000125] 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. [000126] In some embodiments, provided herein is a host cell transformed with a nucleic acid molecule encoding a fusion protein for targeted degradation of an erythroid-enriched or erythroid specific protein as described herein. 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. [000127] In 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 fusion 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 lymphatic 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 ^ cells, among others. [000128] While introduction of nucleic acid encoding the fusion proteins 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 as discussed above, can be used to introduce sequence encoding the fusion protein into a cell expressing the target protein (including, but not limited to BCL11A, ZNF410 LRF) to thereby promote degradation of the target protein. Such cells include, for example, erythroid cells or erythroid progenitor cells in which inhibition of BCL11A can induce expression of HbF. [000129] 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. [000130] 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. [000131] 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. [000132] “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. [000133] 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. [000134] In some embodiments, the hematopoietic progenitor cells of the erythroid lineage have the cell surface marker characteristic of the erythroid lineage: CD71 and Ter119. [000135] It is contemplated that a construct or vector encoding a fusion protein as described herein 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. [000136] 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 the protein targeted for degradation in the target hematopoietic progenitor, erythroid progenitor or erythroid cells. When the fusion protein introduced targets BCL11A, such expression can thereby increase HbF expression in the cells and/or their progeny. [000137] In other embodiments, the degradation-targeting fusion protein can be produced, e.g., as a further fusion with a cell-penetrating peptide to thereby facilitate introduction of the degradation-targeting fusion protein to a target cell. [000138] 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. [000139] 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. [000140] 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. [000141] 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).” [000142] 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. [000143] 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. [000144] 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. [000145] 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. [000146] 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. [000147] 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; ^-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. [000148] 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. [000149] 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. [000150] In some embodiments, cell-penetration peptides or cell-penetrating peptides (CPPs) can be used as a transmembrane drug delivery agent for delivery of degradation-targeting fusion polypeptide 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. [000151] Delivery of a fusion protein as described herein, or nucleic acids encoding them can include the use of lipid complexes or lipid nanoparticles complexed or loaded with the fusion protein 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 [000152] As used herein, the terms "administering," "introducing" and "transplanting" are used interchangeably in the context of the placement of an agent, e.g. a targeted protein degradation fusion protein or composition 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., a fusion protein as 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. [000153] 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. [000154] In one embodiment, the agent (e.g., 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 fusion protein or composition described herein, or including nucleic acid or a vector encoding a fusion protein or composition described herein, or including an isolated 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. [000155] When provided prophylactically, an agent (e.g., a 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 ^-globin to predominantly ^-globin. Accordingly, the prophylactic administration of, e.g., a modified hematopoietic progenitor cell population can serve to prevent a hemoglobinopathy, as described herein. [000156] 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. [000157] In one embodiment, the term “effective amount" as used herein refers to the amount of an agent (e.g., a fusion protein or composition as 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. [000158] The efficacy of a treatment comprising an agent (e.g., a 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. [000159] 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. [000160] 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. [000161] 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. [000162] 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. [000163] 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. [000164] The treatment as described herein ameliorates one or more symptoms associated with, e.g., 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. [000165] 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. [000166] 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. [000167] The technology provided herein can further be defined by the following numbered paragraphs. [000168] Paragraph 1: A fusion polypeptide comprising a binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and a binding domain that specifically binds an erythroid-enriched polypeptide of interest. [000169] Paragraph 2: The fusion polypeptide of paragraph 1 which mediates degradation of the polypeptide of interest in erythroid cells. [000170] Paragraph 3: The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase specifically binds TRIM10 or TRIM58. [000171] Paragraph 4: The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase comprises an antibody or antigen-binding fragment thereof. [000172] Paragraph 5: The fusion polypeptide of any of the preceding paragraphs, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody. [000173] Paragraph 6: The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds an erythroid-enriched polypeptide of interest comprises an antibody or antigen-binding fragment thereof. [000174] Paragraph 7: The fusion polypeptide of any of the preceding paragraphs, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody. [000175] Paragraph 8: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is selected from BCL11A, LRF and ZNF410. [000176] Paragraph 9: The fusion polypeptide of any of the preceding paragraphs, wherein the E3 ubiquitin ligase is TRIM10 or TRIM58. [000177] Paragraph 10: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [000178] Paragraph 11: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide. [000179] Paragraph 12: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12. [000180] Paragraph 13: The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2. [000181] Paragraph 14: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34. [000182] Paragraph 15: The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids at amino acid number 102 or 108. [000183] Paragraph 16: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65. [000184] Paragraph 17: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO: 41). [000185] Paragraph 18: The fusion polypeptide of any of the preceding paragraphs which further comprises a cell-penetrating peptide. [000186] Paragraph 19: The fusion polypeptide of any of the preceding paragraphs, further comprising a linker peptide between the binding domain that specifically binds an erythroid- enriched E3 ubiquitin ligase and the binding domain that specifically binds an erythroid- enriched polypeptide of interest. [000187] Paragraph 20: A nucleic acid comprising sequence encoding a fusion polypeptide of any of the preceding paragraphs. [000188] Paragraph 21: A nucleic acid of any of the preceding paragraphs, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells. [000189] Paragraph 22: A vector comprising a nucleic acid of any of the preceding paragraphs. [000190] Paragraph 23: The vector of any of the preceding paragraphs, which is a viral vector. [000191] Paragraph 24: The viral vector of any of the preceding paragraphs, which is an AAV vector. [000192] Paragraph 25: A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to an erythroid cell. [000193] Paragraph 26: A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell expressing BCL11A. [000194] Paragraph 27: The method of any of the preceding paragraphs, wherein the targeted degradation of BCL11A is targeted to erythroid cells. [000195] Paragraph 28: A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression. [000196] Paragraph 29: A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder. [000197] Paragraph 30: A fusion polypeptide comprising a TRIM10 or TRIM58 polypeptide fused to a binding domain that specifically binds a target polypeptide. [000198] Paragraph 31: The fusion polypeptide of claim 30, wherein the binding domain that specifically binds a target polypeptide comprises an antigen-binding domain of an antibody. [000199] Paragraph 32: The fusion polypeptide of any of the preceding paragraphs, wherein the binding domain that specifically binds a target polypeptide comprises an scFv, a single domain antibody or a nanobody. [000200] Paragraph 33: The fusion polypeptide of any of the preceding paragraphs, wherein the target polypeptide is expressed in an erythroid cell. [000201] Paragraph 34: The fusion polypeptide of any of the preceding paragraphs, wherein the target polypeptide is selected from BCL11A, LRF and ZNF410. [000202] Paragraph 35: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide. [000203] Paragraph 36: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide. [000204] Paragraph 37: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12. [000205] Paragraph 38: The fusion polypeptide of claim 37, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2. [000206] Paragraph 39: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34. [000207] Paragraph 40: The fusion polypeptide of any of the preceding paragraphs, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids number 102 or 108. [000208] Paragraph 41: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65. [000209] Paragraph 42: The fusion polypeptide of any of the preceding paragraphs, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO: 41). [000210] Paragraph 43: The fusion polypeptide of any of the preceding paragraphs which further comprises a cell-penetrating peptide. [000211] Paragraph 44: The fusion polypeptide of any of the preceding paragraphs, further comprising a linker peptide between the TRIM10 or TRIM58 polypeptide and the binding domain that specifically binds a target polypeptide. [000212] Paragraph 45: A nucleic acid comprising sequence encoding a fusion polypeptide of any one of any of the preceding paragraphs. [000213] Paragraph 46: The nucleic acid of any of the preceding paragraphs, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells. [000214] Paragraph 47: A vector comprising a nucleic acid of any of the preceding paragraphs. [000215] Paragraph 48: The vector of any of the preceding paragraphs, which is a viral vector. [000216] Paragraph 49: The viral vector of claim 48, which is an AAV vector. [000217] Paragraph 50: A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to an erythroid cell. [000218] Paragraph 51: A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell expressing BCL11A. [000219] Paragraph 52: The method of any of the preceding paragraphs, wherein the targeted degradation of BCL11A is targeted to erythroid cells. [000220] Paragraph 53: A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of any of the preceding paragraphs, or a vector of any of the preceding paragraphs to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression. [000221] Paragraph 54: A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any of the preceding paragraphs, a nucleic acid of claim 45 or 46, or a vector of any of the preceding paragraphs to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder. EXAMPLES Example 1:  [000222] As described herein, the invention comes in part from investigation into erythroid specific E3 ubiquitin ligases. Analysis of RNA expression of E3 ligases during erythroid cell maturations revealed specific expression of TRIM10 and TRIM58 during erythroid cell maturation (FIG. 1B). TRIM10 and TRIM58 are members of the tripartite motif family that includes three zinc-binding domains, a RING, a B-box type 1 and a B-box type 2, and a coiled- coil region (FIG 1A). Furthermore, TRIM10 and TRIM58 were found to be enriched in erythroid cells as they are highly expressed in that cell type but expressed at much lower levels across other cell types. Known erythroid cell specific E3 ligase UBE2O was used as a positive control (FIG. 1C). [000223] In order to utilize erythroid specific E3 ubiquitin ligases for targeted protein degradation, proteolysis-targeting chimeras were created using TRIM10 or TRIM58. More specifically, TRIM10 or TRIM58 were fused to the recognition domain of cereblon (CRBN), then CRBN-/- HEK293T cells were co-transfected with the E3 ligase construct and BCL11A tagged with FKBP, which binds to dTAG47 PROTAC (a heterobifuncational molecule including a thalidomide derivative and an FKBP12F36V-selective ligand) (Fig. 2) Following transfection, dTAG47 was added to cells for 48 hrs and cells were collected via fluorescent activated cell sorting (FACS). To determine if TRIM-CRBN fusion can degrade BCL11A-FKBP, Western blot analysis with BCL11A antibody was used. Results indicate that fusions of TRIM10/58 with the recognition domain of CRBN are capable of degrading tagged BCL11A upon addition of dTAG47 PROTAC. Wild-type CRBN (WT-CRBN) cDNA transfected into CRBN-/- 293T cells was used as a positive control. An inactive form of dTAG47 (dTAG47i) is inactive for degradation and showed no effects, indicating that degradation is dependent on recruitment through the CRBN recognition domain (Fig.3). [000224] Next, the degradation of BCL11A protein by TRIM10/58 in HUDEP-2 (erythroid cells) were tested. HUDEP-2 cells were tagged with FKBP, which permits the use of dTAG47 PROTAC for recruitment to CRBN for degradation. As a control, HUDEP-2 cells lacking CRBN were generated by CRISPR/Cas9 editing. CRBN+ and CRBN- cells were infected with lentiviruses expressing CRBN cDNA or TRIM10 fused with the recognition domain of CRBN and cells were cultured with and without dTAG47 PROTAC. Degradation of BCL11A was analyzed by Western blot using BCL11A antibody. The fusion of TRIM10 with the recognition domain of CRBN led to a reduction in BCL11A. In CRBN- cells, degradation of BCL11A does not occur (Fig. 4). These results indicate that when recruited to BCL11A by the recognition domain of CRBN, TRIM10 is capable of degrading BCL11A. In other experiments, no degradation is seen with these cells in the presence of dTAG47. [000225] Finally, it was investigated whether BCL11A could be targeted for degradation by TRIM10/58 using BCL11A nanobodies. In these experiments, 293T cells were transfected with constructs expressing either TRIM10 or TRIM58 fused to a BCL11A-specific nanobody. Analysis by Western blot with BCL11A allowed for detection of endogenous BCL11A. As a control, mutant versions of TRIM10 or TRIM58 (C16A) in which a critical, conserved residue of TRIM proteins is mutated were used. Cells were transfected and assayed 2 days later. Results show marked reduction in BCL11A in cells treated with TRIM10-Nb19 (BCL11A) fusion. Thus, TRIM10 and to a lesser extent TRIM58 are able to direct degradation of wild-type, untagged BCL11A protein.    

Claims

CLAIMS 1. A fusion polypeptide comprising a binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and a binding domain that specifically binds an erythroid-enriched polypeptide of interest.
2. The fusion polypeptide of claim 1 which mediates degradation of the polypeptide of interest in erythroid cells.
3. The fusion polypeptide of claim 1 or claim 2, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase specifically binds TRIM10 or TRIM58.
4. The fusion polypeptide of any one of claims 1-3, wherein the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase comprises an antibody or antigen-binding fragment thereof.
5. The fusion polypeptide of claim 4, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
6. The fusion polypeptide of any one of claims 1-5, wherein the binding domain that specifically binds an erythroid-enriched polypeptide of interest comprises an antibody or antigen-binding fragment thereof.
7. The fusion polypeptide of claim 6, wherein the antibody or antigen-binding fragment thereof comprises an scFv, a single domain antibody or a nanobody.
8. The fusion polypeptide of any one of claims 1-7, wherein the polypeptide of interest is selected from BCL11A, LRF and ZNF410.
9. The fusion polypeptide of any one of claims 1-8, wherein the E3 ubiquitin ligase is TRIM10 or TRIM58.
10. The fusion polypeptide of any one of claims 1-9, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
11. The fusion polypeptide of any one of claims 1-9, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
12. The fusion polypeptide of any one of claims 1-10, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
13. The fusion polypeptide of claim 12, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2.
14. The fusion polypeptide of any one of claims 1-9 or 11, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
15. The fusion polypeptide of claim 14, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids at amino acid number 102 or 108.
16. The fusion polypeptide of any one of claims 1-10 or 12-13, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
17. The fusion polypeptide of any one of claims 1-9, 11 or 14-15, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO: 41).
18. The fusion polypeptide of any one of claims 1-17 which further comprises a cell- penetrating peptide.
19. The fusion polypeptide of any one of claims 1-18, further comprising a linker peptide between the binding domain that specifically binds an erythroid-enriched E3 ubiquitin ligase and the binding domain that specifically binds an erythroid-enriched polypeptide of interest.
20. A nucleic acid comprising sequence encoding a fusion polypeptide of any one of claims 1-19.
21. A nucleic acid of claim 20, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
22. A vector comprising a nucleic acid of claim 20 or 21.
23. The vector of claim 22, which is a viral vector.
24. The viral vector of claim 23, which is an AAV vector.
25. A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any one of claims 1-19, a nucleic acid of claim 20 or 21 or a vector of any one of claims 22-24 to an erythroid cell.
26. A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any one of claims 1-19, a nucleic acid of claim 20 or 21, or a vector of any one of claims 22-24 to a cell expressing BCL11A.
27. The method of claim 26, wherein the targeted degradation of BCL11A is targeted to erythroid cells.
28. A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any one of claims 1-19, a nucleic acid of claim 20 or 21, or a vector of any one of claims 22-24 to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
29. A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any one of claims 1-19, a nucleic acid of claim 22 or 21, or a vector of any one of claims 22-24 to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
30. A fusion polypeptide comprising a TRIM10 or TRIM58 polypeptide fused to a binding domain that specifically binds a target polypeptide.
31. The fusion polypeptide of claim 30, wherein the binding domain that specifically binds a target polypeptide comprises an antigen-binding domain of an antibody.
32. The fusion polypeptide of claim 30 or 31, wherein the binding domain that specifically binds a target polypeptide comprises an scFv, a single domain antibody or a nanobody.
33. The fusion polypeptide of any one of claims 30-32, wherein the target polypeptide is expressed in an erythroid cell.
34. The fusion polypeptide of any one of claims 30-33, wherein the target polypeptide is selected from BCL11A, LRF and ZNF410.
35. The fusion polypeptide of any one of claims 30-34, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc-finger 6 (ZNF6) of the BCL11A polypeptide.
36. The fusion polypeptide of any one of claims 30-34, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest binds BCL11A at an epitope comprised by zinc finger 23 (ZNF23) of the BCL11A polypeptide.
37. The fusion polypeptide of any one of claims 30-35, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds the polypeptide of interest has an amino acid sequence at least 90% identical to SEQ ID NO: 12.
38. The fusion polypeptide of claim 37, wherein amino acid sequence variation relative to SEQ ID NO: 12 occurs at one or more of amino acids according to Table 2.
39. The fusion polypeptide of any one of claims 30-34 or 36, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A has an amino acid sequence at least 90% identical to SEQ ID NO: 34.
40. The fusion polypeptide of claim 39, wherein amino acid sequence variation relative to SEQ ID NO: 34 occurs at one or more of amino acids number 102 or 108.
41. The fusion polypeptide of any one of claims 30-35 or 37-38, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has an amino acid sequence selected from SEQ ID NOs: 43-50, CDR2 has an amino acid sequence selected from SEQ ID NOs: 51-59; and CDR3 has an amino acid sequence selected from SEQ ID NOs 60-65.
42. The fusion polypeptide of any one of claims 30-34, 36 or 39-40, wherein the polypeptide of interest is BCL11A, and the binding domain that specifically binds BCL11A comprises a single-domain antibody in which: CDR1 has the amino acid sequence SIFVNNAM (SEQ ID NO: 37); CDR2 has the amino acid sequence ELVAAISASGGSTYY (SEQ ID NO: 38); and CDR3 has a sequence selected from ADQDVYPYEYW (SEQ ID NO: 39), ADQDGYPYEYW (SEQ ID NO: 40), and ADQDVYPYEYL (SEQ ID NO:
43. The fusion polypeptide of any one of claims 30-42 which further comprises a cell- penetrating peptide.
44. The fusion polypeptide of any one of claims 30-43, further comprising a linker peptide between the TRIM10 or TRIM58 polypeptide and the binding domain that specifically binds a target polypeptide.
45. A nucleic acid comprising sequence encoding a fusion polypeptide of any one of claims 30-44.
46. The nucleic acid of claim 45, wherein the sequence encoding the fusion polypeptide is operatively linked to regulatory sequences that permit expression in erythroid cells.
47. A vector comprising a nucleic acid of claim 45 or 46.
48. The vector of claim 47, which is a viral vector.
49. The viral vector of claim 48, which is an AAV vector.
50. A method of erythroid-specific, targeted degradation of a protein of interest, the method comprising introducing a fusion polypeptide of any one of claims 30-44, a nucleic acid of claim 45 or 46, or a vector of any one of claims 47-49 to an erythroid cell.
51. A method of targeted degradation of BCL11A, the method comprising introducing a fusion polypeptide of any one of claims 30-44, a nucleic acid of claim 45 or 46, or a vector of any one of claims 47-49 to a cell expressing BCL11A.
52. The method of claim 51, wherein the targeted degradation of BCL11A is targeted to erythroid cells.
53. A method of promoting fetal hemoglobin (HbF) expression in adult erythroid cells, the method comprising introducing a fusion polypeptide of any one of claims 30-44, a nucleic acid of claim 45 or 46, or a vector of any one of claims 47-49 to a cell, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression.
54. A method of treating a hemoglobinopathy disorder, the method comprising introducing a fusion polypeptide of any one of claims 30-44, a nucleic acid of claim 45 or 46, or a vector of any one of claims 47-49 to a subject in need thereof, wherein the protein of interest is BCL11A, and wherein the fusion polypeptide promotes the degradation of BCL11A in erythroid cells, thereby promoting HbF expression to treat the hemoglobinopathy disorder.
PCT/US2023/066674 2022-05-06 2023-05-05 Compositions and methods for targeted protein degradation Ceased WO2023215877A1 (en)

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