WO2024086518A2 - Enrichissement de types de cellules cliniquement pertinents à l'aide de récepteurs - Google Patents

Enrichissement de types de cellules cliniquement pertinents à l'aide de récepteurs Download PDF

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WO2024086518A2
WO2024086518A2 PCT/US2023/076969 US2023076969W WO2024086518A2 WO 2024086518 A2 WO2024086518 A2 WO 2024086518A2 US 2023076969 W US2023076969 W US 2023076969W WO 2024086518 A2 WO2024086518 A2 WO 2024086518A2
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cell
transmembrane receptor
chimeric transmembrane
domain
cells
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Michael Kyle CROMER
Matthew H. PORTEUS
Carsten CHARLESWORTH
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The Board Of Trustees Of The Leland Stanford Junior University
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  • hemoglobinopathies such as sickle cell anemia and beta-thalassemia using allogeneic hematopoietic stem cell transplantation (allo-HSCT)
  • allo-HSCT allogeneic hematopoietic stem cell transplantation
  • a number of gene therapies and genome editing strategies have been developed to address this need, by correcting a patient’s own HSCs ex vivo and then transplanting the cells back into the patient via autologous-HSCT (auto-HSCT).
  • auto-HSCT autologous-HSCT
  • Using a patient’s own cells thereby minimizes the risk of immune rejection or graft-versus-host disease (GvHD).
  • Introducti on of truncated EPOR to HSCs can impart a dramatic erythroid bias to edited HSCs.
  • tEPOR truncated EPOR
  • These EPOR truncations were also effectively paired with genome editing-based beta-thalassemia correction strategy in order to enrich for RBCs harboring a corrective edit.
  • this system relies on activation of the truncated receptor by endogenous EPO cytokine levels, with little ability to titrate the effect post-transplantation.
  • the present disclosure provides a chimeric transmembrane receptor polypeptide comprising: an extramembrane dimerizer domain, wherein the extramembrane dimerizer domain induces dimerization of the chimeric transmembrane receptor polypeptide upon recognition of a dimerization signal; a transmembrane domain; and an intramembrane domain, wherein the intramembrane domain is configured to induce activation of one or more intramembrane signal pathways upon dimerization of the chimeric transmembrane receptor polypeptide in a modified primary human cell comprising the chimeric transmembrane receptor polypeptide, and wherein the one or more intramem brane signaling pathways promote survival, proliferation, and/or differentiation of the modified primary human cell.
  • the dimerization signal is a pharmaceutically acceptable small molecule dimerization signal.
  • the extramembrane dimerizer domain comprises an FKBP domain, an mFRB domain, an HSV-TK dimerization domain, a rapamycin -inducible dimerization domain, a rapalogue -inducible dimerization domain, or a combination thereof.
  • the extramembrane dimerizer domain comprises an FKBP domain and the small molecule dimerization signal comprises AP20187 (BB dimerizer).
  • the mtramembrane domain comprises an EPOR intracellular domain, an SCF or CD 117 intracellular domain, a TPOR or MPL intracellular domain, an EGFR intracellular domain, an RET intracellular domain, a CSF1R intracellular domain, an IGF1R intracellular domain, or a combination thereof.
  • the intramembrane domain comprises an EPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary human cell upon recognition of the dimerization signal.
  • the intramembrane domain comprises a TPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary' human cell upon recognition of the dimerization signal.
  • the intramembrane domain comprises an SCF intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary human cell upon recognition of the dimerization signal.
  • the extramembrane dimerizer domain is immediately adjacent to the transmembrane domain. In some embodiments, the extracellular domain does not bind erythropoietin (EPO).
  • EPO erythropoietin
  • the chimeric transmembrane receptor polypeptide further comprises a signal peptide.
  • tire signal peptide promotes membrane localization of the chimeric transmembrane receptor polypeptide.
  • the signal peptide comprises an IL6 signal peptide, an EPOR signal peptide, a lysozyme C signal peptide, an angiotensinogen signal peptide, an RNASE1 signal peptide, an RNASE3 signal peptide, or a modified human albumin signal peptide.
  • the chimeric transmembrane receptor polypeptide further comprises a linker peptide.
  • the linker peptide comprises the amino acid sequence GGGGS.
  • the chimeric transmembrane receptor polypeptide comprises an amino acid sequence having at least 80% identity to at least one of SEQ ID NOS: 1 to 3 or 7 to 11.
  • the present disclosure provides a recombinant nucleic acid encoding any of the chimeric transmembrane receptor polypeptides described herein.
  • the present disclosure provides a DNA construct comprising a promoter operably linked to the recombinant nucleic acid.
  • the promoter is an endogenous EPOR promoter, an endogenous HBA1 promoter, an endogenous 7POR promoter, a constitutive SFFV promoter, a constitutive PGK promoter, or a constitutive UbC promoter.
  • a vector comprising any 7 of the recombinant nucleic acids described herein or any of the DNA constructs described herein.
  • the present disclosure provides a host cell comprising any of the recombinant nucleic acids described herein, any of the DNA constructs described herein, or any of the vectors described herein.
  • the recombinant nucleic acid, DNA construct, or vector is integrated into the EPOR locus, the CCR5 locus, or the HBA1 locus.
  • the host cell is a eukaryotic cell. In some embodiments, the host cell is a primary human cell.
  • the host ceil is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC).
  • the host cell was derived from a patient who is a carrier of an allele that causes a genetic disorder.
  • the genetic disorder is beta- thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • the genetic disorder is a hemoglobinopathy.
  • the hemoglobinopathy is beta- thalassemia or sickle cell disease (SCD).
  • SCD sickle cell disease
  • the genome of the host cell is edited to contain a corrected version of the allele that causes the genetic disorder.
  • the present disclosure provides a method of inducing erythroid differentiation of a hematopoietic stem and progenitor ceil (HSPC), the method comprising contacting an HSPC that comprises a recombinant nucleic acid encoding a chimeric transmembrane receptor polypeptide with the dimerization signal.
  • HSPC hematopoietic stem and progenitor ceil
  • the present disclosure provides a method of tuning red blood cell levels in a patient, the method comprising: (i) editing an HSPC so that it can express at least one of the chimeric transmembrane receptor polypeptides described herein; (ii) transferring the resulting HSPC to the patient; (iii) administering a first quantity of the dimerization signal to the patient; (iv) monitoring red blood cell levels in the patient; and (v) administering a second quantity of the dimerization signal to the patient that is the same, less, or more than the first quantity of dimerization signal.
  • the present disclosure provides a method of increasing the proportion of red blood cells with an altered version of an allele associated with a genetic disorder, the method comprising: (i) creating an edited HSPC by introducing an altered version of the allele associated with a genetic disorder and a polynucleotide that encodes at least one of the chimeric transmembrane receptor polypeptides described herein into an HSPC; (ii) transferring the edited HSPC to the patient; and (iii) administering the dimerization signal to the patient.
  • the HSPC is derived from the patient’s own cells.
  • the HSPC is derived from an allogeneic donor’s cells.
  • the genetic disorder is beta-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • SCD sickle cell disease
  • SCID severe combined immunodeficiency
  • mucopolysaccharidosis type 1 Cystic Fibrosis
  • Gaucher disease Krabbe disease
  • X-CGD X-linked chronic granulomatous disease
  • the present disclosure provides a method of genetically modifying a primary human cell, the method comprising introducing into the cell a chimeric transmembrane receptor polypeptide described herein.
  • the method comprises: (i) introducing into the cell a site-directed nuclease (SDN) targeted to a cleavage site at a genetic locus of interest; and (li) introducing a homologous repair template into the cell, wherein the homologous repair template comprises a nucleotide sequence that is homologous to the locus of interest, wherein the site-directed nuclease cleaves the locus at the cleavage site, and the homologous repair template is integrated at the site of the cleaved locus by homology directed repair (HDR).
  • HDR homology directed repair
  • the SDN is an RNA-guided nuclease and the method further comprises introducing into the cell a single guide RNA (sgRNA) targeting the cleavage site, wherein the sgRNA directs the RNA-guided nuclease to the cleavage site.
  • sgRNA single guide RNA
  • the sgRNA comprises 2’-O-methyl-3'- phosphorothioate (MS) modifications at one or more nucleotides.
  • the MS modifications are present at the terminal nucleotides of the 5' and 3 ! ends.
  • the RNA-guided nuclease is Cas9.
  • the sgRNA and RNA- guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • the RNP is introduced into the cell by electroporation.
  • the homologous repair template is introduced into the cell using an adeno-associated virus serotype 6 (AAV6) vector.
  • AAV6 adeno-associated virus serotype 6
  • the primary human cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC).
  • PSC pluripotent stem cell
  • the locus of interest is a gene selected from the group consisting of Erythropoietin Receptor (EPOR), Hemoglobin Subunit Beta (HBB), C- C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha I Stimulator Of Interferon Response cGAMP Interactor
  • EPOR Erythropoietin Receptor
  • HBB Hemoglobin Subunit Beta
  • CCR5 C- C Motif Chemokine Receptor 5
  • IL2RG Interleukin 2 Receptor Subunit Gamma
  • cGAMP Interactor Hemoglobin Subunit Alpha I Stimulator Of Interferon Response
  • the present disclosure provides a method of treating a genetic disorder in a human subject in need thereof, the method comprising: (i) providing an isolated primary’ cell from the subject; (ii) genetically’ modifying the primary’ cell using the method described herein, wherein the integration of the homologous donor template at the locus of interest in the cell alters an alleleat the locus that is associated with the genetic disorder or leads to the expression of a therapeutic protein in the cell that is absent or deficient in the subject; and (iii) reintroducing the genetically’ modified cell into the subject.
  • the genetic disorder is beta-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • SCD sickle cell disease
  • SCID severe combined immunodeficiency
  • mucopolysaccharidosis type 1 Cystic Fibrosis
  • Gaucher disease Krabbe disease
  • X-CGD X-linked chronic granulomatous disease
  • the present disclosure provides a method of generating a population of red blood ceil in vitro, the method comprising: (i) editing one or more HSPCs to express at least one of the chimeric transmembrane receptor polypeptides as disclosed herein; and (ii) contacting the one or more HSPCs with a dimerization signal.
  • the dimerization signal is a small molecule dimerization signal.
  • the small molecule dimerization signal is AP20187 (BB dimerizer).
  • step (ii) further comprises contacting the one or more HSPCs with erythropoietin (EPO).
  • the method is conducted in a bioreactor.
  • FIG. 1 Diagram of novel AAV integration vectors that place coding regions from FKBP (taken from Martin et al. Nature Communications, 202.0) and EPOR in various orientations. Each of the initial constructs targeted the CCR5 safe harbor site, integration at which was mediated by cutting with a Cas9 complexed with established CCR5 gRNA and
  • the integration cassette was comprised of a strong, constitutive SFFV promoter, followed by the FKBP-EPOR coding sequence, then followed by a 2A-YFP-bGH reporter coding sequence.
  • FIG. 2 Schematic showing insertion of chimeric receptors into HSPCs and screening for BB dimerizer-dependent activation ofRBC differentiation in the absence of EPO.
  • Primary’ human CD34+-enriched HSPCs were plated and expanded in HSC expansion media, and then edited using a CRISPR-Cas/AAV platform to introduce FKBP-EPOR. constructs into the HSPCs at the target genomic locus (e.g., the CCR5 safe harbor site). Erythroid differentiation and RBC production were monitored for 16 days in cells from the same donor that were edited as a single treatment in the presence or absence of BB dimerizer (AP20187). Although not indicated in the diagram, day 16 samples exposed to BB dimerizer had many more cells than day 16 samples without exposure to BB dimerizer because erythroid differentiation is associated with dramatic expansion of cells.
  • FIG. 3 Flow cytometry schematics of WT HSPCs transformed with a mock cassete.
  • Cells were stained at day 14 of RBC differentiation with the standard erythroid differentiation protocol. After using antibodies for CD34, CD45, CD71, and GPA, cells were analyzed by flow cytometry with (right) and without (left) incubation in the presence of BB dimerizer.
  • FIG. 4 Flow cytometry schematics of WT HSPCs transformed with FKBP-EPOR chimeras 1.4 and 1 .5. Cells were stained at day 14 of RBC differentiation with the standard erythroid differentiation protocol. After using antibodies for CD34, CD45, CD71, and GPA, cells were analyzed by flow cytometry with (FIG. 4A) and without (FIG. 4B) incubation in the presence of BB dimerizer.
  • FIG. 5 Graph showing fold increase in GFP expression in the presence of BB dimerizer relative to the absence of BB dimerizer in live cells containing FKBP-EPOR chimeras 1.4 and 1.5.
  • FIG. 6 Flow cytometry schematics of second generation of FKBP-EPOR chimeras.
  • Cells were maintained in RBC medium with or without BB, as indicated.
  • FIG. 6A show's the effects of adding a native EPOR signal peptide (SP) to the N-terminus of the receptor encoded by the 1.5 integration cassette (MKC-121) and integrating at the CCR5 locus.
  • FIG. 6B show's the effects of adding an IL6 signal peptide to the N-terminus of the receptor encoded by the 1.5 integration cassette (MKC120) and integrating at the CCR5 locus.
  • SP native EPOR signal peptide
  • MKC-121 1.5 integration cassette
  • FIG. 6B show's the effects of adding an IL6 signal peptide to the N-terminus of the receptor encoded by the 1.5 integration cassette (MKC120) and integrating at the CCR5 locus.
  • MKC120 1.5 integration cassette
  • 6C show's the effects of a cassette with the same orientation of FKBP and EPOR as 1 ,5, but using truncated EPOR (tEPOR), integrated at the strong RBC-specific safe harbor locus zlHBAl (MKC125) (Cromer, et al. Nature Medicine 202.1).
  • tEPOR truncated EPOR
  • FIG. 7 Direct comparison of the flow cytometry schematics of first generation (with cassette 1.5) and second generation (with cassette 2.1) FKBP-EPOR chimeras. Ceils were maintained in RBC medium with or without BB dimerizer. To determine the relative potency of adding the IL6 signal peptide, compared to the effective 1.5 chimera from the first- generation vector designs, HSPCs were edited with each vector and analyzed by flow cytometry following RBC differentiation without EPO and with (FIG. 7A) or without BB dimerizer (FIG. 7B).
  • FIG. ⁇ Flow' cytometry schematics of cells that were edited to contain the IL6-signal peptide containing version of vector 1 .5 (FIG. 8A) or the native EPOR signal peptide- containing version of vector 1.5 (FIG. 8B) and kept in HSPC ex vivo culture media without EPO (Dever, et al. Nature 2016) with or without BB dimerizer. Cells were maintained at 100K cells/mL for 7 days post-editing in this media and then stained for tire RBC antibody panel.
  • FIGS. 9 and 10 show flow cytometry schematics of cells which contain an IL6- containing polypeptide with the FKBP extracellular domain attached to a truncated EPOR (tEPOR) integrated at the HBA 1 locus (MKC 135). Cells were incubated without EPO and with (FIGS. 9 A and 10A) or without (FIGS. 9B and 10B) BB dimerizer after RBC differentiation without Epo.
  • tEPOR truncated EPOR
  • FIG. 11 and Table 1 further describe constructs and cell lines used in the experiments described herein.
  • FIG. 12 shows a summary of data from flow cytometry' experiments with some of the edited HSPCs after differentiation in RBC media without EPO and +/-BB dimerizer.
  • FIG. 13 show's hemoglobin tetramer HPLC data indicating that cells edited with iEPOR version 2.1, 2.2, or 2.3 can produce normal hemoglobin with addition of BB alone equivalent to unedited cells + EPO. Cells edited with iEPOR 2.2 become ‘’leaky” and produce high quantities of hemoglobin even in the absence of EPO and BB.
  • FIG. 14 illustrates a bicistronic expression vector (MCK144) that combines a beta- thalassemia correction scheme with inducible EPOR expression. This cassette may both correct the molecular pathology of beta-thalassemia and promote RBC differentiation of edited cells.
  • FIG. 14A shows a diagram of vectors used to edit WT primary human HSPCs.
  • FIG. 14B shows flow cytometry schematics of the differentiated ceils incubated with (right) or without (middle) BB dimerizer. A mock sample without a chimeric receptor transgene is shown for comparison on the left.
  • FIG. 15 depicts the effects of pairing a second-generation RBC drive with a beta- thalassemia correction strategy.
  • FIG. 15A depicts the second-generation vector used in this study.
  • FIG. 15B show's % RBC differentiation of edited cells at day 14 as determined by flow cytometry-. The middle and right panels depict % targeted alleles (equivalent to the percentage of edited cells that express RBC markers) as determined by ddPCR in two separate WT HSPC donors over the course of RBC differentiation without EPO and +/-BB dimerizer.
  • the ddPCR assay was set up to quantify the frequency of unedited ceils at the integration site compared to a genomic reference probe near the HBA1 locus, which was validated on unedited ceils. The inverse of this was assumed to be the frequency of edited cells.
  • FIG. 15C shows hemoglobin tetramer HPLC data indicating BB alone can generate functional RBCs with hemoglobin production equivalent to unedited cells + EPO.
  • FIG. 16 describes the effects of editing WT human HSPCs that containing inducible chimeric truncated EPOR (IL6-FKBP-div.tEPOR) (MKC145) whose coding region was integrated at the endogenous EPOR locus and whose expression is under the control of the EPOR promoter.
  • FIG. 16A depicts the overall scheme that could be used for integrating iEPOR, itEPOR, or div.itEPOR into the endogenous EPOR locus.
  • Div.itEPOR (encoded by SEQ ID NO: 2.2.) is an inducible chimeric receptor with a truncated EPOR that has a codon- diverged sequence relative to native tEPOR due to the incorporation of silent mutations. The presence of silent mutations limits homology to the native sequence so as to prevent premature recombination that would result in incomplete insertion of the integration cassette. As determined by ddPCR and flow cytometry, we observe a BB dimerizer-dependent increase in the number of GFP+ cells in cells with this chimeric transgene integration at the EPOR locus, and many of these are CD34-CD45-CD71-f- RBCs (compare FIGS. I6B and 16C).
  • FIG. 17 shows flow cytometry schematics of WT human HSPCs that were edited with inducible truncated EPOR (IL6-FKBP-tEPOR) whose coding region was integrated at the OCRS locus and whose expression was under the control of the PGK promoter (MKC158).
  • IL6-FKBP-tEPOR inducible truncated EPOR
  • MKC158 PGK promoter
  • FIG. 18 show's HPLC profiles of fetal and adult hemoglobin (HbF and HbA, respectively) within edited and unedited HSPCs either with or without induction.
  • FIG. ISA shows HPLC profile of HbF and HbA from WT unedited umbilical cord blood-derived HSPCs induced with Epo.
  • FIGS. 18B and 18C show' hemoglobin HPLC profiles of HSPCs grown with (black) or without (gray) BB dimerizer after editing with the indicated inducible chimeric receptor construct or with a mock cassette. Since some samples had overlapping profiles with and without BB dimerizer (e.g., MKC120 and Mock sample), insets show the HPLC profile of the corresponding sample without BB dimerizer.
  • BB dimerizer e.g., MKC120 and Mock sample
  • FIG. 19 shows bulk RNA-seq results of -18,000 genes with assigned expression values (as normalized read counts) to compare transcriptional response to native EPOR + EPO vs. iEPOR + BB after genome editing of healthy donor HSPCs and in vitro RBC differentiation.
  • Native EPOR and iEPOR expression levels are annotated for Mock “unedited’’ cells at dO and dI4 of RBC diff (Mock dO and Mock d!4 t EPO) followed by cells edited with iEPOR version 2.3 expressed by strong-RBC specific promoter HBA1 (HBA1 (iEPOR) + BB), endogenous EPOR promoter (EPOR(iEPOR) + BB), and constitutive-expressing hPGK promoter (PGK(iEPOR) + BB).
  • HBA1 HBA1 (iEPOR) + BB
  • EPOR(iEPOR) + BB endogenous EPOR promoter
  • PGK(iEPOR) + BB constitutive-expressing hPGK promoter
  • FIG. 20 describes IL6-FKBP chimeras that could place SCF, TPOR, and EGFR signaling under control of BB dimerizer.
  • FIG. 20 depicts three vector cassettes that were cloned and packaged into AAV DNA repair vectors to be used to test other inducible signaling receptor cassettes.
  • the top cassette encodes a chimeric FKBP-SCF-IC receptor.
  • the middle cassette encodes a chimeric FKBP-TPOR-IC receptor.
  • Tire bottom cassette encodes a chimeric FKBP-EGFR-IC receptor.
  • FIG. 21 show's flow cytometry' schematics of WT primary human HSPCs that were edited with chimeric TPOR and maintained m HSPC media without EPO and with (FIG. 21B) or without (FIG. 21 C) BB dimeri zer for 7 days post-editing relative to a mock control (FIG. 21A).
  • Cells were stained with an HSC-specific antibody panel developed by the Ravindra
  • FIG. 22 summarizes flow' cytometry schematics for WT primary human HSPCs that w'ere edited to express an inducible chimeric TPOR (IL6-FKBP-TPOR or iTPOR) receptor or an inducible chimeric SCF (IL6-FKBP-SCF or iSCF) receptor and maintained in HSPC media with and without BB dimerizer for 7 days post-editing.
  • FIG. 22A show's a BB-dependent increase in GFP expression in cells edited with either an inducible chimeric TPOR receptor or an inducible chimeric SCF receptor relative to cells edited with a mock control.
  • FIGS. 22B and 22C show the targeted allele frequency for cells edited with either the BB-inducible chimeric TPOR receptor or the BB-inducible chimeric SCF receptor, respectively.
  • FIG. 23 shows editing efficiency of the PGK-driven iEPORv2.3 in iPSCs with or without AZD, a small molecule used to increase genome editing frequency, indicating high editing rates with the PGK-driven iEPORv2.3 integration cassette in human iPSCs.
  • FIG. 24A depicts an iPSC>HSC>RBC differentiation workflow using STEMdiff Hematopoietic Kit.
  • FIG. 24B shows no major differences in cell proliferation between unedited ceils cultured with BB E BB; and iEPOR-edited cells cultured without BB (-BB) at the first stage of differentiation (iPSOHI’C).
  • FIG. 24C shows a dramatic increase of RBC differentiation in iEPOR-edited cells cultured +BB during stage 1 iPSC>HPC, indicating that some frequency of early differentiation driven by BB-mduced iEPOR signaling is occurring at this stage.
  • FIG. 25A depicts an iPSC>HSC>RBC differentiation workflow using in-house differentiation protocol for CD34s.
  • FIG. 25B shows iEPOR-edited cells cultured with BB but without EPO (-EPO/+BB) lead to an intermediate cell expansion in comparison to cells cultured with EPO over the second stage of differentiation (HPC>RBC), indicating that BB can be used to replace EPO in the media and lead to substantial RBC production.
  • FIG. 25B also show's the highest cell production in iEPOR-edited cells cultured with EPO and BB (+EPO/+BB), indicating that BB stimulation of iEPOR leads to an elevated boost even on top of natural EPO stimulation .
  • FIG. 26 shows RBC proliferation and differentiation differences between cells with or without EPO, indicating that in absence of EPO, only iEPOR-edited cells with BB (+BB) are able to effectively differentiate and proliferate.
  • the present disclosure provides methods and compositions for tunable differentiation of a hematopoietic stem and progenitor ceil (HSPC) in response to a small molecule.
  • HSPC hematopoietic stem and progenitor ceil
  • this is an FDA-approved orally bioavailable small molecule, e.g., AP20187, also referred to as “B/B homodimerizer,” or “BB dimerizer.”
  • the methods and compositions use a CRISPR-Cas system to introduce into cells a chimeric transmembrane receptor polypeptide that dimerizes in response to an extracellular dimerization signal. Dimerization triggers a signaling cascade, the consequence of which is increased survival, increased proliferation, and/or increased erythroid differentiation.
  • the HSPC is also genetically modified using a CRISPR-Cas system to correct an allele that causes a genetic disorder.
  • the methods and compositions described herein may provide an improved means for treating a genetic disorder.
  • Current treatments for genetic disorders using edited HSPCs require a devastating myeloablation regimen to clear out the bone marrow in order for edited HSPCs to sufficiently engraft.
  • the methods and compositions described herein allow for selective differentiation of edited HS PCs such that lower levels of engraftment are sufficient to treat a disorder. Therefore, the methods and compositions described herein overcome at least one significant recognized obstacle to treating genetic disorders.
  • nucleic acids sizes are given in either kilobases (kb), base pairs (bp), or nucleotides (nt). Sizes of single-stranded DNA and/or RNA can be given in nucleotides. These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given m kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
  • Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al.. Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion -exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983).
  • HPLC high performance liquid chromatography
  • any reference to “about X” specifically indicates at least the values X, 0.8X, 0.8 IX, 0.82X, 0.83X, 0.84X, 0.85X, 0.86X, 0.87X, 0.88X, 0.89X, 0.9X, 0.9 LX, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, 1.1X, 1.1 IX, 1.12X, 1.13X, 1.14X, 1.15X, 1.16X, 1.17X, 1.18X, 1.19X, and 1.2X.
  • “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X ”
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SXPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al.. Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J Biol. Chem.
  • gene means the segment of DNA involved in producing a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • the promoter can be a heterologous promoter.
  • an “expression cassete” or “cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression cassette may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.
  • the promoter can be a heterologous promoter.
  • a “heterologous promoter” refers to a promoter that would not be so operably linked to the same polynucleotide as found in a product of nature (e.g., in a wild-type organism).
  • a first polynucleotide or polypeptide is “heterologous” to an organism or a second polynucleotide or polypeptide sequence if the first polynucleotide or polypeptide originates from a foreign species compared to the organism or second polynucleotide or polypeptide, or, if from the same species, is modified from its original form.
  • a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence).
  • Polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full -length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • the terms “expression” and “expressed” refer to the production of a transcriptional and/or translational product, e.g., of a tEPOR encoding mRNA or an encoded tEPOR protein. In some embodiments, the term refers to the production of a transcriptional and/or translational product encoded by a gene or a portion thereof.
  • the level of expression of a DN A molecule in a cell may be assessed on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell.
  • “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or -where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. In some cases, conservatively modified variants of a protein can have an increased stability, assembly, or activity as described herein.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single -letter codes.
  • amino acid residues are numbered according to their relative positions from the left most residue, which is numbered 1 , in an unmodified wild-type polypeptide sequence.
  • the terms “identical” or percent “identity,” in the context of describing two or more polynucleotide or amino acid sequences, refer to two or more sequences or specified subsequences that are the same. Two sequences that are “substantially identical” have at least 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or 100%) identity, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection where a specific region is not designated.
  • polynucleotide sequences this definition also refers to the complement of a test sequence.
  • amino acid sequences in some cases, the identity exists over a region that is at least about 50 amino acids or nucleotides in length, or more preferably over a region that is 75-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative pantmeters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST 2.0 algorithm and the default parameters discussed below are used.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence maybe compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • HSPs high scoring sequence pairs
  • These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity’ X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Set. USA 89: 10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Set. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • CRISPR-Cas refers to a class of bacterial systems for defense against foreign nucleic acids, CRISPR-Cas systems are found in a wide range of bacterial and archaeal organisms. CRISPR-Cas systems fall into two classes with six types, I, II, III, IV, V, and VI as well as many sub-types, with Class 1 including types I and III CRISPR systems, and Class 2 including types II, IV, V and VI; Class 1 subtypes include subtypes I-A to 1-F, for example.
  • Endogenous CRISPR-Cas systems include a CRISPR locus containing repeat clusters separated by non-repeating spacer sequences that correspond to sequences from viruses and other mobile genetic elements, and Cas proteins that carry out multiple functions including spacer acquisition, RNA processing from the CRISPR locus, target identification, and cleavage.
  • Cas3 providing the endonuclease activity
  • Cas9 all carried out by a single Cas, Cas9.
  • a “homologous repair template” or “donor template” refers to a polynucleotide sequence that can be used to repair a double stranded break (DSB) in the DNA, e.g., a CRISPR/Cas9-mediated break at a CCR5 locus as induced using the herein-described methods and compositions.
  • Tire homologous repair template comprises homology to the genomic sequence surrounding the DSB, i.e., comprising CCR5 homology aims.
  • two distinct homologous regions are present on the template, with each region comprising at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 400-1000, 500-900, or more nucleotides of homology with the corresponding genomic sequence.
  • the templates comprise two homology aims comprising, e.g., about 900 nucleotides of homology, with one arm extending upstream starting at. the translation start site, and the other arm extending downstream from the sgRNA target site.
  • the repair template can be present in any form, e.g., on a plasmid that is introduced into the cell, as a free floating doubled stranded DNA template (e.g..
  • Tire templates of the present disclosure erm also comprise a transgene, e.g., a chimeric transmembrane receptor transgene and optionally a therapeutic transgene as described herein.
  • homologous recombination or “HR” refers to insertion of a nucleotide sequence during repair of double-strand breaks in DNA via homology-directed repair mechanisms. This process uses a “donor template” or “homologous repair template” with homology to nucleotide sequence in the region of the break as a template for repairing a double-strand break. The presence of a double-stranded break facilitates integration of the donor sequence.
  • Tire donor sequence may be physically integrated or used as a template for repair of the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence, dins process is used by a number of different gene editing platforms that create the double-strand break, such as meganucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the C-RISPR-Cas9 gene editing systems.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • HR involves double-stranded breaks induced by CRISPR-Cas9.
  • EPOR erythropoietin receptor
  • EPO erythropoietin
  • EPOR refers to a polynucleotide (e.g., gene, locus, transgene, coding sequence, cDNA, expression cassette) encoding EPOR.
  • JAK2 tyrosine kinase Upon binding of EPO, EPOR activates JAK2 tyrosine kinase, which in turn activates different intracellular pathways such as Ras/MAP kinase, PI3 kinase, and STAT transcription factors.
  • EPOR. is a member of the cytokine receptor family, and the EPOR gene is located on human chromosome 19p ( 19p 13.2 ) .
  • Truncated EPOR refers to forms of the EPO receptor, or to polynucleotides encoding the receptor forms, that lack a portion or all of the receptor’s cytoplasmic domain.
  • a tEPOR lacks the 70 C- terminal ammo acids of full-length EPOR.
  • a tEPOR lacks all 236 amino acids of the cytoplasmic domain.
  • a tEPOR lacks, e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 10-236, 10-50, 50-60, 60-70, 65-75, 70-80, 80-90, 90-100, 100-150, 150-200, or 200-236 amino acids.
  • a tEPOR lacks a binding site and/or does not interact with the tyrosine phosphatase SFIP-1 (or SHPTP-1 ), which normally plays a role in inhibiting EPOR signaling.
  • a coding sequence (e.g., gene or transgene) encoding a tEPOR comprises a nonsense mutation in exon 7 or exon 8, and/or encodes any of the herein- described forms of truncated EPOR.
  • Nonsense mutations causing the expression of truncated EPOR act as dominant mutations that render cells hypersensitive to EPO, leading to an ability to undergo effective proliferation and differentiation in the presence of reduced amounts of EPO, and to show enhanced levels of proliferation and differentiation in the presence of normal EPO levels.
  • An exemplary tEPOR cDNA is shown herein as SEQ ID NO: 21 .
  • lEPOR can refer to any nucleotide sequence comprising about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more homology to SEQ ID NO: 21 or a subsequence thereof.
  • iEPOR and “itEPOR” proteins comprise a signal peptide (e.g., IL6) and FKBP and EPOR polypeptides, or fragments thereof, in the same orientation as FKBP and EPOR polypeptides in 1.5.
  • iTPOR and iSCF are used to describe proteins comprising a signal peptide and the orientation of FKBP and EPOR polypeptides as in 1.5, with polypeptides from TPOR or SCF instead of EPOR.
  • hematopoietic stem and progenitor cell refers to a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), or a population of hematopoietic stem cells and hematopoietic progenitor cells.
  • the present disclosure provides a chimeric transmembrane receptor polypeptide comprising: an extramembrane dimerizer domain, wherein the extramembrane dimerizer domain induces dimerization of the chimeric transmembrane receptor polypeptide upon recognition of a dimerization signal; a transmembrane domain; and an intramembrane domain, wherein the intramembrane domain is configured to induce activation of one or more intramembrane signal pathways upon dimerization of the chimeric transmembrane receptor polypeptide in a modified primary human cell comprising the chimeric transmembrane receptor polypeptide, and wherein the one or more intramembrane signaling pathways promote survival, proliferation, and/or differentiation of the modified primary human cell.
  • a chimeric polypeptide may, for example, comprise fragments of distinct proteins and contain functional properties derived from each of the distinct proteins.
  • the dimerization signal is a pharmaceutically acceptable small molecule dimerization signal.
  • the dimerization signal is an orally bioavailable small molecule.
  • the small molecule dimerization signal comprises AP20187, AP21967, ganciclovir, rapamycin or an analog thereof.
  • the small molecule dimerization signal comprises AP20187.
  • AP20187 is a cell permeable small molecule approved by the FDA that is orally bioavailable and can be used to induce dimerization of FK506-bindmg protein (FKBP).
  • the extramem brane dimerizer domain comprises an FKBP domain, or a variant or a fragment thereof.
  • the chimeric transmembrane receptor polypeptide extramembrane domain can include or consist of a heterodimer FKBP/FRB domain.
  • the terms “variant,” and “fragment,” refer to a polypeptide related to a wild-type polypeptide, for example, either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function.
  • Variants and fragments of a polypeptide can include one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild-type polypeptide.
  • a variant or fragment can include at least 50%, e.g., 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence, structure, activity, and/or function of the corresponding wild-type polypeptide.
  • the extramembrane dimerizer domain comprises an mFRB domain, an HSV-TK dimerization domain, a rapamycin -inducible dimerization domain, a rapalogue-inducible dimerization domain, or a combination thereof.
  • the mFRB domain can heterodimerize with the FKBP domain using AP21967;
  • the HSV-TK domain can homodimerize with ganciclovir;
  • the FKBP domain can homodimerize with AP20187, AP21967, rapamycin or its various analogs (i.e., rapaiogs).
  • the intramembrane domain comprises an EPOR intracellular domain, a stem cell factor (SCF) intracellular domain or a myeloprolifereatie leukemia virus oncogene (MPL) intracellular domain, a thrombopoietin (TPOR) intracellular domain or a CD117 intracellular domain, an epidermal growth factor (EGFR) intracellular domain, an RET intracellular domain, a macrophage colony-stimulating factor 1 receptor (CSF1R) intracellular domain, an insulin-like growth factor (IGF1R) intracellular domain, or a combination thereof
  • the intramembrane domain comprises an EPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary human cell upon recognition of the dimerization signal.
  • the intramembrane domain comprises a TPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary human cell upon recognition of the dimerization signal.
  • the intramembrane domain comprises an SC'F intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary' human cell upon recognition of the dimerization signal.
  • the extramembrane dimerizer domain is immediately adjacent to the transmembrane domain. As shown in FIG. 5, only a subset of the chimeric FKBP-EPOR polypeptides (1.4 and 1.5) caused BB-induced differentiation of HSPCs.
  • both of these chimeric polypeptides contained an extracellular dimerizer domain immediately adjacent to the transmembrane domain ,
  • the immediately adjacent domains are separated by only a linker peptide sequence, but in other contexts immediately adjacent domains may contain multiple linker peptide sequences, e.g., two linker peptide sequences, three linker peptide sequences, four linker peptide sequences, five linker peptide sequences, or six linker peptide sequences.
  • Construct 1 ,5 was subsequently used to create second generation constructs since it is a smaller cassette, which can be more easily included in an AAV repair template, and it fully removes the extracellular domain of EPOR (as indicated in FIG. 5), ensuring that it is no longer responsive to EPO protein.
  • the chimeric transmembrane receptor polypeptide further comprises a signal peptide.
  • the signal peptide promotes cell surface membrane localization of the chimeric transmembrane receptor polypeptide.
  • the signal peptide comprises an IL6 signal peptide (such as SEQ ID NO: 4), an EPOR signal peptide (such as SEQ ID NO: 5), a lysozyme C signal peptide, an angiotensinogen signal peptide, an RNASE 1 signal peptide, an RNASE3 signal peptide, or a modified human albumin signal peptide.
  • Both the IL-6 and EPOR signal peptides (indicated as SP) enhanced BB-dependent differentiation as shown in FIGS. 6A and 6B.
  • these constructs were able to induce BB-driven differentiation in Porteus HSPC ex vivo culture media (media known to preserve HSPC stem-ness; Dever et al.. Nature 2016), as shown in FIGS. 8A and 8B. This suggests that this is a strong, pro-RBC differentiation strategy that is activated by BB dimerizer.
  • BB-dependent differentiation was also observed after editing and differentiation of HSPCs with constructs comprising iSCF (IL-6-FKBP-SCF) and iTPOR (IL-6-FKBP-TPOR) cassettes, as shown in FIGS. 20, 21A-21C and 22A.
  • iSCF IL-6-FKBP-SCF
  • iTPOR IL-6-FKBP-TPOR
  • the chimeric transmembrane receptor polypeptide further comprises a linker peptide.
  • the provided chimeric transmembrane receptor polypeptide can optionally include one or more linker peptide sequences.
  • the chimeric transmembrane receptor polypeptide includes two linker peptide sequences. In some embodiments, the receptor polypeptide includes more than two linker peptide sequences.
  • Linker peptide sequences can be found, for example, between elements of the chimeric proteins described herein.
  • one or more linker peptide sequences may connect signal peptides to the proteins (e.g., signal peptide -linker- 1.5), as well as FKBPs to EPOR in various orientations.
  • signal peptides e.g., signal peptide -linker- 1.5
  • FKBPs FKBPs to EPOR in various orientations.
  • one or more linker peptide sequences can be incorporated on each end of the FKBP.
  • the linker peptide comprises the amino acid sequence GGGGS.
  • Linker sequences suitable for use with the provided chimeric transmembrane receptor polypeptide include, for example, those consisting of glycine (G) and serine (S).
  • at least one of the one or more linker peptide sequences of the provided chimeric transmembrane receptor polypeptide is a GGS linker peptide sequence.
  • each of the one or more linker peptide sequences is GGS.
  • at least one of the one or more linker peptide sequences of the chimeric transmembrane receptor polypeptide is a GGSGGSGGS linker peptide sequence.
  • each of the one or more linker peptide sequences is GGSGGSGGS. In some embodiments, at least one of the one or more linker peptide sequences of the chimeric transmembrane receptor polypeptide is a GS linker sequence. In some embodiments, each of the one or more linker peptide sequences is GS. In some embodiments, at least one of the one or more linker peptide sequences of the chimeric transmembrane receptor polypeptide is a GSGSGS linker peptide sequence. In some embodiments, each of the one or more linker peptide sequences is GSGSGS.
  • linker peptide sequences suitable for use with the provided chimeric transmembrane receptor polypeptide include, for example, IgG hinge linker peptide sequences.
  • at least one of the one or more linker peptide sequences of the chimeric transmembrane receptor polypeptide is a wild-type IgG4 ESKYGPPCPPCP linker peptide sequence.
  • each of the one or more linker peptide sequences is ESKYGPPCPPCP.
  • at least one of the one or more linker peptide sequences of the chimeric transmembrane receptor polypeptide is a mutated IgG4 ESKYGPPAPPAP linker peptide sequence.
  • each of the one or more linker peptide sequences is ESKYGPPAPPAP.
  • the chimeric transmembrane receptor polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOS: 1 to 3 or 7 to 11.
  • the present disclosure provides a recombinant nucleic acid encoding any of the chimeric transmembrane receptor polypeptide described herein.
  • the present disclosure provides a DNA construct comprising a promoter operably linked to the recombinant nucleic.
  • the promoter is an endogenous EPOR promoter, an endogenous HBA1 promoter, an endogenous TPOR promoter, a constitutive SFFV promoter, a constitutive PGK promoter, or a constitutive UbC promoter. Integration at the HBA1 locus is predicted to elicit strong, erythroid-specific expression, and did show enhanced BB-dependent expression of inducible chimeric tEPOR, as shown in FIG. 6C. Of course, the overall expression of the transgene can vary depending on the promoter used to express the transgene.
  • Also provided is a vector comprising any of the recombinant nucleic acids described herein or any of the DNA constructs described herein.
  • the present disclosure provides a host cell comprising any of the recombinant nucleic acids described herein, any of the DNA constructs described herein, or any of the vectors described herein.
  • the recombinant nucleic acid, DNA construct, or vector is integrated into the EPOR locus, the CCR5 locus or the HBA1 locus.
  • the inducible chimeric receptor coding region is integrated at the 3’ end of the endogenous EPOR locus.
  • FIG. 16A One such scheme is depicted in FIG. 16A and involves cutting near the endogenous EPOR gene and inserting the inducible chimeric transgene.
  • the cDNA encoding the iEPOR. gene or fragment thereof could be codon- diverged relative to the native EPOR sequence by incorporating silent mutations.
  • integration of an inducible codon -diverged truncated EPOR receptor transgene resulted in an obeservable BB dimerizer-dependent increase in GFP+ cells relative to the samples without BB dimerizer (compare FIGS. 16B and 16C).
  • the GFP+ cells appear to be CD34-CD45-CD71+ RBCs.
  • the RBCs produced by cells containing inducible chimeric transgenes are functionally normal in terms of hemoglobin production.
  • FIG. 18A shows the profile of fetal hemoglobin (HbF) and adult hemoglobin (HbA) in unedited umbilical cord blood-derived HSPCs (CB Mock) that were differentiated in RBC media with Epo.
  • HbF and HbA profiles in edited cells containing integrated chimeric receptor transgenes with BB dimerizer displayed HbF and HbA production that is equivalent in total amount and ratio of HbF:HbA to the CB Mock sample with EPO (compare FIGS. 17B and 17C to 17A).
  • the hemoglobin profiles closely resembled those from cells with a mock cassette without either EPO or BB dimerizer. Therefore, in some instances, the native EPO signaling is replaced with BB-inducible signalling to create functionally normal RBCs.
  • the host cell is a eukaryotic cell. In some embodiments, the host cell is a primary human cell. In some embodiments, the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC) . In some embodimen ts, the host cell was derived from a patient who is a carrier of an allele that causes a genetic disorder.
  • PSC pluripotent stem cell
  • the genetic disorder is beta- thalassemia, sickle cell disease (SCO), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • the genetic disorder is a hemoglobinopathy.
  • the hemoglobinopathy is beta- thalassemia or sickle cell disease (SCO).
  • the genome of the host cell is edited to contain a corrected version of the allele that causes the genetic disorder.
  • the present disclosure provides a method of inducing erythroid differentiation of an HSPC, the method comprising contacting an HSPC that comprises a recombinant nucleic acid encoding a chimeric transmembrane receptor polypeptide with the dimerization signal.
  • the present disclosure provides a method of tuning red blood cell levels in a patient, the method comprising: (i) editing an HSPC so that it can express at least one chimeric transmembrane receptor polypeptide described herein; (ii) transferring the resulting HSPC to the patient; (iii) administering a first quantity of tire dimerization signal to the patient; (iv) monitoring red blood cell levels in the patient; and (v) administering a second quantity of the dimerization signal to the patient that is the same, less, or more than the first quantity of dimerization signal.
  • the present disclosure provides a method of increasing the proportion of red blood ceils with a corrected version of an allele that causes a genetic disorder, the method comprising: (i) creating an edited HSPC by introducing a corrected version of the allele associated with a genetic disorder introducing a construct that encodes at least one chimeric transmembrane receptor polypeptide described herein; (iii) transferring the edited HSPC to the patient; and (iv) administering the dimerization signal to the patient.
  • the HSPC is derived from the patient’s own cells.
  • the HSPC is derived from an allogeneic donor’s cells.
  • the genetic disorder is beta-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • SCD sickle cell disease
  • SCID severe combined immunodeficiency
  • mucopolysaccharidosis type 1 Cystic Fibrosis
  • Gaucher disease Krabbe disease
  • X-CGD X-linked chronic granulomatous disease
  • the present disclosure uses CRISPR guide sequences that specifically direct the cleavage of a EPOR, CCR5, HBA1, or HBB gene by RNA-guided nucleases, in particular within coding sequences encoding the EPOR cytoplasmic domain.
  • the present disclosure provides a CRISPR-Cas/AAV6-mediated genome editing method that can achieve high rates of targeted integration at these loci.
  • the chimeric transmembrane receptor transgene may encode a fragment of a extramembrane domain from one cell surface receptor and a fragment of an intracellular domain from the same or a different cell surface receptor.
  • the chimeric transmembrane receptor transgene may comprise the coding region for a fragment of FKBP and either EPOR or tEPOR.
  • the chimeric transmembrane receptor transgene may comprise the coding region for a fragment of FKBP and a fragment of either SCF, TPOR, or EGFR.
  • the chimeric transmembrane receptor transgene may also encode a signal peptide.
  • the chimeric transmembrane receptor transgene may also encode a reporter protein such as GFP or YFP.
  • cleavage by the RN A-guided nuclease at the sgRNA target site can occur at one or both copies of a target locus in a cell.
  • the cleavage of a target locus will lead to an indel that will result in the expression of chimeric transmembrane receptor transgene in the cell, i.e., under the control of the endogenous target gene promoter.
  • cleavage of target gene locus in the presence of a donor template leads to integration of a chimeric transmembrane receptor transgene at the target locus, and consequently to the expression of chimeric transmembrane receptor transgene in the cell under the control of the endogenous EPOR promoter.
  • cleavage of a target sequence in a safe- harbor locus such as CCR5, HBA I, HBB, or EPOR locus in the presence of a donor template leads to integration of a chimeric transmembrane receptor transgene and optionally a therapeutic transgene encoding a protein at the safe harbor locus.
  • the chimeric transmembrane receptor transgene cDNA and/or therapeutic transgene can be under the control of a heterologous promoter such as SP'F'V, PGK1 , or UBC.
  • the integrated chimeric transmembrane receptor transgene cDNA and/or therapeutic transgene is under the control of the endogenous promoter of the safe-harbor locus, e.g., the CCR5, HBA1, HBB, or EPOR promoter.
  • the single guide RNAs (sgRNAs) of the present disclosure target a safe-harbor locus such as CCR5. HBA1, or HBB or the EPOR locus.
  • sgRNAs interact with a site-directed nuclease such as Cas9 and specifically bind to or hybridize to a target nucleic acid within the genome of a cell, such that the sgRNA and the site-directed nuclease co-localize to the target nucleic acid in the genome of the cell.
  • the sgRNAs as used herein comprise a targeting sequence comprising homology (or complementarity) to a target DNA sequence at, e.g., &HBA /, HBB, or CCR5 locus, and a constant region that mediates binding to Cas9 or another RNA-guided nuclease.
  • the sgRNA can target any sequence within the target gene adjacent to a PAM sequence.
  • the targeted sequence can be within a coding sequence or a non-coding sequence of the gene.
  • the target sequence comprises one of the sequences shown as SEQ ID NOS: 23-28, or a sequence having, e.g., at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to, e.g., comprising 1, 2, 3, or more nucleotide substitutions, additions, or subtractions relative to, one of SEQ ID NOS: 2.3- 28.
  • the guide RNA target sequence comprises the sequence of SEQ ID NO: 23 or SEQ ID NO: 24, respectively, or a sequence having, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to, e.g,, comprising 1, 2, 3, or more nucleotide substitutions, additions or subtractions relative to, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the guide RNA target sequence comprises the sequence of any one of SEQ ID NOS: 25-27, or a sequence having, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to, e.g., comprising 1 , 2, 3, or more nucleotide substitutions, additions or subtractions relative to any one of SEQ ID NOS: 25-27.
  • the targeting sequence of the sgRNAs may be, e.g., 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or 15-25, 18-22, or 19-21 nucleotides in length, and shares homology with a targeted genomic sequence, in particular at a position adjacent to a CRISPR PAM sequence.
  • the sgRNA targeting sequence is designed to be homologous to the target DM A, i.e., to share the same sequence with the non-bound strand of the DNA template or to be complementary' to the strand of the template DNA that is bound by the sgRNA.
  • the homology or complementarity of the targeting sequence can be perfect (i.e., sharing 100% homology or 100% complementarity to the target DNA sequence) or the targeting sequence can be substantially homologous (i.e., having less than 100% homology or complementarity, e.g., with 1-4 mismatches with the target DNA sequence).
  • Each sgRNA also includes a constant region that interacts with or binds to the site- directed nuclease, e. g, , Cas9.
  • the constant region of an sgRNA can be from about 70 to 250 nucleotides in length, or about 75-100 nucleotides in length, 75-85 nucleotides in length, or about 80-90 nucleotides in length, or 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides m length.
  • the overall length of the sgRN A can be, e.g., from about 80-300 nucleotides in length, or about 80-150 nucleotides in length, or about 80-120 nucleotides in length, or about 90-110 nucleotides in length, or, e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides m length.
  • crRNAs two-piece gRNAs
  • crtracrRNAs two-piece gRNAs
  • the sgRNAs comprise one or more modified nucleotides.
  • the polynucleotide sequences of the sgRNAs may also comprise RNA analogs, derivatives, or combinations thereof.
  • the probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone (e.g., phosphorothioates).
  • the sgRNAs comprise 3’ phosphorothiate intemucleotide linkages, 2’-O-methyl- 3 ’-phosphoacetate modifications, 2 ’-fluoro-pyrimidines, S-constrained ethyl sugar modifications, or others, at one or more nucleotides.
  • the sgRNAs comprise 2'-O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides (see, e.g., Hendel et al. (2015) Nat. Biotech. 33(9):985-989, the entire disclosure of which is herein incorporated by reference).
  • the 2'-O-methyl-3'- phosphorothioate (MS) modifications are at the three terminal nucleotides of the 5' and 3' ends of the sgRNA.
  • the sgRNAs can be obtained in any of a number of ways.
  • primers can be synthesized in the laboratory using an oligo synthesizer, e.g., as sold by Applied Biosystems, Biolytic Lab Performance, Sierra Biosystems, or others.
  • primers and probes with any desired sequence and/or modification can be readily ordered from any of a large number of suppliers, e.g., ThermoFisher, Biolytic, IDT, Sigma-Aldritch, GeneScript, etc.
  • any CRISPR-Cas nuclease can be used in the method, i.e., a CRISPR-Cas nuclease capable of interacting with a guide RNA and cleaving the DNA at the target site as defined by the guide RNA,
  • the nuclease is Cas9 or Cpfl.
  • the nuclease is Cas9.
  • the Cas9 or other nuclease used in the present methods can be from any source, so long that it is capable of binding to an sgRNA as described herein and being guided to and cleaving the specific target (e.g., CCR5. HBAI, HBB, or EPOR) sequence targeted by the targeting sequence of the sgRNA.
  • the Cas9 is from Streptococcus pyogenes .
  • CRISPR/Cas or CRISPR/Cpfl systems that target and cleave DNA at, e.g., the CCR5, HBA1, or HBB locus.
  • An exemplary CRISPR/Cas system comprises (a) a Cas (e.g.. Cas9) or Cpfl polypeptide or a nucleic acid encoding said polypeptide, and (b) an sgRNA that hybridizes specifically to CCR5, (or HBA / , HBB, or other safe-harbor locus, or the EPOR locus), or a nucleic acid encoding said guide RNA.
  • the nuclease systems described herein further comprise a donor template as described herein.
  • the CRISPR/Cas system comprises an RNP comprising an sgRNA targeting CCR5 (or HBA 1 or HBB, or other safe harbor locus, or the EPOR locus) and a Cas protein such as Cas9.
  • the Cas9 is a high fidelity (HiFi) Cas9 (see, e.g., Vakulskas, C. A. et al., Nat. Med. 24, 1216-1224 (2016)).
  • CRISPR/Cas9 platform which is a type II CRISPR/Cas system
  • CRISPR/Cas9 platform which is a type II CRISPR/Cas system
  • alternative systems exist including type I CRISPR/Cas systems, type III CRISPR/Cas systems, and type V CRISPR/Cas systems.
  • Various CRISPR/Cas9 systems have been disclosed, including Streptococcus pyogenes Cas9 (SpCas9), Streptococcus thermophilus Cas9 (StCas9), Campylobacter jejuni Cas9 (CjCas9) and Neisseria cinerea Cas9 (NcCas9) to name a few.
  • Alternatives to the Cas system include the Francisella novicida Cpfl (FnCpfl),
  • Acidaminococcus sp. Cpfl (AsCpfl), and Lachnospiraceae bacterium ND2006 Cpfl (LbCpfl) systems.
  • Any of the above CRISPR systems may be used to induce a single or double stranded break at the CCR5 locus (or, e.g., the HBA1 or HBB, or other safe harbor locus) to carry out the methods disclosed herein.
  • the guide RNA and nuclease can be introduced into the cell using any suitable method, e.g. , by introducing one or more polynucleotides encoding the guide RNA and the nuclease into the cell, e.g., using a vector such as a viral vector or delivered as naked DNA or RNA, such that the guide RNA and nuclease are expressed in the cell.
  • a vector such as a viral vector or delivered as naked DNA or RNA
  • one or more polynucleotides encoding the sgRNA, the nuclease or a combination thereof are included in an expression cassette.
  • the sgRNA, the nuclease, or both sg ⁇ RNA and nuclease are expressed in the cell from an expression cassette.
  • the sgRNA, the nuclease, or both sgRNA and nuclease are expressed in tire cell under the control of a heterologous promoter.
  • one or more polynucleotides encoding the sgRNA and the nuclease are operatively linked to a heterologous promoter.
  • the gm do RNA and nuclease are assembled into ribonucleoproteins (RNPs) prior to delivery' to the cells, and the RNPs are introduced into the cell by, e.g., electroporation.
  • RNPs are complexes of RNA and RNA-binding proteins.
  • the RNPs comprise the RNA-binding nuclease (e.g., Cas9) assembled with the guide RNA (e.g., sgRNA), such that the RNPs are capable of binding to the target DNA (through the gRNA component of the RNP) and cleaving it (via the protein nuclease component of the RNP).
  • RNA-binding nuclease e.g., Cas9
  • guide RNA e.g., sgRNA
  • an RNP for use in the present methods can comprise any of the herein-described guide RNAs and any ofthe herein-described RNA-guided nucleases.
  • Animal ceils mammalian cells, preferably human cells, modified ex vivo, in vitro, or in vivo are contemplated. Also included are cells of other primates; mammals, including commercially relevant mammals, such as cattle, pigs, horses, sheep, cats, dogs, mice, rats; birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • the cell is an embryonic stem cell, a stem cell, a progenitor cell, a pluripotent stem cell, an induced pluripotent stem (iPS) cell, a somatic stem cell, a differentiated cell, a mesenchymal stem cell or a mesenchymal stromal cell, a neural stem cell, a hematopoietic stem cell or a hematopoietic progenitor cell, an adipose stem cell, a keratinocyte, a skeletal stem cell, a muscle stem cell, a fibroblast, an NK cell, a B-cell, a T cell, or a peripheral blood mononuclear cel! (PBMC).
  • PBMC peripheral blood mononuclear cel!
  • the cells are CD34 + hematopoietic stem and progenitor cells (HSPCs), e.g., cord blood-derived (CB), adult peripheral blood-derived (PB), or bone marrow' derived HSPCs.
  • HSPCs hematopoietic stem and progenitor cells
  • CB cord blood-derived
  • PB adult peripheral blood-derived
  • bone marrow' derived HSPCs bone marrow' derived HSPCs
  • HSPCs cati be isolated from a subject, e.g., by collecting mobilized peripheral blood and then enriching the HSPCs using the CD34 marker.
  • the cells are from a subject with a genetic condition involving erythroid cells (e.g., alpha-thalassemia, beta- thalassemia, sickle cell disease), or from a subject with a condition that could be treated with genetically modified HSPCs expressing a beneficial and/or therapeutic protein (e.g. , hemophilia B, phenylketonuria, mucopolysaccharidosis type 1, Gaucher disease, Krabbe disease).
  • a beneficial and/or therapeutic protein e.g. , hemophilia B, phenylketonuria, mucopolysaccharidosis type 1, Gaucher disease, Krabbe disease.
  • a method is provided of treating a subject with any of the herein-described conditions or disorders (e.g., alpha-thalassemia, beta-thalassemia, sickle cell disease, hemophilia B, phenylketonuria, Gaucher disease, Krabbe disease) comprising genetically modifying a plurality of HSPCs isolated from the subject so as to integrate a therapeutic transgene tor the particular condition or disorder (e.g., a transgene encoding a- globin, ⁇ -globin, factor IX, phenylalanine hydroxylase (PAH), iduronidase, glucocerebrosidase, galactocerebrosidase, and the like), and also to effect the expression of a chimeric transmembrane receptor transgene as described herein, and reintroducing the HSPCs into the subject.
  • a therapeutic transgene tor the particular condition or disorder e.g., a transgene encoding
  • the therapeutic transgene is a full-length (e.g., from start codon to stop codon, including introns) transgene comprising a corrective (e.g. , wild-type) sequence of an endogenous gene containing one or more deleterious mutations in the HSPCs or encoding a protein that is deficient in the HSPCs.
  • a corrective e.g. , wild-type sequence of an endogenous gene containing one or more deleterious mutations in the HSPCs or encoding a protein that is deficient in the HSPCs.
  • HSPCs expressing a chimeric transmembrane receptor transgene and comprising a therapeutic transgene or other beneficial genetic modification proliferate more rapidly in vivo and become enriched relative to equivalent HSPCs not expressing a chimeric transmembrane receptor transgene, e.g., in cells from the subject that have not been genetically modified using the present methods.
  • the cells to be modified are preferably derived from the subject’s own cells.
  • the mammalian cells are autologous cells from the subject to be treated with the modified cells.
  • the cells are allogeneic, i.e., isolated from an HLA-matched or HLA -compatible, or otherwise suitable, donor.
  • cells are harvested from the subject and modified according to the methods disclosed herein, which can include selecting certain cell types, optionally expanding the cells and optionally culturing the cells, and which can additionally include selecting cells that contain a chimeric transmembrane receptor transgene integrated into the CCR5 (or HBA1 or HBB, or other safe harbor) locus, or the EPOR locus, and/or cells that have been modified to express a therapeutic or otherwise beneficial transgene.
  • such modified cells are then reintroduced into the subject.
  • nuclease systems to produce the modified host cells described herein, comprising introducing into the cell (a) an RNP of the present disclosure that targets and cleaves DNA at the CCR5 (or HBA1, or HBB, or other safe harbor) locus, or the EPOR locus, and optionally (b) a homologous donor template or vector as described herein.
  • Each component can be introduced into the cell directly or can be expressed in the cell by introducing a nucleic acid encoding the components of said one or more nuclease systems.
  • the present methods target integration of a chimeric transmembrane receptor transgene, i.e., at a safe harbor locus such as CCR5, HBA 1 , or HBB, or EPOR, in a host cell ex vivo.
  • a chimeric transmembrane receptor transgene i.e., at a safe harbor locus such as CCR5, HBA 1 , or HBB, or EPOR
  • the present methods can comprise (a) introducing a donor template comprising a therapeutic transgene encoding a protein and the chimeric transmembrane receptor transgene at a safe harbor locus in the genome of the cell, e.g., to introduce a therapeutic genetic modification (such as the introduction of an HBA1,HBA 2, HBB, PDGFB, FIX, LDLR, PAH, 1DUA, GBA, or GALC transgene or vector into the cell at the safe harbor locus), optionally after expanding said cells, or optionally before expanding said cells, and (b) optionally culturing the cell.
  • a therapeutic genetic modification such as the introduction of an HBA1,HBA 2, HBB, PDGFB, FIX, LDLR, PAH, 1DUA, GBA, or GALC transgene or vector into the cell at the safe harbor locus
  • the first and second homology regions of the donor template flank both the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • the donor template is a bicistronic cassette comprising an internal ribosome entry site (IRES) between the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • IRES internal ribosome entry site
  • the donor template is a bicistronic cassette comprising a nucleic acid sequence encoding a 2A cleavage peptide (e.g., a member of the 2A peptide family such as a T2A, P2A, E2A, or F2A cleavage peptide) between the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • a 2A cleavage peptide e.g., a member of the 2A peptide family such as a T2A, P2A, E2A, or F2A cleavage peptide
  • Exemplary nucleic acid sequences encoding T2.A and P2A cleavage peptides are shown as SEQ ID NOS: 30 and 31, respectively.
  • the 2A cleavage peptide is a T2A or P2A cleavage peptide.
  • the 2A cleavage peptide is a peptide having sequence similarity and functional interchangeability to a T2A or P2A cleavage peptide, such as an E2A or F2A cleavage peptide.
  • the therapeutic transgene is 5’ of the IRES sequence or the sequence encoding the 2A cleavage peptide and the chimeric transmembrane receptor transgene is 3’ of the IRES sequence or the sequence encoding the 2A cleavage peptide.
  • the first homology region is 5’ of the therapeutic transgene and the second homology region is 3’ ofthe chimeric transmembrane receptor transgene.
  • the chimeric transgene is 5’ of the IRES sequence or the sequence encoding the 2 A cleavage peptide and the therapeutic transgene is 3’ of the IRES sequence or the sequence encoding the 2A cleavage peptide.
  • the first homology region is 5’ of the chimeric transmembrane receptor transgene and the second homology region is 3’ of the therapeutic transgene.
  • the present methods can further comprise (a) introducing a second guide RNA and donor template into the cell, e.g., to introduce a second, therapeutic genetic modification (such as the introduction of an HBA 1 , HBA .':.
  • the disclosure herein contemplates a method of producing a modified mammalian host cell, the method comprising introducing into a mammalian cell: (a) an RNP comprising a Cas nuclease such as Cas9 and an sgRNA as described herein, and optionally (b) a homologous donor template or vector as described herein.
  • the nuclease can produce one or more single stranded breaks within the targeted (e.g., CCR5, HBA1 , or HBB) locus, or the EPOR locus, or a double-stranded break within the targeted locus.
  • the targeted locus is modified by homologous recombination with a donor template or vector to result in insertion of the transgene into the locus.
  • the methods can further comprise (c) selecting cells that contain the integrated transgene at the targeted locus.
  • i53 Canny et al.
  • HDR homology directed repair
  • NHEJ non-homologous end-joining
  • transgenes including large transgenes, capable of expressing functional proteins, including enzymes, cytokines, antibodies, and cell surface receptors are known in the art (See, e.g., Bak and Porteus, Cell Rep. 2017 Jul 18; 20(3): 750- 756 (integration of EGFR); Kanojia et al., Stern Cells. 2015 Oct;33(l()):2985-94 (expression of anti-Her2 antibody); Eyquem et al., Nature.
  • the transgene to be integrated which is comprised by a polynucleotide or donor construct, can be any chimeric transmembrane receptor transgene whose gene product can provide chimeric transmembrane receptor expression in red blood cells or other cells of the erythroid lineage, and particularly provide inducible differentiation and/or drive the elevated proliferation of the modified cells relative to equivalent cells lacking the chimeric transmembrane receptor.
  • the transgene could be integrated at the EPOR locus.
  • the transgene could be integrated a genomic location outside of the EPOR locus.
  • the transgene could be integrated using an EPOR left homology arm comprising SEQ ID NO: 18.
  • the transgene could be integrated using an EPOR right homology arm comprising SEQ ID NO: 19.
  • a chimeric transmembrane receptor transgene and optionally a therapeutic transgene encoding a protein is integrated at a safe harbor locus such as CCR5, HBA1, or HBB.
  • the donor template further comprises a therapeutic transgene.
  • a therapeutic transgene includes HBA1 (hemoglobin subunit alpha 1; see, e.g., NCBI Gene ID No. 3039), HBA2 (hemoglobin subunit alpha 2; see, e.g., NCBI Gene ID No. 3040), HBB (hemoglobin subunit beta; see, e.g., NCBI Gene ID No. 3043), PDFGB (platelet-derived growth factor subunit B; see, e.g., NCBI Gene ID No.
  • 1DUA alpha-L-iduronidase; see, e.g., NCBI Gene ID No, 3425
  • PAH phenylalanine hydroxylase; see, e.g., NCBI Gene ID No. 5053
  • Factor IX or FIX:, see, e.g., NCBI Gene ID NO. 2158
  • Hyperactive Factor IX Padua or the Padua Variant (see, e.g., Simioni et al., (2009) NEJM 361: 1671-1675; Cantore et al. (2012) Blood 120:4517-4520; Monahan et al., (2015) Hum. Gene.
  • the first and second homology aims of the donor template flank both the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • the donor template is a bicistronic cassette comprising an interna] ribosome entry' site (IRES) between the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • the donor template is a bicistronic cassette comprising a nucleic acid sequence encoding a 2A cleavage peptide (e.g., a member of the 2A peptide family such as a T2A, P2A, E2A, or F2A cleavage peptide) between the therapeutic transgene and the chimeric transmembrane receptor transgene.
  • a 2A cleavage peptide e.g., a member of the 2A peptide family such as a T2A, P2A, E2A, or F2A cleavage peptide
  • the therapeutic transgene is 5’ of the IRES sequence or the sequence encoding the 2A cleavage peptide and the chimeric transmembrane receptor transgene is 3 ’ of the IRES sequence or the sequence encoding the 2 A cleavage peptide.
  • the first homology arm is 5’ of the therapeutic transgene and the second homology arm is 3’ of the chimeric transmembrane receptor transgene.
  • the chimeric transmembrane receptor transgene is 5’ of the IRES sequence ortho sequence encoding the 2A cleavage peptide and the therapeutic transgene is 3’ of the IRES sequence or the sequence encoding the 2A cleavage peptide.
  • the first homology arm is 5’ of the chimeric transmembrane receptor transgene and the second homology arm is 3’ of the therapeutic transgene.
  • the IRES sequence comprises SEQ ID NO: 29.
  • the T2A cleavage peptide coding sequence comprises SEQ ID NO: 30.
  • the P2A cleavage peptide coding sequence comprises SEQ ID NO: 31.
  • the bicistronic construct encodes an HBB polypeptide comprising SEQ ID NO: 6.
  • the bicistronic construct encodes an iEPOR polypeptide comprising SEQ ID NO: 7.
  • a second donor template that comprises a therapeutic transgene, e.g., an HBA 1, HBA2, HBB, PDGFB, IDUA, GBA, FIX, LDLR, PAH, or GALC transgene.
  • a therapeutic transgene e.g., an HBA 1, HBA2, HBB, PDGFB, IDUA, GBA, FIX, LDLR, PAH, or GALC transgene.
  • expression of a chimeric transmembrane receptor transgene, and/or a second transgene such as an HBA1, HBA2, HBB, IDUA, PDGFB, GBA, FIX, LDLR, PAH, or GALC transgene is driven by a heterologous promoter such as HBA1, HBA2, HBB, PGK1, SFFV, or UBC.
  • a chimeric transmembrane receptor transgene, and/or a second transgene such as an HBA 1 , HBA2, HBB, IDUA, PDGFB, GBA, FIX, LDLR, PAH, or GALC transgene, is driven by an endogenous promoter such asHBAl, HBA2, or HBB.
  • the transgene in the homologous repair template is codon- optimized, e.g., comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, or more homology to the corresponding wild-type coding sequence or cDNA, or a fragment thereof such as in the case of a truncated EPOR.
  • a transgene as used herein may also comprise optional elements such as introns, WPREs, polyA regions, UTRs (e.g., 5’ or 3’ UTRs).
  • the template comprises a polyA sequence or signal, e.g., a bovine growth hormone polyA sequence or a rabbit beta-globin polyA sequence, at the 3’ end of the cDNA,
  • a Woodchuck Hepatitis Virus Posttranscriptional Regulator ⁇ ' Element is included within the 3’UTR of the template, e.g., between the 3" end of the coding sequence and the 5’ end of the polyA sequence, so as to increase the expression of the transgene .
  • WPRE sequence can be used; See, e.g., Zufferey et al, (1999) J. Virol. 73(4):2886-2892; Donello, et al. ( 1998). J Virol 72: 5085-5092; Loeb, et al. (1999). Hum Gene Ther 10: 2295- 2305; the entire disclosures of which are herein incorporated by reference).
  • the transgene is flanked within the polynucleotide or donor construct by sequences homologous to the target genomic sequence.
  • the transgene is flanked by one sequence homologous to the region 5’ to the cleavage site (e.g., starting at or around the guide RNA target sequence and running upstream) and a second sequence homologous to the region 3’ of the site of cleavage (e.g., starting at or around the guide RNA target site and running downstream).
  • the donor template comprises a left homology sequence comprising the sequence shown as SEQ ID NO: 12 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 12 or a subsequence thereof.
  • the donor template comprises a right homology sequence comprising the sequence shown as SEQ ID NO: 13 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 13 or a subsequence thereof.
  • the donor template comprises a left homology sequence comprising the sequence shown as SEQ ID NO: 14 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 14 or a subsequence thereof.
  • the donor template comprises a right homology sequence comprising the sequence shown as SEQ ID NO: 15 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 15 or a subsequence thereof.
  • the donor template comprises a left homology sequence comprising the sequence shown as SEQ ID NO: 16 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 16 or a subsequence thereof.
  • the donor template comprises a right homology sequence comprising the sequence shown as SEQ ID NO: 17 or a subsequence thereof, or to a sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to SEQ ID NO: 17 or a subsequence thereof.
  • a chimeric transmembrane receptor transgene replaces all or part of a safe harbor gene such as CCR5, HBAI, or HBB such that its expression is driven by the endogenous CCR5, HBAI, ox HBB promoter.
  • a chimeric transmembrane receptor transgene replaces all or part of a safe harbor gene such as CCR5, HBAI, or HBB such that its expression is driven by the endogenous CCR5, HBAI, ox HBB promoter.
  • a chimeric transmembrane receptor transgene and optionally a therapeutic transgene such as
  • HBAI, HBA2, HBB, PDGFB, FIX, LDLR, PAH, 1DUA, GBA, or GALC is integrated into a safe harbor locus such as CCR5, HBAI, or HBB wherein the expression of the transgene is driven by a heterologous promoter such as HBA 1 , HBA2, HBB, PGK1 , SFFV, or UBC.
  • a part or a fragment of the target gene is replaced by the transgene.
  • the whole coding sequence of the target gene is replaced by the transgene.
  • the coding sequence and regulatory’ sequences of the transgene is replaced by the transgene.
  • the target gene sequence replaced by the transgene comprises an open reading frame. In some embodiments, the target gene sequence replaced by the transgene comprises an expression cassette. In some embodiments, the target gene sequence replaced by the transgene comprises a sequence that transcribes into a precursor mRNA, In some embodiments, the target gene sequence replaced by the transgene comprises a 5’ UTR, one or more introns, one or more exons, and a 3’ UTR.
  • the 5’ (or left) homology arm is at least lOObp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, lOOObp or more in length.
  • the 5’ homology arm is l OObp, 150bp, 200bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 450bp, or greater than 500bp in length.
  • the 5’ homology arm is at least 400bp in length.
  • the 5’ homology arm is at least SOObp, 600bp, 700bo, 800bp, 900bp, or lOOObp in length. In some embodiments, the 5’ homology arm is at least 850bp in length. In some embodiments, the 5’ homology ami is 400 - 500 bp.
  • the 5’ homology arm is 400-5 OObp, 400-55 Obp, 400-600bp, 400-65 Obp, 400- 700bp, 400-750bp, 400-800bp, 400-850bp, 400-900bp, 400-950bp, 400-1000bp, 400-1 lOObp, 400-1200bp, 400-1 SOObp, 400-1400bp, 450-500bp, 450-550bp, 450-600bp, 450-650bp, 450- 700bp, 450-750bp, 450-800bp, 450-850bp, 450-900bp, 450-950bp, 450-1000bp, 450-1 l OObp, 450-1200bp, 450-1450bp, 500-600bp, 500-650bp, 500-700bp, 500-750bp, 500- 800bp, 500-850bp, 500-900bp, 500-1000bp, 450-1
  • the 3 ’ (or right) homology arm is at least 1 OObp, 200bp, 3 OObp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1 OOObp or more in length.
  • the 3’ homology arm is l OObp, 150bp, 200bp, 250bp, 275bp, 300bp, 325bp, 350bp, 375bp, 400bp, 450bp, or greater than 500bp in length.
  • the 3’ homology arm is at least 400bp in length.
  • the 3’ homology arm is at least SOObp, 600bp, 700bo, 800bp, 900bp, or 1 OOObp in length. In some embodiments, the 3’ homology arm is at least 850bp in length. In some embodiments, the 3’ homology arm is 400 - SOObp.
  • the 3’ homology arm is 400-5 OObp, 400-55 Obp, 400-600bp, 400-65 Obp, 400- 700bp, 400-750bp, 400-800bp, 400-850bp, 400-900bp, 400-950bp, 400-1000bp, 400-1 lOObp, 400-1200bp, 400-1300bp, 400-1400bp, 450-500bp, 450-550bp, 450-600bp, 450-650bp, 450- 700bp, 450-750bp, 450-800bp, 450-850bp, 450-900bp, 450-950bp, 450-1000bp, 450-1 lOObp, 450-1200bp, 450-1450bp, 500-600bp, 500-650bp, 500-700bp, 500-750bp, 500- 800bp, 500-850bp, 500-900bp, 500
  • the provided chimeric transmembrane receptor polypeptide can optionally include one or more linker peptide sequences.
  • the chimeric transmembrane receptor polypeptide includes two linker peptide sequences.
  • the receptor polypeptide includes more than two linker peptide sequences.
  • the polynucleotide is introduced using a recombinant adeno-associated viral vector (rAAV).
  • the rAAV can be from serotype 1 (e.g., an rAAVl vector), 2 (e.g., an rAAV2 vector), 3 (e.g,, an rAAV3 vector), 4 (e.g., an rAAV4 vector), 5 (e.g., an rAAV.5 vector), 6 (e.g., an rAAV6 vector), 7 (e.g., an rAAV7 vector), 8 (e.g., an rAAV8 vector), 9 (e.g., an rAAV9 vector), 10 (e.g., an rAAVIO vector), or 11 (e.g., an rAAVl l vector).
  • serotype 1 e.g., an rAAVl vector
  • 2 e.g., an rAAV2 vector
  • 3 e.g, an rAAV3 vector
  • 4 e.g., an rAAV4 vector
  • 5 e.g.
  • tire vector is an rAAV 6 vector.
  • the donor template is single stranded, double stranded, a plasmid or a DNA fragment.
  • plasmids comprise elements necessary for replication, including a promoter and optionally a 3’ UTR.
  • vectors comprising (a) one or more nucleotide sequences homologous to the EPOR locus, and (b) a chimeric transmembrane receptor transgene as described herein.
  • the vector can be a viral vector, such as a retroviral, lentiviral (both integration competent and integration defective lentiviral vectors), adenoviral, adeno- associated viral or herpes simplex viral vector.
  • Viral vectors may further comprise genes necessary for replication of the viral vector.
  • the targeting construct comprises: (1) a viral vector backbone, e.g., an AAV backbone, to generate virus; (2) arms of homology to the target site of at least 200 bp but ideally at least 400 bp or at least 900 on each side to assure high levels of reproducible targeting to the site (see, Porteus, Annual Review of Pharmacology and Toxicology, Vol.
  • a transgene encoding a functional alpha globin protein and capable of expressing the functional alpha globin protein, a polyA sequence, and optionally a WPRE element; and optionally (4) an additional marker gene to allow for enrichment and/or monitoring of the modified host cells.
  • Any AAV known m the art can be used.
  • the primary AAV serotype is AAV6.
  • the vector, e.g., rAAV 6 vector, comprising the donor template is from about 1-2 kb, 2-3 kb, 3-4 kb, 4-5 kb, 5-6 kb, 6-7 kb, 7-8 kb, or larger.
  • Suitable marker genes are known in the art and include Myc, HA, FLAG, GFP, YFP, truncated NGFR, truncated EGFR, truncated CD20, truncated CD 19, as well as antibiotic resistance genes.
  • the homologous repair template and/or vector e.g., AAV6
  • the inserted construct can also include other safety switches, such as a standard suicide gene into the locus ⁇ e.g., iCasp9) in circumstances where rapid removal of cells might be required due to acute toxicity.
  • a standard suicide gene into the locus e.g., iCasp9
  • the present disclosure provides a robust safety switch so that any engineered cell transplanted into a body can be eliminated, e.g., by removal of an auxotrophic factor. This is especially important if the engineered cell has transformed into a cancerous cell.
  • the present methods allow for the insertion of the donor template in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more cells, e.g., HSPCs from a subject.
  • the CRISPR-mediated systems as described herein are assessed in primary HSPCs, e.g., as derived from mobilized peripheral blood or from cord blood.
  • the HSPCs can be WT primary HSPCs (e.g., for initial testing of the system) or from patient-derived HSPCs (e.g., for pre-clinical in vitro testing).
  • the HSPCs are cultured in vitro and allowed to differentiate into RBCs to confirm the elevated rate of proliferation relative to unmodified cells (as measured, e.g., by a co-culture experiment in the presence or absence of EPO as described in Example 1 or by other methods of determining proliferation rate such as BrdU incorporation or by monitoring the number of cells in a culture over time) prior to the reintroduction of HSPCs into a subject.
  • a plurality of modified HSPCs can be reintroduced into the subject.
  • the HSPCs are introduced by intrafemoral injection, such that they can populate the bone marrow and differentiate into, e.g., red blood cells.
  • the HSPCs are introduced by intravenous injection.
  • the HSPCs are induced to initiate differentiation into red blood cells in vitro, and tire modified erythroid lineage cells are then re-introduced into the subject.
  • a genetic condition or disorder e.g., alpha-thalassemia, beta-thalassemia, sickle cell disease, hemophilia B, phenylketonuria, mucopolysaccharidosis type 1, Gaucher disease, Krabbe di sease, and the like
  • the method comprising genetically modifying HSPCs from the individual so as to provide a beneficial effect (e.g., by introducing a therapeutic transgene for correcting a mutation underlying the condition or disorder, or for providing to the individual a protein replacement therapy) and also such that they express chimeric transmembrane receptor transgene and are therefore enriched in vivo following reintroduction of the cells to the individual.
  • the present methods allow for the efficient integration of a donor template comprising a therapeutic transgene and a chimeric transmembrane receptor transgene at a safe harbor locus.
  • expression of the therapeutic transgene and the chimeric transmembrane receptor transgene causes an enrichment of genetically modified HSPCs in a population of HSPCs, e.g., over the course of red biood cell differentiation, as compared to expression of the therapeutic transgene in the absence of expression of the chimeric transmembrane receptor transgene.
  • the present methods allow for the insertion of the donor template in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more HSPCs in a population of HSPCs, e.g., over the course of red blood cell differentiation.
  • expression of the therapeutic transgene and the chimeric transmembrane receptor transgene increases the proportion of genetically modified HSPCs in a population of HSPCs by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, e.g., over the course of red blood cell differentiation, as compared to expression of the therapeutic transgene in the absence of expression of the chimeric transmembrane receptor transgene.
  • expression of the therapeutic transgene and the chimeric transmembrane receptor transgene increases a level of adult hemoglobin tetramers in the genetically modified HSPCs by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to expression of the therapeutic transgene in the absence of expression of the chimeric transmembrane receptor transgene.
  • compositions and kits for use of the modified cells including pharmaceutical compositions, therapeutic methods, and methods of administration.
  • pharmaceutical compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any animals.
  • a pharmaceutical composition comprising a modified autologous host cell as described herein.
  • the modified autologous host cell is genetically engineered to comprise an integrated chimeric transmembrane receptor transgene at a safe harbor locus such as CCR5, HBA1, or HBB, or the EPOR locus, as well as optionally a second, therapeutic genetic modification as described herein (e.g., a therapeutic transgene integrated at the safe harbor locus).
  • the modified host, cell of the disclosure herein may be formulated using one or more excipients to, e.g. -. (1) increase stability; (2) alter the biodistribution (e.g., target the cell line to specific tissues or cell types); (3) alter the release profile of an encoded therapeutic factor.
  • Formulations of the present disclosure can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • pharmaceutical composition refers to compositions including at least one active ingredient (e.g., a modified host cell) and optionally one or more pharmaceutically acceptable excipients.
  • Pharmaceutical compositions of the present disclosure may be sterile.
  • Relative amounts of the active ingredient may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • the composition may include between 0.1% and 99% (w/w) of the active ingredient.
  • the composition may include between 0.1 % and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5- 80%, or at least 80% (w/w) active ingredient.
  • Excipients include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by' reference in its entirety).
  • Tire use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect, or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry' starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the modified host, cells of the present disclosure included in the pharmaceutical compositions described above may be administered by any delivery route, systemic delivery or local delivery, which results in a therapeutically effective outcome.
  • delivery route systemic delivery or local delivery, which results in a therapeutically effective outcome.
  • these include, but are not limited to, enteral, gastroenteral, epidural, oral, transdermal, intracerebral, intracerebroventricular, epicutaneous, intradermal, subcutaneous, nasal, intravenous, intra- arterial, intramuscular, intracardiac, intraosseous, intrathecal, intraparenchymal, intraperitoneal, intravesical, intravitreal, intracavernous), interstitial, intra-abdominal, intralymphatic, intramedullary, intrapulmonary, intraspinal, intrasynovial, intrathecal, intratubular, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, soft tissue, and topical.
  • a subject undergoes a conditioning regime before cell transplantation.
  • a conditioning regime may involve administration of cytotoxic agents.
  • the conditioning regime may also include immunosuppression, antibodies, and irradiation.
  • conditioning regimens include antibody-mediated conditioning (see, e.g., Czechowicz et al., 318(5854) Science 1296-9 (2007); Palchaudari el al., 34(7) Nature Biotechnology 738- 745 (2.016); Chhabra et al., 10:8(351) Science Translational Medicine 351ral05 (2016)) and CAR T-mediated conditioning (see, e.g., Arai et al., 26(5) Molecular Therapy 1181-1197 (2016); each of which is hereby incorporated by reference in its entirety).
  • conditioning needs to be used to create space in the brain for microglia derived from engineered hematopoietic stem cells (HSCs) to migrate in to deliver the protein of interest (as in recent gene therapy trials for ALD and MLD).
  • Tire conditioning regimen is also designed to create niche ‘‘space’ 1 to allow the transplanted cells to have a place in the body to engraft and proliferate.
  • niche space In HSPC transplantation, for example, tire conditioning regimen creates niche space in the bone marrow for the transplanted HSPCs to engraft. Without a conditioning regimen, the transplanted HSCs cannot engraft.
  • compositions including the modified host cell of the present disclosure are directed to methods of providing pharmaceutical compositions including the modified host cell of the present disclosure to target tissues of mammalian subjects, by contacting target tissues with pharmaceutical compositions including the modified host cell under conditions such that they are substantially retained in such target tissues.
  • pharmaceutical compositions including the modified host ceil include one or more cell penetration agents, although “naked” formulations (such as without cell penetration agents or other agents) are also contemplated, with or without pharmaceutically acceptable excipients.
  • the present disclosure additionally provides methods of administering modified host cells in accordance with the disclosure to a subject in need thereof.
  • the pharmaceutical compositions including the modified host cell, and compositions of the present disclosure may be administered to a subject using any amount and any route of administration effective for preventing, treating, or managing the condition or disorder, e.g., alpha-thalassemia, beta- thalassemia, sickle cell disease, etc.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • Idle subject may be a human, a mammal, or an animal.
  • the specific therapeutically or prophylactically effective dose level for any particular individual will depend upon a variety of factors including the condition or disorder being treated and the severity of the condition or disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration; the duration of the treatment; drugs used in combination or coincidental with the specific modified host cell employed; and like factors well known in the medical arts.
  • modified host cell pharmaceutical compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from, e.g., about 1 x 10 4 to 1 x 10 ⁇ 1 x 10 5 to 1 x 10 b , 1 x 10 6 to 1 x 10 7 , or more modified cells to the subject, or any amount sufficient to obtain the desired therapeutic or prophylactic, effect.
  • the desired dosage of the modified host cells of the present disclosure may be administered one time or multiple times.
  • delivery of the modified host cell to a subject provides a therapeutic effect for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, I year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 2.1 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years.
  • the modified host cells may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents, or medical procedures, either sequentially or concurrently .
  • each agent will be administered at a dose and/or on a time schedule determined for that agent.
  • kits comprising compositions or components of the present disclosure, e.g., sgRNA, Cas9, RNPs, i53, and/or homologous templates, as well as, optionally, reagents for, e.g., the introduction of the components into cells.
  • the kits can also comprise one or more containers or vials, as well as instructions for using the compositions in order to modify cells and treat subjects according to the methods described herein.
  • the methods and compositions disclosed herein can be used to produce red blood cells (RBCs) in vitro, in small, medium, or large scale as needed.
  • the method comprises: (i) editing one or more HSPCs to express at least one of the chimeric transmembrane receptor polypeptides as disclosed herein; and (ii) contacting the one or more HSPCs with a dimerization signal.
  • the dimerization signal is a small molecule dimerization signal.
  • the small molecule dimerization signal is AP20187 (BB dimerizer).
  • step (ii) further comprises contacting the one or more HSPCs with erythropoietin (EPO).
  • the method is conducted in a bioreactor to produce RBCs at large scale.
  • pre-harvest RBC sample aliquots may be taken to establish cell counts, viability, cell characterization, e.g., FACs analysis, purity, and/or other general release criteria for the cells.
  • post-harvest sample aliquots may be taken to establish cell counts and/or viability.
  • the populations of RBCs are then transferred to one or more IV bags or other suitable vessels and cryopreserved in a controlled rate freezer until the cells are ready for use.
  • cells are frozen in 50% plasmalyte and 50% Cryostor 10; 50/40/10 (XVIVO/HABS/DMSO); Crytostor 5 or Cryostor 10.
  • bags e.g., 10 to 250 mL capacity
  • Infusion bags can be stored in the freezer until needed.
  • the method disclosed herein can be used generate RBCs at a total cell number that is appropriate for the treatment of a subject in need thereof.
  • the treatment is a blood transfusion.
  • the scale of said method can result in a production of from about 1 million RBCs to about 200 million RBCs.
  • the scale of said method can result in a production of about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 20 million, about 50 million, about 100 million, about 150 million, or about 200 million RBCs.
  • Illustrative embodiments of cell culture bags include, but are not limited to, MACS® GMP Cell Expansion Bags, MACS® GMP Cell Differentiation Bags, EXP-PakTM Cell Expansion Bio-Containers, VueLifeTM bags, KryoSureTM bags, KryoVueTM bags, Lifecell® bags, PermaLifeTM bags, X-FoldTM bags, Si-CultureTM bags, Origen biomedical cryobags, and VectraCellTM bags.
  • cell culture bags comprise one or more of the following characteristics: gas permeability (materials have suitable gas transfer rates for oxygen, carbon dioxide and nitrogen); negligible water loss rates (materials are practically impermeable to water); chemically and biologically inert (materials do not react with the vessel contents), and retention of flexibility' and strength in various conditions (materials enable vessel to be microwaved, treated with UV irradiation, centrifuged, or used within a broad range of temperatures, e.g., from ”100° C to +100° C).
  • Exemplary- large-scale volumes of the cell culture vessel contemplated herein include, without limitation, volumes of about 10 mL, about 25 mb, about 50 ml,, about 75 mb, about 100 mL, about 150 mb, about 250 mb, about 500 mL, about 750 mL, about 1000 mL, about 1250 mL, about 1500 mL, about 1750 mL, about 2000 mL, or more, including any intervening volume.
  • intervening volumes between 10 mL and 25 mb include 11 mL, 12 mb, 13 mL, 14 mL, 15 mb, 16 mL, 17 mb, 18 mb, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, and 24 mb.
  • the volume of the cell culture vessel is from about 100 mb to about 500 L.
  • tire method of manufacturing is prepared in a volume of at least 10L - 500L.
  • the method is prepared in a volume of at least 10L, 20L, SOL, 100L, 250L, or SOOL.
  • Example 1 FKBP-EPOR chimeras place EPOR signaling under the control of AP20187 (BB dimerizer)
  • HSPCs Two days post-editing, we placed HSPCs in differentiation media, which is comprised of three phases of media over the course of 14 days (Dulmovits et al., 2016). Then HSPCs were cultured in RBC media without EPO and split into media with or without BB dimerizer. We hypothesized that RBCs should only be generated in media with BB dimerizer and the vast majority of these RBCs would be YFP+. To determine whether we achieved BB- dependent activation of each receptor, we compared the ability of cells from the same donor that were edited as a single treatment to differentiate into erythroid cells in the presence or absence of BB dimerizer. (FIG. 2).
  • BB dimerizer-driven RBC differentiation in the presence of BB dirnerizer for constructs 1.4 and 1.5, which place the FKBP domain immediately adjacent to the transmembrane domain on the extracellular side of the receptor.
  • About 11-12% of all cells in the samples in the absence of BB dirnerizer are GFP+ at day 14, suggesting that baseline editing has occurred, yet none of these differentiate into RBCs (FIG . 4B).
  • about 19-27% of all cells in samples with BB dirnerizer are GFP+ and nearly 100% of RBCs (CD34- CD45-CD71+ cells) are GFP+ (FIG. 4A).
  • BB dirnerizer can act as a substitute for EPO cytokine and drive RBC differentiation of cells containing chimeric receptors.
  • FIG. 7A shows a representative comparison of original 1 .5 to an IL6-containing version, 2.1 .
  • BB-driven activation of our inducible EPOR cassettes promote differentiation into erythroid cells. This indicates that we have developed a strong, pro-RBC differentiation strategy that is activated by a small molecule, the BB dimerizer, rather than BB dimerizer simply acting as a proliferation switch that is guided toward RBC differentiation by the cytokines included in the RBC differentiation media.
  • these chimeric EPOR monomers may incidentally collide and activate some degree of EPOR signaling even in the absence of BB dimerizer.
  • BB-independent, constitutively active chimeric EPOR has perhaps limited utility, we include the data here to demonstrate how quickly several design-build-test cycles can achieve extremely high-functioning signaling receptors.
  • the resulting cells displayed a dramatic BB dimerizer-dependent increase in the number of CD34-CD45- CD71+ RBCs (compare with and without BB dimerizer, FIGS. 16B and 16C, respectively).
  • Other variations that are likely to work comprise cutting the 3’ UTR and integrating iEPOR or itEPOR there in order to keep endogenous EPOR intact.
  • FIG. 18A shows unedited umbilical cord blood-derived HSPCs from a donor (“CB Mock”) that were differentiated in RBC media with Epo. Fetal and adult hemoglobin are labeled as HbF and HbA, respectively.
  • 18B shows the corresponding HPLC profiles of hemoglobin extracted from cells incubated with (black) or without (gray) BB dimerizer after editing to introduce SSF-IL6- FKBP-EPOR integrated at the CCR5 locus (MCK120), SSFV-SP-FKBP-EPOR integrated at the CCR5 locus (MKC121 ), SSFV-IL6-FKBP-tEPOR integrated at the CCR5 locus (MKC13I), IL6-FKBP-tEPOR integrated at the HBAI locus (MKC135), HBB-IRES-IL6- FKBP-tEPOR integrated at the HBAI locus (MKC144).
  • RNA-Seq was performed to compare transcriptional response to native EPOR+EPO vs. iEPOR+BB. About 18,000 genes are RNA sequenced with assigned expression values shown as normalized read counts in FIG. 19.
  • Native EPOR and iEPOR expression levels are annotated for Mock ‘"unedited” cells at dO and d 14 of RBC diff (Mock dO and Mock dl4+EPO) followed by cells edited with iEPOR version 2.3 expressed by strong-RBC specific promoter HBAI (HBA1 (iEPOR) +BB), endogenous EPOR promoter (EPOR(iEPOR) +BB), and constitutive- expressing hPGK promoter (PGK(iEPOR) +BB), as shown in FIG. 19.
  • HBAI HBA1 (iEPOR) +BB
  • EPOR(iEPOR) +BB endogenous EPOR promoter
  • PGK(iEPOR) +BB constitutive- expressing hPGK promoter
  • PCA Principal Component Analysis
  • Example 4 Engineering complex signaling pathways that are tunable with small molecules
  • HSPC chimerism in the BM must yield equivalent RBC chimerism in the bloodstream.
  • We also believe that our efforts to engineer tunable, inducible elements into living cells will lend an unprecedented level of control to cells post-transplant. These approaches could be immediately paired with current viral-based gene therapies as well as CRISPR-based genome editing strategies to reduce the threshold for curative levels of corrected HSPC chimerism in the BM.
  • our inducible erythroid bias strategies could also be integrated into the allo-HSCT workflow' to yield tunable RBC production from donor- derived HSPCs.
  • a patient’s own HSPC or an HSPC from an allogeneic donor could be edited to correct an allele that causes a hemoglobinopathy such as beta-thalassemia or sickle cell anemia as well as to express a chimeric EPO receptor that is inducible by an orally available or locally administered small molecule, so edited HSPC could then be engrafted into the patient’s bone marrow and, upon exposure to the small molecule, be allowed to preferentially expand and differentiate into a population of edited erythroid cells, thereby treating the hemoglobinopathy.
  • FIG. 25B Top panels of FIG. 26 show the data in FIG. 25B split up between -EPO and +EPO conditions.
  • iEPOR-edited cells +BB are able to effectively differentiate and acquire RBC markers (FIG. 26).
  • each dish was transfected with a standard polyetbylenimine (PEI) transfection of 6pg ITR-containing plasmid and 22p.g pDGM6, which contains the AAV6 cap genes, AAV2 rep genes, and Ad5 helper genes.
  • PEI polyetbylenimine
  • cells were purified using AAVPro Purification Kits (All Serotypes)(Takara Bio USA, Mountain View, CA, USA) as per manufacturer’s instructions.
  • AAV6 vectors were titered using ddPCR to measure number of vector genomes per ⁇ L as previously described 2 .
  • CD34 + HSPCs were sourced from fresh cord blood, frozen cord blood, and Plerixafor- and/or G-CSF-mobilized peripheral blood (AllCells, Alameda, CA, USA and STEMCELL Technologies, Vancouver, Canada).
  • CD34 + HSPCs were cultured at 1x 10 s cells/mL in StemSpan SFEM II (STEMCELL Technologies, Vancouver, Canada) base medium supplemented with stem cell factor (SCF)( lOOng/mL), thrombopoietin (TPO)( lOOng/mL), FLT3-ligand (lOOng/mL), IL-6 (lOOng/mL), UMI71 (35nM), streptomycin (20mg/mL), and penicillin (20U/mL).
  • SCF stem cell factor
  • TPO thrombopoietin
  • FLT3-ligand lOOng/mL
  • IL-6 lOOng/mL
  • UMI71 35nM
  • Chemically modified Cas9 sgRNAs were purchased from Synthego (Menlo Park, CA, USA) and TriLink BioTechnologies (San Diego, CA, USA) and were purified by high- performance liquid chromatography (HPLC). Tire sgRNA modifications added were the 2'-O- metliyl-3'-phosphorothioate at the three terminal nucleotides of the 5' and 3' ends described previously 9 .
  • the target sequences for human sgRNAs were as follows: HBAlsg'.
  • CD34 + HSPCs were harvested at day 5 and erythrocytes at day 16 post-targeting. Cells were analyzed for viability using ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA, USA) and reporter expression was assessed using the FACS Aria II system (BD Biosciences, San Jose, CA, USA). The data was subsequently analyzed using FlowJo (FlowJo LLC, Ashland, OR, USA). In vitro differentiation of CD 34* HSPCs into erythrocytes
  • HSPCs derived from healthy donors or polycythemia vera patients were cultured for 14-16 days at 37°C and 5% CO2 in SFEM II medium (STEMCELL Technologies, Vancouver, Canada).
  • SFEMII base medium was supplemented with lOOU/mL penicillin-streptomycin, lOng/mL SCF, Ing/mL IL-3 (PeproTech, Rocky Hill, NJ, USA), 3U/mL erythropoietin (eBiosciences, San Diego, CA, USA), 200pg/mL transferrin (Sigma- Aldrich, St.
  • days 0-7 day 0 being 2 days post-targeting
  • days 7- days were maintained at 1 x 10 5 cells/mL
  • IL-3 was removed from the culture.
  • days 1 1—16 days were cultured at GIO 6 cells/mL, and transferrin was increased to I mg/mL within the culture medium.
  • HSPCs subjected to the above erythrocyte differentiation were analyzed at day 14 for erythrocyte lineage -specific markers using a FACS Ana II (BD Biosciences, San Jose, CA, USA). Edited and non-edited cells were analyzed by flow cytometry' using the following antibodies: 11CD45 V450 (HI30; BD Biosciences, San Jose, CA, USA), CD34 APC (561; BioLegend, San Diego, CA, USA), CD71 PE-Cy7 (OKT9; Asymetrix, Santa Clara, CA, USA), and CD235a PE (GPA)(GA-R2; BD Biosciences, San Jose, CA, USA).
  • HSPCs were harvested and QuickExtract DNA extraction solution (Epicentre, Madison, WI, USA) was used to collect gDN A. Primers were then used to amplify the region surrounding the predicted cut site and/or deletion. PCR reactions were then run on a 1% agarose gel and appropriate bands were cut and gel-extracted using a GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions. Gel-extracted amplicons were then Sanger sequenced and resulting chromatograms were used as input for indel frequency analysis by TIDE as previously described 13 . Allelic targeting analysis by ddPCR
  • HSPCs were harvested and QuickExtract DNA extraction solution (Epicentre, Madison, WI, USA) was used to collect gDNA.
  • gDNA was then digested using BamHI-HF as per manufacturer’s instructions (New England Biolabs. Ipswich, MA, USA).
  • BamHI-HF as per manufacturer’s instructions
  • the percentage of targeted alleles within a cell population was measured by ddPCR using the following reaction mixture: l-4pL of digested gDNA input, lOpL ddPCR SuperMix for Probes (No dUTP)(Bio-Rad, Hercules, CA, USA), primer/probes (1:3.6 ratio: Integrated DNA Technologies, Coralville, Iowa, USA), volume up to 20uL with H 2 O.
  • ddPCR droplet were then generated following the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA): 20pL of ddPCR reaction, 70 ⁇ L droplet generation oil, and dOgL of droplet sample.
  • Thermocycler (Bio-Rad, Hercules, CA, USA) settings were as follows: 1. 98°C (lOmin), 2. 94°C (30s), 3. 57.3°C (30s), 4. 72°C (I.75min)(retum to step 2 x 40-50 cycles), 5. 98°C (10 min).
  • Analysis of droplet samples was done using the QX200 Droplet Digital PCR System (Bio-Rad, Hercules, CA, USA). To determine percentage of alleles targeted, the number of Poisson-corrected integrant copies/mL were divided by the number of Poisson-corrected reference DNA copies/mL. mRNA analysis
  • RNA input levels of the RBC-specific reference gene GPA was determined in each sample using the following primers and HEX/ZEN/BBFQ- labelled hydrolysis probes purchased as custom-designed PrirneTime qPCR Assays from Integrated DNA Technologies (Coralvilla, IA, USA): forward: 5'- ATATGC AGC CACTC CTAG A GCTC - 3 ’ , reverse : 5 ’ -
  • HSPCs subjected to erythrocyte differentiation were lysed using water equivalent to three volumes of pelleted cells, fire mixture was incubated at room temperature for 15 mm, followed by 30s sonication .
  • centrifugation was performed at 13,000 RPM for 5min.
  • HPLC analysis of hemoglobins in their native form were analyzed on a weak cation-exchange PolyCAT A column (100 x 4.6-mm, 3pm, 1 ,000A) (PolyLC Inc., Columbia, AID, USA) using a Shimadzu UFLC system at room temperature.
  • Mobile phase A (MPA) consists of 20mM Bis-tris +2mM KCN, pH 6.96.
  • MPB Mobile phase B
  • Clear hemolysate was diluted four times in MPA, and then 20pL was injected onto the column.
  • a flow rate of 1.5mL/min and the following gradients were used in time (min)/%B organic solvent: (0/10%; 8/40%; 17/90%; 20/10%; 30/stop).
  • Embodiment 1 A chimeric transmembrane receptor polypeptide comprising: an extramembrane dimerizer domain, wherein the extramembrane dimerizer domain induces dimerization of the chimeric transmembrane receptor polypeptide upon recognition of a dimerization signal; a transmembrane domain; and an intramembrane domain, wherein the intramembrane domain is configured to induce activation of one or more intramembrane signal pathways upon dimerization of the chimeric transmembrane receptor polypeptide in a modified primary human cell comprising the chimeric transmembrane receptor polypeptide, and wherein the one or more intramembrane signaling pathways promote survival, proliferation, and/or differentiation of the modified primary' human cell.
  • Embodiment 2 The chimeric transmembrane receptor polypeptide of embodiment 1, wherein the extramembrane dimerizer domain comprises an FKBP domain, an mFRB domain, an HSV-TK dimerization domain, a rapamycin-inducible dimerization domain, a rapalogue- inducible dimeri zation domain, or a combination thereof.
  • Embodiment 3 The chimeric transmembrane receptor polypeptide of embodiment 1 or 2, wherein the dimerization signal is a pharmaceutically acceptable small molecule dimerization signal.
  • Embodiment 4 The chimeric transmembrane receptor polypeptide of embodiment 3, wherein the extramem brane dimerizer domain comprises an FKBP domain and wherein the small molecule dimerization signal comprises AP20187.
  • Embodiment 5 The chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 4, wherein the intramembrane domain comprises an EPOR intracellular domain, a c-KIT/stem cell factor (SCF) receptor intracellular domain, a thrombopoietin receptor (TPOR) intracellular domain, an epidermal growth factor (EGFR) intracellular domain, , an RET intracellular domain, a CSF1 R intracellular domain, an IGF1R intracellular domain, or a combination thereof.
  • SCF c-KIT/stem cell factor
  • TPOR thrombopoietin receptor
  • EGFR epidermal growth factor
  • RET RET intracellular domain
  • CSF1 R intracellular domain
  • IGF1R intracellular domain
  • Embodiment 6 the chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 5, wherein the intramembrane domain comprises an EPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary human cell upon recognition of the dimerization signal.
  • Embodiment 7 The chimeric transmembrane receptor polypeptide of any 7 one of embodiments 1 to 5, wherein the intramembrane domain comprises a TPOR intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary- human cell upon recognition of the dimerization signal.
  • Embodiment 8 The chimeric transmembrane receptor polypeptide of any- one of embodiments 1 to 7, wherein the intramembrane domain comprises an SCF intracellular domain and wherein dimerization of the chimeric transmembrane receptor polypeptide induces activation of intramembrane signal pathways resulting in increased survival, increased proliferation, and/or increased erythroid differentiation of the modified primary- human cell upon recognition of the dimerization signal.
  • Embodiment 9 Tire chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 8, wherein the extramembrane dimerizer domain is immediately adjacent to the transmembrane domain.
  • Embodiment 10 The chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 9, wherein the chimeric transmembrane receptor polypeptide further comprises a signal peptide.
  • Embodiment 11 The chimeric transmembrane receptor polypeptide of embodiment
  • Embodiment 12 The chimeric transmembrane receptor polypeptide of embodiment
  • the signal peptide comprises an IL6 signal peptide, an EPOR signal peptide, a lysozyme C signal peptide, an angiotensinogen signal peptide, an RNASE 1 signal peptide, an RNASE3 signal peptide, or a modified human albumin signal peptide.
  • Embodiment 13 The chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 12, wherein tire chimeric transmembrane receptor polypeptide further comprises a linker peptide.
  • Embodiment 14 The chimeric transmembrane receptor polypeptide of embodiment
  • linker peptide comprises the amino acid sequence GGGGS.
  • Embodiment 15 The chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 14, wherein the chimeric transmembrane receptor polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOS: 1 to 3 and 7 to
  • Embodiment 16 A recombinant nucleic acid encoding the chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 15.
  • Embodiment 17 A DNA construct comprising a promoter operably linked to the recombinant nucleic acid of embodiment 16.
  • Embodiment 18 The DNA construct of embodiment 17, wherein the promoter is an endogenous EPOR promoter, an endogenous HBA1 promoter, an endogenous TPOR promoter, a constitutive SFFV promoter, a constitutive PGK promoter, or a constitutive UbC promoter.
  • Embodiment 19 A vector comprising the recombinant nucleic acid of embodiment 16 or the DNA construct of embodiment 17 or 18.
  • Embodiment 20 A host ceil comprising the recombinant nucleic add of embodiment 16, the DNA construct of embodiment 17 or 18, or the vector of embodiment 19.
  • Embodiment 21 The host cell of embodiment 20, wherein the recombinant nucleic acid, DNA construct, or vector is integrated into the CCR5 locus, the HBA 1 locus, or the EPOR locus.
  • Embodiment 22 The host cell of embodiment 20, wherein the host cell is a eukaryotic cell.
  • Embodiment 23 The host cell of embodiment 20 or 21 , wherein the host cell is a primary’ human cell.
  • Embodiment 24 The host cell of any one of embodiments 20 to 23, wherein the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC).
  • the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC).
  • PSC pluripotent stem cell
  • Embodiment 25 The host cell of any one of embodiments 20 to 24, wherein the host cell is a hematopoietic stem and progenitor cell (HSPC).
  • HSPC hematopoietic stem and progenitor cell
  • Embodiment 26 The host cell of any one of embodiments 20 to 25, wherein the host cell was derived from a patient who is a carrier of an allele that causes a genetic disorder.
  • Embodiment 27 The host cell of embodiment 26, wherein the genetic disorder is beta-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1 , Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • the genetic disorder is beta-thalassemia, sickle cell disease (SCD), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1 , Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • Embodiment 28 The host, cell of embodiment 26 or 27, wherein the genetic disorder is a hemoglobinopathy.
  • Embodiment 2.9 The host cell of embodiment 28, wherein the hemoglobinopathy is beta-thalassemia or sickle cell disease.
  • Embodiment 30 The host cell of any one of embodiments 25 to 29, wherein the genome of the host cell is edited to alter the allele associated with the genetic disorder.
  • Embodiment 31 A method of inducing erythroid differentiation of an HSPC, the method comprising contacting the HSPC of embodiment 25 with the dimerization signal.
  • Embodiment 32 A method of tuning red blood cell levels in a patient, the method comprising:
  • Embodiment 33 A method of increasing the proportion of red blood cells with an altered version of an allele associated with a genetic disorder, the method comprising:
  • Embodiment 34 The method of embodiment 32 or 33, wherein the HSPC is derived from the patient’s own cells.
  • Embodiment 35 The method of embodiment 32 or 33, wherein the HSPC is derived from an allogeneic donor's cells.
  • Embodiment 36 The method of embodiment 35, wherein the genetic disorder is beta- thalassemia, sickle cell disease (SCO), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • Embodiment 37 A method of genetically modifying a primary human cell, the method comprising introducing into the cell a chimeric transmembrane receptor polypeptide of any one of embodiments 1 to 15.
  • Embodiment 38 The method of embodiment 37, the method further comprising:
  • SDN site-directed nuclease
  • homologous repair template comprises a nucleotide sequence that is homologous to the locus of interest, wherein the site-directed nuclease cleaves the locus at the cleavage site, and the homologous repair template is integrated at the site of the cleaved locus by homology directed repair (HDR),
  • HDR homology directed repair
  • Embodiment 39 The method of embodiment 38, wherein the SDN is an RNA-guided nuclease and the method further comprises introducing into the cell a single guide RNA (sgRNA) targeting the cleavage site, wherein the sgRNA directs the RNA-guided nuclease to the cleavage site.
  • sgRNA single guide RNA
  • Embodiment 40 The method of embodiment 39, wherein the sgRNA comprises 2'- O-methyl-3'-phosphorothioate (MS) modifications at one or more nucleotides.
  • MS 2'- O-methyl-3'-phosphorothioate
  • Embodiment 41 The method of embodiment 40, wherein the MS modifications are present at the terminal nucleotides of the 5' and 3’ ends.
  • Embodiment 42 The method of any one of embodiments 39 to 41, wherein the RNA- guided nuclease is Cas9.
  • Embodiment 43 The method of any one of embodiments 39 to 42, wherein the sgRNA and RNA-guided nuclease are introduced into the cell as a ribonucleoprotein (RNP).
  • RNP ribonucleoprotein
  • Embodiment 44 The method of embodiment 43, wherein the RNP is introduced into the cell by electroporation.
  • Embodiment 45 The method of any one of embodiments 38 to 44, wherein the homologous repair template is introduced into the cell using an adeno-associated virus serotype 6 (AAV6) vector.
  • AAV6 adeno-associated virus serotype 6
  • Embodiment 46 The method of any one of embodiments 38 to 45, wherein the primary- human cell is a hematopoietic stem cell, a hematopoietic progenitor ceil, a T cell, a B cell, an airway basal stem cell, or a pluripotent stem cell (PSC).
  • AAV6 adeno-associated virus serotype 6
  • Embodiment 47 The method of any one of embodiments 38 to 46, wherein the locus of interest is a gene selected from the group consisting of Erythropoietin Receptor (EPOR), Hemoglobin Subunit Beta (HBB), C-C Motif Chemokine Receptor 5 (CCR5), Interleukin 2 Receptor Subunit Gamma (IL2RG), Hemoglobin Subunit Alpha 1 (HBA1), Stimulator Of Interferon Response cGAMP Interactor 1 (STING1) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
  • EPOR Erythropoietin Receptor
  • HBB Hemoglobin Subunit Beta
  • CCR5 C-C Motif Chemokine Receptor 5
  • IL2RG Interleukin 2 Receptor Subunit Gamma
  • HBA1 Stimulator Of Interferon Response cGAMP Interactor 1
  • STING1 Stimulator
  • Embodiment 48 A method of treating a genetic disorder in a human subject in need thereof, the method comprising:
  • Embodiment 49 The method of embodiment 48, wherein tire genetic disorder is beta- thalassemia, sickle cell disease (SCO), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • tire genetic disorder is beta- thalassemia, sickle cell disease (SCO), severe combined immunodeficiency (SCID), mucopolysaccharidosis type 1, Cystic Fibrosis, Gaucher disease, Krabbe disease, X-linked chronic granulomatous disease (X-CGD), or a combination thereof.
  • Embodiment 50 A method of generating a population of red blood cell in vitro, the method comprising:
  • Embodiment 51 The method of embodiment 50, wherein tire dimerization signal is a small molecule dimerization signal.
  • Embodiment 52 The method of embodiment 51 , wherein the small molecule dimerization signal is AP20187 (BB dimerizer).
  • Embodiment 53 The method of any one of embodiments 50 to 52, wherein step (ii) further comprises contacting the one or more HSPCs with erythropoietin (EPO).
  • EPO erythropoietin
  • Embodiment 54 Tire method of any one of embodiments 50 to 53, wherein the method is conducted in a bioreactor.

Abstract

La présente divulgation concerne des procédés et des compositions pour la différenciation accordable de cellules souches et progénitrices hématopoïétiques (HSPC) en réponse à de petites molécules à l'aide de HSPC génétiquement modifiées qui expriment des récepteurs transmembranaires chimériques.
PCT/US2023/076969 2022-10-17 2023-10-16 Enrichissement de types de cellules cliniquement pertinents à l'aide de récepteurs WO2024086518A2 (fr)

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