Modified CD90 expression in therapeutic hematopoietic stem and progenitor cells and uses thereof

Abstract

Modified CD90 expression in therapeutic hematopoietic stem and progenitor cells (HSPCs) and uses thereof are described. CD90 expression is modified in therapeutic HSPCs to protect the therapeutic HSPCs from CD90-targeted therapeutics, for example, in the treatment of a blood cancer. HSPCs can additionally be modified to correct a genetic defect, such as beta globin in sickle cell disease.

Description

F053-0197PCT / 24-151 -WO-PCTMODIFIED CD90 EXPRESSION IN THERAPEUTIC HEMATOPOIETIC STEM AND PROGENITOR CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application No. 63 / 746,059 filed on January 16, 2025, the entire contents of which are incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under HL136135 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The current disclosure describes modified CD90 expression in therapeutic hematopoietic stem and progenitor cells (HSPCs) and uses thereof. The CD90 expression is modified in therapeutic HSPCs to protect the therapeutic HSPCs from CD90-targeted therapeutics. Therapeutic HSPCs can also be modified to correct a genetic defect, such as beta globin in sickle cell disease. After the modified therapeutic HSPCs have been administered to a subject, the subject can be administered a CD90-targeted therapeutic to kill HSPCs presenting native, but not modified, CD90.BACKGROUND OF THE DISCLOSURE

[0004] Hematopoietic cell transplantation (HOT) is a treatment for a variety of disorders including cancer, hemoglobinopathies, and primary immune deficiencies. HOT includes infusing healthy hematopoietic stem and progenitor cells (HSPCs) into a patient whose bone marrow or immune system is damaged or defective. Particularly, HCT is used to treat malignant and nonmalignant diseases that affect the hematopoietic system, including leukemia, lymphoma, myeloma, aplastic anemia, thalassemia, sickle cell anemia, and severe combined immunodeficiency. HCT is usually accompanied by a preparative or conditioning regimen. A conditioning regimen may include chemotherapy, monoclonal antibody therapy, and radiation to the entire body. Unfortunately, condition regimens can have toxic effects and thus limit the use of HCT.SUMMARY OF THE DISCLOSURE

[0005] A small subset of CD34+ hematopoietic stem and progenitor cells (HSPCs) express CD90 and these cells are responsible for short- and long-term engraftment of immune cells following HCT. While CD90 expression is dispensable in established adult HSPCs, its expression is essential in embryonic blood development.

[0006] The present disclosure describes modified CD90 expression in therapeutic HSPCs and uses thereof. The CD90 expression is modified in therapeutic HSPCs to protect the modified HSPCs from CD90-targeted therapeutics. In particular embodiments, HSPCs are modified to correct a genetic defect, such as beta globin in sickle cell disease. Those same HSPCs can be modified to knock down, knock out, or modify CD90 to remove or reduce CD90 binding with a CD90-targeted therapeutic. After the modified HSPCs have been administered to a subject, the subject can be administered a CD90-targeted therapeutic which will kill HSPCs presenting native CD90 but leave genetically modifiedF053-0197PCT / 24-151 -WO-PCTtherapeutic HSPCs unaffected.

[0007] In particular embodiments, therapeutic HSPCs are genetically modified to have modified expression of CD90 and to express a therapeutic payload. In particular embodiments, modified expression of CD90 includes a knock down of CD90 expression, a knockout of CD90 expression, or a modification of CD90 such that a CD90-targeted therapeutic does not bind the modified CD90.

[0008] In particular embodiments, the knock down of CD90 expression includes introducing anti-CD90 short hairpin RNA (shRNA). In particular embodiments, the shRNA includes the sequence set forth in SEQ ID NO: 4.

[0009] In particular embodiments, the knockout of CD90 expression includes introducing guide RNA (gRNA) and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 5 and the gene editing component includes a nuclease such as Cas9 or Cpf1. In particular embodiments, the gRNA includes the sequence set forth in SEQ ID NO: 1 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in human cells. In particular embodiments, the gRNA includes the sequence set forth in SEQ ID NO: 2 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in Rhesus macaque cells.

[0010] In particular embodiments, the modification of CD90 includes a modification such that the 5E10 antibody does not bind the modified CD90. In particular embodiments, this modification of CD90 such that the 5E10 antibody does not bind the modified CD90 includes introducing a gRNA and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 6 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in humans or Rhesus macaque cells.

[0011] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position F80. In particular embodiments, the mutation at position F80 is an F80P or F80S mutation. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody binding to CD90 includes a mutation at position F80. In particular embodiments, the mutation at position F80 is an F80P or F80S mutation.

[0012] In particular embodiments, the modification of CD90 includes a modification such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90. In particular embodiments, this modification of CD90 such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90 includes introducing a gRNA and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 7 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in humans cells.

[0013] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position D98. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90 includes a mutation at position D98. In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position E99. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90 includes a mutation at position E99. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90F053-0197PCT / 24-151 -WO-PCTincludes a mutation at position D98 and E99.

[0014] In particular embodiments, the therapeutic payload includes one or more of globin family genes (e.g., of γ-globin, β-globin, and / or α-globin); yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40 (e.g., soluble CD40); CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; coagulation factor genes (factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII)); CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancG (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; interleukins (IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, IL7RA); antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; and ZAP70.

[0015] Particular embodiments include administering genetically modified therapeutic HSPCs to a subject. After administration, HSPCs expressing native CD90 can be killed by administering a CD90-targeted therapeutic. In particular embodiments, the CD90-targeted therapeutic includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor (e.g., anti-CD90 chimeric antigen receptor (CAR)). In particular embodiments, an anti-CD90 CAR includes a binding domain having a variable heavy chain set forth in SEQ ID NO: 19 and a variable light chain set forth in SEQ ID NO: 21. In particular embodiments, an anti-CD90 CAR includes a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.BRIEF DESCRIPTION OF THE FIGURES

[0016] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0017] FIGs. 1 A-1 D. CD90 ablation by short hairpin (sh)RNA and adenine base editing (ABE) do not adversely impact engraftment potential of postnatal and adult hematopoietic stem cells (HSCs). (1A) Experimental schematic for the ex vivo transduction of human umbilical cord blood-derived CD34+HSPCs, intrahepatic (IH) injection 50,000 cells intoF053-0197PCT / 24-151 -WO-PCTneonatal NBSGW mice, and longitudinal / terminal assessment of engraftment in blood and tissues. Mice were transplanted with either scramble-shRNA (circle) or CD90-shRNA (square) transduced human CD34+HSPCs. (1B) Longitudinal flow-cytometric tracking of mCherry expression within total human white blood cells (WBCs) (huCD45+), B cells (CD19 / 20*), and monocytes (CD14+) in the peripheral blood (PB). (1C) Representative flow-cytometric assessment of mScarlet expression in human WBCs isolated from the bone marrow. (1D) Frequency of mScarlet+cells within phenotypically defined human HSPC subsets engrafted in the murine bone marrow (BM) assessed by flowcytometry.

[0018] FIGs. 2A-2E. CD90-KD in cell lines and human HSPCs engrafted into NBSGW mice. (2A) Comparison of three anti-CD90 shRNAs for the knock down of CD90 in K562 cells. K562 cells were transduced with scramble or shRNA encoding lentiviral vectors and expression of mCherry as well as downregulation of CD90 on the cell surface measured by flow-cytometry on day 5 post transduction. (2B) Longitudinal flow-cytometric tracking of total human chimerism (huCD45+), phenotypic human monocytes (CD14+of huCD45+), granulocytes (CD15+of huCD45+), B cells (CD 19 / 20* of huCD45j, T cells (CD3+of huCD45+), and NK cells (CD 16 / 56* of huCD45j in the PB. (2C) Longitudinal flow-cytometric tracking of mCherry expression within T cells (CD3*), NK cells (CD16 / 56*) and granulocytes (CD15+) in the PB complementing graphs in FIG. 1B. (2D) Frequency of total human chimerism (huCD45+) and phenotypically defined human HSPC subsets engrafted in the murine BM assessed by flow-cytometry. (2E) Testing of sgRNAs for the knockout of CD90 with and without HBG-175 editing in human and nonhuman primate primary CD34+HSPCs and cell lines. sgRNA were electroporated into cells and the allelic base editing efficiency determined by EditR analysis of Sanger sequencing.

[0019] FIGs. 3A-3D. CD90 ablation by shRNA and ABE do not adversely impact engraftment potential of postnatal and adult HSCs. (3A) Protospacer and PAM design for ablation of CD90 expression through ABE-mediated mutation of the start codon. (3B) Assessment of allelic based editing efficiencies at the on-target adenine A6 (black) and bystander positions A2 / A3 (white) and A9 / 10 (dashed) in human G-CSF-mobilized CD34+HSPCs from two independent healthy donors using EditR on Sanger sequencing data. (3C) Experimental schematic for the ex vivo editing of human G-CSF-mobilized CD34+HSPCs, intravenous / retroorbital (IV) injection of 200,000 cells into 8-12-week-old adult NBSGW mice, and longitudinal / terminal assessment of engraftment in blood and tissues. Mice were transplanted with either mock electroporated (Ctr, grey) or CD90-KO HSPCs. (3D) CD90-KO in cell lines and human HSPCs engrafted into NBSGW mice. Longitudinal flow-cytometric tracking of phenotypic human granulocytes (CD15+of huCD45*), T cells (CD3+of huCD45*), and NK cells (CD 16 / 56* of huCD45+).

[0020] FIGs. 4A-4M. Multilineage long-term engraftment of CD90-KC HSCs in nonhuman primate (NHP). (4A) Assessment of allelic based editing efficiencies at the on-target adenine A6 (black) and bystander positions A2 / A3 (white) and A9 / 10 (dashed) in steady-state BM CD34+HSPCs from two independent healthy NHP donors using EditR on Sanger sequencing data. (4B) Experimental design for the autologous transplantation of ex vivo gene-modified CD90-KC HSCs into busulfan-conditioned NHPs. Base editor mRNA and sgRNAs for CD90 and HBG were deliveredF053-0197PCT / 24-151 -WO-PCTvia electroporation and gene-edited cells frozen. 72 hours after busulfan conditioning, cryopreserved cells were thawed at bedside and infused intravenously (IV). Animals stayed on long-term follow-up monitoring of CBC counts and PB and BM assessments using flow-cytometry and genomics. (4C) Representative flow-cytometric assessment of CD34+HSPCs for the expression of CD90 and CD45RA post-MACS enrichment (24 hours before electroporation) and 24 hours post-editing for both animals. (4D) Summary of animal and infusion product characteristics. Total cell numbers per kg body weight were determined by combining infusion product cell counts, flow-cytometric data, and the animal’s weight at time of infusion. Allelic base editing efficiencies were determined on day 5 post-editing using EditR on Sanger sequencing data. (4E) Pharmacokinetic analysis of busulfan metabolism after administration in both RM measured in the serum. AUC: Area under the curve. (4F) Neutrophil and platelet counts in the PB determined on an automated CBC counter. Dashed horizontal lines represent threshold(s) for neutrophil (>500 neutrophils / pil) and platelet (lower line: 25K platelets / pil; upper line: >50K platelets / pil) recovery. Grey zones represent the normal range in healthy NHPs. (4G) Phenotypic composition of the PB over time assessed by flow-cytometry. (4H) (top plots) Representative flowcytometric assessment of CD90 expression on total WBCs, granulocytes, and monocytes, (lower two graphs) Frequency of CD90- granulocytes and monocytes over time assessed by flow-cytometry. (41) (top plots) Representative flow cytometric data showing the reactivation of HbF expression in red blood cells (RBCs) in the PB. (bottom graph) Longitudinal tracking of the HbF expression in RBCs. (4J) (top two graphs) Longitudinal assessment of allelic base editing efficiencies at the CD90 and HBG1 / 2 loci in PB WBCs using next-generation sequencing (NGS). (bottom two graphs) Allelic base editing efficiencies at the CD90 and HBG1 / 2 loci in FACS-purified lineages determined by NGS. (4K) Flow-cytometric assessment of BM WBCs from A240483 months-post-transplant and quantification of phenotypic HSPC subsets. (4L) (left graph) Percentage colony formation (number of colonies / number of seeded cells) of FACS-purified HSPC subsets from the BM of A24048 3 months-post-transplant. Abbreviations: CFU = colonyforming unit; G = granulocyte; M = monocyte / macrophage; BFU = burst forming unit; E = erythrocyte; MIX = myeloid + erythroid, (right graph) Total number of cells grown in secondary CFC assays after replating 5% of cells from primary CFC assays. (4M) Allelic base editing efficiencies at the CD90 and HBG1 / 2 loci in FACS-purified BM HSPC subsets determined by NGS.

[0021] FIGs. 5A-5E. Quality control of gene-edited NHP HSCs before infusion and long-term follow-up of animals after transplantation in NHPs. (5A) Percentage colony formation (number of colonies I number of seeded cells) of FACS-purified HSPC subsets from the infusion product for both animals. Abbreviations: CFU = colony-forming unit; G = granulocyte; M = monocyte / macrophage; BFU = burst forming unit; E = erythrocyte; MIX = myeloid + erythroid. (5B) Automated complete blood cell counts in the PB over time. Grey zones represent the normal range in healthy NHPs. (5C) Representative flow-cytometric gating of hematopoietic subsets based on cell surface marker expression in the PB (top row). CD90 expression on phenotypic subsets before editing (middle row) and after editing (bottom row) in A23048. (5D) Flow-cytometric assessment of BM WBCs from A23150 at necropsy one-month post-transplant and quantification of phenotypic HSPC subsets. (5E) (left graph) Percentage colony formation (number of colonies / numberF053-0197PCT / 24-151 -WO-PCTof seeded cells) of FACS-purified HSPC subsets from the BM of A23150 at necropsy one-month post-transplant. Abbreviations: CFU = colony-forming unit; G = granulocyte; M = monocyte / macrophage; BFU = burst forming unit; E = erythrocyte; MIX = myeloid + erythroid, (right graph) Total number of cells grown in secondary CFC assays after replating 5% of cells from primary CFC assays.

[0022] FIG. 6. Representative sequencing analysis of Rhesus Macaque cells transfected with ABE8E and gRNA targeted to Rhesus Thy 1 exon2 locus (ATG20-R, see SEQ ID NO: 2). Asterisk denotes intended site of base edit.

[0023] FIGs. 7A-7D. A virus-like particle (VLP) screen identified an epitope that is required for binding the CD905E10 mAb (7 A) Protospacer sequences targeting exon 3 of Thy1 (expresses the extracellular domain) were cloned into a guide RNA expression library and used for producing a VLP adenine base editing screen library. CD90-expressing jurkat cells were transduced with the library and the cells that lost 5E10 clone binding were sorted. (7B) The population that were negative for 5E10 mAb staining were sorted, expanded, and sorted again. (7C) Sanger sequencing reveals that the cells sorted in (7B) had a single amino acid change: F80P. Portion of wildtype CD90 protein (SEQ ID NO: 153) and coding sequence (SEQ ID NO: 41) and portion of 5E10 antibody protein (SEQ ID NO: 154) and coding sequence (SEQ ID NO: 42). (7D) The level of loss of binding to 5E10 clone corresponds to the editing efficiency

[0024] FIGs. 8A-8C. F80 residue is located at the interaction site between the 5E10 clone scFv and CD90 membrane glycoprotein. (8A) CD90 amino acid sequence. The membrane glycoprotein is underlined and the F80 residue is underlined and in bold. (8B) The quaternary interface of the 5E10 clone-CD90 membrane protein complex. The F80 residue is circled in zoom windows. (8C) Cryo-EM structure of the 5E10 scFv and CD90 membrane glycoprotein and the 5E10 scFv and CD90 membrane glycoprotein quaternary structure fitted into the cryo-EM model.

[0025] FIGs. 9A, 9B. The F80P mutation confers protection against the CD90-directed CAR T cells. (9A) Schematic of CAR T cell targeting WT CD90 surface protein. Jurkat cells with WT CD90 expression are susceptible whereas jurkat cells edited to carry the F80P mutation are protected. (9B) Percent survival over time of the WT and F80P jurkat cells against CAR T cells targeting CD90. WT cells were almost completely eliminated after 72 hours. F80P cells were protected in a competitive assay where both WT and F80P cells were plated in the same well.

[0026] FIGs. 10A-10C. (10A) F80P GFP and (10B) F80P mScarlet. (10C) Estimation plot: Unpaired t-test.

[0027] FIG. 11. Editing with different guide RNAs.

[0028] FIG. 12. Editing with Guide 65 leads to two distinct 5E10-negative population suggestive of mono- and bi-allelic edits.

[0029] FIGs. 13A-13F. Human CD34+ HSPCs were electroporated ex vivo to edit the F80P epitope of CD90. Gene edited cells were then injected into NBSGW mice to monitor engraftment and multilineage differentiation in comparison to unmodified / mock cells. (13A) Depicts and experimental schematic used herein. (13B) Depicts allele frequencies at different time points. (13C) Depicts the human chimerism and multilineage potentials. Mock and edited cells demonstrated comparable engraftment potential and multilineage output and persisted long-term in blood, bone marrow (BM), and the spleen. (13D) Epitope editing and engraftment of NHP HSCs. A rhesus macaque wasF053-0197PCT / 24-151 -WO-PCTtransplanted with F80P-edited CD34+ HSPCs after busulfan conditioning and monitored over time for engraftment and recovery. CD90-F80P editing did not show any impact on neutrophil and platelet recovery (first two graphs, complete cell counts). Ablation of the epitope was visible by flow-cytometry on myeloid CD11b+ cells (flow plot). Epitope edited cells lost the ability to bind the antibody clone 5E10, whereas a second antibody recognizing an unedited epitope was able to label cells confirming presentation of the protein on the cell surface. Gene-editing persisted in WBCs over time as determined by NGS (right plot). (13E) Bio-layer interferometry (BLI) response curves across the indicated analyte concentrations (inset at right) for CD90wtanalytes binding to probe-captured 5E10 (top) or MAB20671 (bottom) antibodies; mutant CD90N60Qanalytes binding to 5E10 (top) or MAB20671 (bottom) antibodies; and mutant CD90F61Sanalytes binding to 5E10 (top) or MAB20671 (bottom) antibodies. Red lines show the global kinetic fits using a 1:1 binding model and the fitting R2 is indicated. (13F) CD90 antibody-drug conjugate (ADC). Nonhuman primate CD34+cells were isolated from an animal previously transplanted with F80P-ed ited HSPCs. CD34+cells were cultured ex vivo for 24 hours and exposed to either toxin alone or the CD90-5E10-toxin ADC. The number of viable CD90+cells was assessed using flow-cytometry after 72 hours. As expected, toxin alone did not cause any effect on either unedited wildtype cells nor the F80P-edited CD90 cells (eCD90). However, ADC treatment did lead to selective killing of nonprotected wild type cells, whereas edited CD90 cells were protected and enriched.

[0030] FIGs. 14A-14G. In vitro validation of CAR90. (14A) Backbone structure of a CD90 CAR (top). The aCD90 scFv was designed in four variants using either a Whitlow (CAR90.1 and CAR90.2) or G4S (CAR90.3 and CAR90.4) linker in between the variant heavy (VH) and variant light (VL) chains. VH-Linker-VL was used for CAR90.1 and CAR90.3, whereas VL-Linker-VH was used for CAR90.2 and CAR90.4. (14B) Representative, indirect flow-cytometric assessment of CAR expression on human CD3+T cells detecting either the co-expressed truncated EGFR (tEGFR, left plot) or staining the Whitlow / G4S linker (right plot). (14C) Assessment of function and on- / off-target specificity for all four CD90CAR constructs carrying out killing assays in the Incucyte imaging system. CD90CAR T cells and GFP+on-target (CD90+, top row) or off-target (CD90-, bottom row) HEK293T cells were mixed at different effector to target (E: T) ratios (see pattern of lines in legend) and outgrowth of HEK293T cells measured based on the intensity of GFP signal over time. All plotted datapoints are relative values normalized to the GFP intensity at the start of the experiments. Lines representative of technical triplicates. (14D) Assessment of CAR-mediated cytokine production in response to the co-culture with human G-CSF-mobilized CD34+HSCPs using a cytokine bead array kit from BD and read out by flow-cytometry. (14E) Evaluation of T-cell activation staining for CD25 surface expression on CD90CAR T cells (left two graphs) and loss of H SC-associated markers (middle two graphs) on human G-CSF-mobilized CD34+HSCPs after 72 hours by flow cytometry. All values are shown as mean fluorescence intensity (MFI) relative to untreated controls. Representative flow cytometric plots of co-cultured T cells / CD90CAR T cells with human CD34+HSCPs after 72 hours (flow plots). (14F) Total number of primary colonies derived from FACS-purified CD34+HSPC cultured without T cells, in presence with untreated T cells, or CD90CAR T cells (top graph). Total number of colonies grown in secondary CFC assays after replating 5% of cells from primary CFC assays (middle graph). Total number ofF053-0197PCT / 24-151 -WO-PCTcells grown within secondary CFC assays (bottom graph). (14G) Co-culture of CAR90.2 T cells with CD90- ML-1 cells (left) and CD90+Jurkat cells (right). Killing of tumor cells was determined by flow-cytometry quantifying viable ML 1 / Jurkat cells via FSC / SSC comparing tumor cells alone (black), tumor cells in co-culture with untreated? cells (purple), or CD90CAR.2 T cells (blue) at different E: T ratios.

[0031] FIGs. 15A-15D. In vitro killing assays of CD90CAR T cells on cell lines and primary human cells. (15A) HEK 293T were transduced with VSV-G pseudotyped lentiviral particles stably integrating a codon-optimized CD90 cDNA for permanent expression. Ectopic expression of CD90 on HEK cells was determined by flow-cytometry. (15B) Representative still images of T-cell mediated killing collected on the Incucyte co-culturing CAR90 T cells with either CD90-expressing HEK293T (top row) or WT HEK293T cells lacking CD90 expression (bottom row). HEK293T cells co-express GFP. (15C) Assessment of CAR-mediated cytokine production in response to the co-culture with either CD90-expressing HEK293T (top row) or WT HEK293T cells lacking CD90 expression (bottom row) using a cytokine bead array kit from BD and read out by flow-cytometry. (15D) Assessment of CAR-mediated cytokine production in response to the co-culture with human G-CSF-mobilized CD34+HSCPs from a second healthy donor using a cytokine bead array kit from BD and read out by flow-cytometry.

[0032] FIGs. 16A-16H. CD90-KC protects human HSPCs in vivo and enriches gene-edited cells. (16A) Human G-CSF-mobilized CD34+HSPCs were multiplex gene-edited ex vivo to knock out CD90 and at the HBG-175 promotor to reactive HbF production. Gene-modified HSPCs were retro-orbital ly injected into adult NBSGW mice (8-12 weeks old) and followed for 8-10 weeks to confirm human engraftment in the PB. In parallel, CAR90 T cells were manufactured and 8-10 weeks post-humanization retro-orbitally injected into mice. Mice were followed for 2 weeks to monitor CAR T-cell growth in the PB and comprehensively analyzed at necropsy for tissue engraftment and persistence and / or enrichment of gene-modified HSPCs in the BM. (16B) Pre-infusion validation of CAR90 T cells by flow cytometry measuring expression of tEGFR on CD3+T cells. (16C) Longitudinal flow-cytometric assessment of total human chimerism (huCD45+), phenotypic human B cells (CD19 / 20* of huCD45+), myeloid cells [granulocytes + monocytes] (CD33+of huCD45+), and T cells (CD3+of huCD45j in the PB. The blue arrow indicates the time of CAR90 infusion in CAR-treated mice. (16D) Flow-cytometric assessment of CAR90 T cells in mice over time (top left graph), CD4 / CD8 ratio of CAR T cells at week 10 (top right), and representative flow plots of Whitlow-linker expression on human CAR90 T cells in the PB of mice (bottom). (16E) Flow-cytometric quantification of total human CD3+T cell and CAR90 T cells in the spleen (left) and BM (right). (16F) Frequency of human WBCs (huCD45+) and phenotypically defined human HSPC subsets within human WBCs engrafted in the murine BM assessed by flow-cytometry. (16G) (first graph) Percentage colony formation (number of colonies I number of seeded cells) of FACS-purified human CD34+HSPC from the BM humanized mice, (second graph) Erythro-myeloid differentiation potential of human CD34+HSPC in primary CFCs. (third graph) Percentage secondary colony formation after replating 5% of cells from primary CFC assays, (fourth graph) Erythro-myeloid differentiation potential of human CD34+HSPC in secondary CFCs. Abbreviations: CFU = colony-forming unit; G = granulocyte; M = monocyte / macrophage; BFU = burst forming unit; E =F053-0197PCT / 24-151 -WO-PCTerythrocyte; MIX = myeloid + erythroid. (16H) Assessment of allelic base editing efficiencies at CD90 (left two graphs) and the HBG1 / 2 promoter (right two graphs) in bulk cells harvested from primary and secondary CFC assays using next-generation sequencing (NGS). Numbers within graph indicate the fold-increase in the mean frequency of allelic editing comparing control and CAR90-treated mice.

[0033] FIGs. 17A-17H. CAR90 facilitates clearance of CD90+tumor cells in vivo. (17A) Human G-CSF-mobilized CD34+HSPCs were gene-edited ex vivo to knock out CD90. Gene-modified HSPCs were retro-orbitally injected into adult NBSGW mice (8-12 weeks old) and followed for 8-10 weeks to confirm human engraftment in the PB. After 8-10 weeks, mice were retro-orbitally injected with 1,000,000 CD90+Jurkat cells. In parallel, CAR90 T cells were manufactured and retro-orbitally injected into mice 4 days after tumor cell administration. Mice were followed for up to 4 weeks to monitor tumor growth via live imaging (I VIS) of Luciferase - Jurkat cells, CAR T cell growth in the PB by flow-cytometry, and comprehensively at necropsy for tissue engraftment and persistence and / or enrichment of gene-modified HSPCs in the BM. (17B) Pre-infusion validation of CAR90 T cells by flow cytometry measuring expression of tEGFR on CD3+T cells. (17C) Longitudinal flow-cytometric assessment of total human chimerism (huCD45+), phenotypic human B cells (CD19 / 20+of huCD45+), myeloid cells [granulocytes + monocytes] (CD33+of huCD45+), and T cells (CD3+of huCD45+) in the PB. The vertical red line indicates the time of tumor injection. The vertical blue line indicates the time ofCAR90 infusion in CAR-treated mice. (17D) Flow-cytometric assessment of CAR90 T cells in mice over time (top left graph), CD4 / CD8 ratio of CAR T cells at week 10 (top right), and representative flow plots of Whitlowlinker expression on human CAR90 T cells in the PB of mice (bottom). (17E) Flow-cytometric quantification of total human CD3+T cell and CAR90 T cells in the spleen (left) and BM (right). (17F) IVIS images of mice over time to assess growth and biodistribution of Luciferase* Jurkat cells in mice receiving untreated (UTD) (left) or CAR90 T cells (right). (17G) Longitudinal tracking of total bioluminescence (left) and Kaplan-Meier survival curves (right) of mice receiving untreated (UTD) or CAR90 T cells. (17H) Frequency of human WBCs (huCD45+) and phenotypically defined human HSPC subsets within human WBCs engrafted in the murine BM assessed by flow-cytometry.

[0034] FIGs. 18A-18C. Schematics summarizing the overall strategy for ex vivo gene therapy for either HSC enrichment or cancer immunotherapy. (18A) Depicts a schematic for traditional ex vivo gene therapy that is therapeutic only (i.e., tumor / cancer killing with no HSC protection). (18B) Depicts a schematic for ex vivo gene therapy that is therapeutic plus protective / shielding (i.e., tumor / cancer killing and HSC enrichment / protection). (18C) Depicts a schematic for a next-generation HSC-Targeted ex vivo gene therapy that is therapeutic plus protective / shielding (i.e., tumor / cancer killing and HSC enrichment / protection).

[0035] FIGs. 19A-19C. Schematics summarizing the overall strategy for ex vivo and in vivo gene therapy for either enrichment or cancer immunotherapy. (19A) Depicts a schematic for traditional ex vivo gene therapy resulting in the post-transplant enrichment of gene-modified cells that is therapeutic only (i.e., no protection or enrichment of blood or organs / tissues). (19B) Depicts a schematic for next-generation ex vivo gene therapy resulting in the post-transplant enrichment of gene-modified cells that is therapeutic plus protective / shielding (i.e., protection / enrichment of blood, noF053-0197PCT / 24-151 -WO-PCTprotection of organs / tissues). (19C) Depicts a schematic for in vivo gene therapy resulting in post-transplant enrichment of gene-modified cells that is both therapeutic plus protective / shielding (i.e., protection / enrichment of blood and organs / tissues).

[0036] FIGs. 20A-20F. Sequences supporting the disclosure. (20A) CD90 knockout (KO) sequences. (20B) CD90 F80P editing sequences. (20C) Antibody / antigen quantitative binding parameters determined by BLI; (20D) Guides for sequencing, shRNA knockdown, and base editing; (20E) Epitope mutation strategies and positions; direction is stand direction; (20F) CD90 CAR sequences and components thereof.DETAILED DESCRIPTION

[0037] Hematopoietic cell transplantation (HCT) is a treatment for a variety of disorders including cancer, hemoglobinopathies, and primary immune deficiencies. HCT includes infusing healthy hematopoietic stem and progenitor cells (HSPCs) into a patient whose bone marrow or immune system is damaged or defective. Particularly, HCT is used to treat malignant and nonmalignant diseases that affect the hematopoietic system, including leukemia, lymphoma, myeloma, aplastic anemia, thalassemia, sickle cell anemia, and severe combined immunodeficiency. HCT is usually accompanied by a preparative or conditioning regimen. A conditioning regimen may include chemotherapy, monoclonal antibody therapy, and radiation to the entire body. Unfortunately, conditioning regimens can have toxic effects and thus limit the use of HCT.

[0038] The present disclosure describes modified CD90 expression in therapeutic hematopoietic stem and progenitor cells (HSPCs) and uses thereof. The CD90 expression is modified in therapeutic HSPCs to protect the therapeutic HSPCs from later-administered CD90-targeted therapeutics. In particular embodiments, therapeutic HSPCs are modified to correct a genetic defect, such as beta globin in sickle cell disease. Those same therapeutic HSPCs can be modified to knock down, knock out, or modify CD90 to remove or reduce CD90 binding with a CD90-targeted therapeutic After the modified therapeutic HSPCs have been administered to a subject, the subject can be administered a CD90-targeted therapeutic which will kill HSPCs presenting native CD90 but leave the genetically modified HSPCs unaffected. Modified therapeutic HSPCs can also be administered to a subject concurrently with or after a CD90-targeted therapeutic has been administered to the subject.

[0039] In particular embodiments, therapeutic HSPCs are genetically modified to have modified expression of CD90 and to express a therapeutic payload. In particular embodiments, modified expression of CD90 includes a knock down of CD90 expression, a knockout of CD90 expression, or a modification of CD90 such that a CD90-targeted therapeutic does not bind the modified CD90.

[0040] In particular embodiments, the knock down of CD90 expression includes introducing anti-CD90 short hairpin RNA (shRNA). In particular embodiments, the shRNA includes the sequence set forth in SEQ ID NO: 4.

[0041] In particular embodiments, the knockout of CD90 expression includes introducing guide RNA (gRNA) and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 5 and the gene editing component includes a nuclease such as Cas9 or Cpf1. In particular embodiments, the gRNAF053-0197PCT / 24-151 -WO-PCTincludes the sequence set forth in SEQ ID NO: 1 and the gene editing component includes an adenine base editor (e.g, ABE8e) for use in human cells. In particular embodiments, the gRNA includes the sequence set forth in SEQ ID NO: 2 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in Rhesus macaque cells.

[0042] In particular embodiments, the modification of CD90 includes a modification such that the 5E10 antibody does not bind the modified CD90. In particular embodiments, this modification of CD90 such that the 5E10 antibody does not bind the modified CD90 includes introducing a gRNA and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 6 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in humans or Rhesus macaque cells.

[0043] In particular embodiments, the modification of CD90 includes a modification such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90. In particular embodiments, this modification of CD90 such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90 includes introducing a gRNA and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 7 and the gene editing component includes an adenine base editor (e g., ABE8e) for use in humans cells.

[0044] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position F80. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody binding to CD90 includes a mutation at position F80. In particular embodiments, the mutation at position F80 is an F80P or F80S mutation.

[0045] In particular embodiments, the modification of CD90 includes a modification such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90. In particular embodiments, this modification of CD90 such that the 5E10 antibody and / or the FAB20671R antibody do not bind the modified CD90 includes introducing a gRNA and a gene editing component. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 7 and the gene editing component includes an adenine base editor (e.g., ABE8e) for use in humans cells.

[0046] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position D98. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90 includes a mutation at position D98. In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position E99. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90 includes a mutation at position E99. In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes a mutation at position D98 and E99. In particular embodiments, a CD90 mutation to eliminate 5E10 antibody and / or FAB20671R antibody binding to CD90 includes a mutation at position D98 and E99.

[0047] In particular embodiments, the therapeutic payload includes one or more of globin family genes (e.g., of γ-globin, β-globin, and / or α-globin); yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40 (e.g.F053-0197PCT / 24-151 -WO-PCTsoluble CD40); CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; coagulation factor genes (factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII)); CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1 ), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; H0XB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; interleukins (IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, IL7RA); antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; and ZAP70.

[0048] Particular embodiments include administering genetically modified therapeutic HSPCs with modified CD90 expression to a subject. HSPCs expressing native CD90 can be killed by administering a CD90-targeted therapeutic. In particular embodiments, the CD90-targeted therapeutic includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor (e.g., anti-CD90 chimeric antigen receptor (CAR)). In particular embodiments, an anti-CD90 CAR includes a binding domain having a variable heavy chain set forth in SEQ ID NO: 19 and a variable light chain set forth in SEQ ID NO: 21. In particular embodiments, an anti-CD90 chimeric antigen receptor includes a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0049] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Modified CD90 Expression; (II) Therapeutic Payload; (III) Cell Sample Collection and Cell Enrichment; (IV) Genetic Engineering Techniques; (V) CD90-Targeted Therapeutics; (VI) Compositions and Formulations; (VI I) Kits; (VIII) Methods of Use; (IX) Exemplary Embodiments; (X) Experimental Example; and (XI) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0050] (I) Modified CD90 Expression. CD90, also referred to as Thy-1, is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein. CD90 is expressed on hematopoietic stem cells (HSC), hematopoietic stem and progenitor cells (HSPCs), and mesenchymal stem / stromal cells (MSC), therapeutically important cell types.

[0051] In particular embodiments, CD90 [Homo sapiens] (GenBank: AAH65559.1) includes the sequence:MNLAISIALLLTVLQVSRGQKVTSLTACLVDQSLRLDCRHENTSSSPIQYEFSLTRETKKHVLFGTVGVPEHTYRSRTN FTSKYNMKVLYLSAFTSKDEGTYTCALHHSGHSPPISSQNVTVLRDKLVKCEGISLLAQNTSWLLLLLLSLSLLQATDFF053-0197PCT / 24-151 -WO-PCTMSL (SEQ ID NO: 43).

[0052] In particular embodiments, CD90 expression is modified (knocked down, knocked out, or the epitope binding domain is mutated) in therapeutic HSPC to reduce or prevent binding of CD90-targeted therapeutics. In particular embodiments, CD90 expression is modified by introducing a nucleotide and / or a gene editing component to a cell.

[0053] The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or combinations thereof, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), guide RNA (gRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNAof any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., WO 97 / 03211 and WO 96 / 39154. A polynucleotide may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0054] In particular embodiments, a gene editing component includes a nuclease, a base editor, or other components needed to perform the genetic engineering techniques described elsewhere herein.

[0055] Particular embodiments provide artificial expression constructs to disrupt CD90 expression The genetic disruption herein can be affected by various agents. In some embodiments, the genetic disruption of the CD90 gene can be effected by an agent such as an inhibitory nucleic acid molecule, such as a short hairpin (shRNA), RNA interference (RNAi) agent, short interfering RNA (siRNA), micro RNA (miRNA), antisense RNA, and / or ribozymes, which can be used to selectively suppress or repress expression of the CD90 gene. shRNA uses RNA interference to degrade target genes or viral single-stranded RNA, which inhibits the production of the virus. shRNA include a region of internal hybridization that creates a hairpin structure. shRNA molecules are processed within the cell to form siRNA which in turn knock down gene expression.

[0056] siRNA technology includes that based on RNAi utilizing a double-stranded RNA molecule having a sequence homologous with the nucleotide sequence of mRNA which is transcribed from the gene, and a sequence complementary with the nucleotide sequence. siRNA generally is homologous / complementary to one region of mRNA which is transcribed from the gene, or may be siRNA including a plurality of RNA molecules which are homologous / complementary to different regions.

[0057] In particular embodiments, CD90 expression can be knocked down by introducing or administering shRNA having the sequence as set forth in SEQ ID NO: 4.F053-0197PCT / 24-151 -WO-PCT

[0058] In particular embodiments, CD90 expression can be knocked out using targeted genetic engineering techniques described elsewhere herein. For example, CD90 expression can be knocked out using the CRISPR / Cas system or a base editing system. In particular embodiments, CD90 expression is knocked out by introducing a nuclease and a guide RNA (gRNA). In particular embodiments, the nuclease includes Cas9 or Cpf1. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 5.

[0059] In particular embodiments, CD90 expression is knocked out by introducing a base editor and a guide RNA (gRNA). In particular embodiments, the base editor includes ABE8e. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 1. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 2.

[0060] Particular embodiments provide artificial expression constructs to modify CD90 expression in therapeutic HSPC such that a CD90-targeted therapeutic does not bind or has reduced binding compared to its binding to wildtype (also referred to herein as native) CD90. In particular embodiments, CD90 is mutated at an epitope to prevent binding to CD90-targeted therapeutics. In particular embodiments, the CD90-targeted therapeutic includes a 5E10 antibody and / or a FAB20671R antibody. In particular embodiments, a method of modifying a CD90 epitope includes introducing a gene editing component and a nucleic acid. In particular embodiments, methods of modifying a CD90 epitope includes introducing gRNA and a base editor. In particular embodiments, the base editor includes a adenine base editor or a cytosine base editor.

[0061] In particular embodiments, modified CD90 does not bind a 5E10 antibody. In particular embodiments, modified CD90 includes an F80P mutation. In particular embodiments, methods of modifying a CD90 epitope includes introducing a gene editing component and a nucleic acid. In particular embodiments, methods of modifying a CD90 epitope includes introducing gRNA and a base editor. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 6. In particular embodiments, the base editor includes a adenine base editor (e.g., ABE8e).

[0062] In particular embodiments, modified CD90 does not bind a FAB20671R antibody. In particular embodiments, modified CD90 includes a D98 and / or E99 mutation. In particular embodiments, methods of modifying a CD90 epitope includes introducing a gene editing component and a nucleic acid. In particular embodiments, methods of modifying a CD90 epitope includes introducing gRNA and a base editor. In particular embodiments, the gRNA includes the sequence as set forth in SEQ ID NO: 7. In particular embodiments, the base editor includes a adenine base editor (e.g., ABE8e).

[0063] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes one or more of L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and T121A.

[0064] In particular embodiments, a CD90 mutation to eliminate binding to a CD90-targeted therapeutic includes oneF053-0197PCT / 24-151 -WO-PCTor more of a CRISPR target (5' to 3’), editing window sequence, and epitope edit as depicted in FIG. 20E for row A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, 02, D2, E2, F2, G2, H2, A3, B3, C3, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, H4, A5, B5, C5, D5, E5, F5, G5, H5, A6, B6, C6, D6, E6, F6, G6, H6, A7, B7, 07, D7, E7, F7, G7, H7, A8, B8, 08, D8, E8, F8, G8, H8, A9, B9, 09, D9, and E9.

[0065] (II) Therapeutic Payloads. A therapeutic payload can include a single gene, multiple genes, or sequences and / or gene editing systems to modify an endogenous sequence. The therapeutic payload can result in a variety of therapeutic expression products such as gene editing systems (CRISPR / Cas system or base editing system), small RNAs (e.g., gRNA), integration elements, or therapeutic genes. The therapeutic payload can include or result in expression of a therapeutic gene.

[0066] Particular examples of therapeutic genes and / or gene products to treat immune deficiencies can include genes associated with Fanconi Anemia (FA) including: FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3). Exemplary genes and proteins associated with FA include: Homo sapiens FANCA coding sequence; Homo sapiens FANCC coding sequence; Homo sapiens FANCE coding sequence; Homo sapiens FANCF coding sequence; Homo sapiens FANCG coding sequence; Homo sapiens FANCA AA; Homo sapiens FANCC AA; Homo sapiens FANCE AA; Homo sapiens FANCF AA; and Homo sapiens FANCG AA.

[0067] Particular examples of therapeutic genes and / or gene products to treat immune deficiencies can include genes associated with SCID including: yC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, and SLC46A1. Exemplary genes and proteins associated with SCID include: exemplary codon optimized Human yC DNA; exemplary native Human yC DNA; exemplary native canine yC DNA; exemplary human yC AA; and exemplary native canine yC AA (91% conserved with human). Exemplary genes and proteins associated with SCID include: Homo sapiens JAK3 coding sequence; Homo sapiens PNP coding sequence; Homo sapiens ADA coding sequence; Homo sapiens RAG1 coding sequence; Homo sapiens RAG2 coding sequence; Homo sapiens JAK3 AA; Homo sapiens PNP AA; Homo sapiens ADA AA; Homo sapiens RAG1 AA; and Homo sapiens RAG2 AA.

[0068] Additional exemplary therapeutic genes can include or encode for clotting and / or coagulation factors such as factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII.

[0069] Additional examples of therapeutic genes and / or gene products include those that can provide a therapeutically effective response against diseases related to red blood cells and clotting. In particular embodiments, the disease is a hemoglobinopathy like thalassemia, or a SCD / trait. Exemplary therapeutic genes for these disorders include F8 and F9.

[0070] Additional examples of therapeutic genes and / or gene products include y-globin; soluble CD40; CTLA; Fas L;F053-0197PCT / 24-151 -WO-PCTantibodies to CD4, CD5, CD7, CD52, etc.; antibodies to IL1, IL2, IL6; an antibody to TOR specifically present on autoreactive T cells; IL4; IL10; IL12; IL13; IL1Ra, sILIRI, sILIRII; sTNFRI; sTNFRII; antibodies to TNF; P53, PTPN22, and DRB1*1501 / DQBr0602; globin family genes; WAS; phox; dystrophin; pyruvate kinase (PK); CLN3; ABCD1; arylsulfatase A (ARSA); SFTPB; SFTPC; NLX2.1; ABCA3; GATA1; ribosomal protein genes; TERC; CFTR; LRRK2; PARK2; PARK / ; PINK1; SNCA; PSEN1; PSEN2; APP; S0D1; TDP43; FUS; ubiquilin 2; C9ORF72 and other therapeutic genes described herein.

[0071] Particular embodiments include inserting or altering a gene selected from ABLI, AKT1, APC, ARSB, BCL11A, BLC1, BLC6, BRCA1, BRIP1, C46, CAS9, C-CAM, CBFAI, CBL, CCR5, CD19, CDA, C-MYC, CRE, CSCR4, CSFIR, CTS-I, CYB5R3, DCC, DHFR, DLL1, DMD, EGFR, ERBA, ERBB, EBRB2, ETSI, ETS2, ETV6, FCC, FGR, FOX, FUSI, FYN, GALNS, GLB1, GNS, GUSB, HBB, HBD, HBE1, HBG1, HBG2, HCR, HGSNAT, H0XB4, HRAS, HYAL1, ICAM-1, iCaspase, IDUA, IDS, JUN, KLF4, KRAS, LYN, MCC, MDM2, MGMT, MLL, MMACI, MYB, MEN-I, MEN-II, MYC, NAGLU, NANOG, NF-1, NF-2, NKX2.1, NOTCH, OCT4, p16, p21, p27, p57, p73, PALB2, RAD51C, ras, at least one of RPL3 through RPL40, RPLPO, RPLP1, RPLP2, at least one of RPS2 through RPS30, RPSA, SGSH, SLX4, SOX2, VHL, and / or WT-I.

[0072] In various embodiments of the present disclosure, a therapeutic gene includes a globin gene, wherein the globin protein encoded by the globin gene is selected from a y-globin, a |3-globin, and / or an a-globin. Globin genes useful with the present disclosure can include, e.g., one or more regulatory sequences such as a promoter operably linked to a nucleic acid sequence encoding a globin protein. As those of skill in the art will appreciate, each of γ-globin, β-globin, and / or α-globin is a component of fetal and / or adult hemoglobin and is therefore useful in various vectors disclosed herein. In particular embodiments, the therapeutic gene is used in the treatment of sickle cell disease.

[0073] (III) Cell Sample Collection and Cell Enrichment. Methods of cell sample collection and enrichment are known by those skilled in the art. In particular embodiments, cells are derived from humans, for example a patient to be treated. Cells can be derived from cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate (e.g., Rhesus macaque), or pig. In particular embodiments, cells are derived from humans, for example a patient to be treated.

[0074] Exemplary sources of hematopoietic stem progenitor cells (HSPCs) include bone marrow, peripheral blood, and umbilical cord blood. Pre-treatment with cytokines, such as G-CSF (granulocyte colony-stimulating factors), can induce cells to be released from the bone marrow compartment. For example, growth factors GM-CSF (granulocytemacrophage colony-stimulating factor) and G-CSF can be administered to a subject to mobilize existing HSPC in the bone marrow niches to the peripheral blood in vivo in order to increase the fraction of HSPCs circulating in the blood.

[0075] HSPCs can be isolated by any methods known in the art, e.g. as described herein and in U. S. Pat. No.7,510,877, U. S Patent Publication No. 20090215083 and 20090169523, and WO / WO / 2009 / 129288 and WO / 2005 / 030040.

[0076] Cells can be obtained directly by removal from the bone marrow using a needle and syringe or by collectionF053-0197PCT / 24-151 -WO-PCTfrom peripheral blood using a heparinized syringe.

[0077] In some embodiments, cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In particular embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. Washing can be accomplished using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In particular embodiments, cells can be re-suspended in a variety of biocompatible buffers after washing.

[0078] Cell isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and / or simultaneously. In particular embodiments, the isolation, culture, and / or engineering of the different cell populations is carried out from the same starting composition or material, such as from the same sample.

[0079] In particular embodiments, HSPC can be enriched for and / or isolated based on cell-marker based positive and / or negative selection. In positive selection, cells having bound cellular markers are retained for further use. In negative selection, cells not bound by a capture agent, such as an antibody to a cellular marker are retained for further use. In some examples, both fractions can be retained for a further use. In particular embodiments, HSPCs can be enriched for and / or isolated using positive selection for CD34, CD90, and / or CD133. In particular embodiments, HSPCs can be enriched for and / or isolated using negative selection using CD38 and CD45RA.

[0080] Cell separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells but need not result in a complete removal of all such cells.

[0081] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0082] In some embodiments, an antibody or binding domain for a cellular marker is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ); see also US 4,452,773; US 4,795,698; US 5,200,084; and EPF053-0197PCT / 24-151 -WO-PCT452342.

[0083] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0084] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5): 355- 376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined cell subsets at high purity.

[0085] HSPCs can be manipulated and maintained in culture media. Culture media suitable for the ex vivo culturing of HSPCs are well known in the art, e.g. as disclosed in U. S. Pat. No. 6,030,836, and by J. Hartshorn, et al., “Ex Vivo Expansion of Hematopoietic Stem Cells Using Defined Culture Media” in Cell Technology for Cell Products, Chapter III, pages 221-224. Such culture media include high glucose Dulbecco's Modified Eagles Medium (DMEM) with L-Glutamine which is well known and readily commercially available. The media can be supplemented with recombinant human basic fibroblast growth factor (rhbFGF) and contain sera, such as human serum, and antibiotics. Cell cultures can be maintained in a CO2atmosphere, e.g., 5% to 12%, to maintain pH of the culture fluid, and incubated at 37°C in a humid atmosphere. Suitable chemically defined serum-free media are described in U. S. Ser. No. 08 / 464,599 and WO96 / 39487, and “complete media” are described in U. S. Pat. No. 5,486,359. Chemically defined medium includes a minimum essential medium such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco), supplemented with human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and a mitogen. These media stimulate cell growth without differentiation. As used herein, a mitogen refers to an agent that stimulates cell division of a cell. Such an agent can be a chemical, usually some form of a protein that encourages a cell to commence cell division triggering mitosis. Other examples of culture medium include RPMI 1640, Iscove's modified Dubelcco's media (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically Defined Medium includes a minimum essential medium such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco), supplemented with human serum albumin, human Ex Cyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine and a mitogen is also suitable.F053-0197PCT / 24-151 -WO-PCT

[0086] Commercial culture media for HSPCs are also available, e.g. STEMPRO®-34 SFM from INVITROGEN™ Inc., MACS® HSC-CFU Media from Miltenyi Biotech, STEMSPAN® SFEM Medium and STEMSPAN® CC110-STEMSPAN® Cytokine Cocktail from STEMCELL Technologies, Inc.

[0087] In particular embodiments, commercially-available formulations can be supplemented with 3700 mg / l of sodium bicarbonate and 10 ml / l of a 100x (100 times concentrated) antibiotic-antimycotic cocktail containing 10,000 units of penicillin, 10,000 pg of streptomycin, and 25 pg of amphotericin B / ml utilizing penicillin G (sodium salt), streptomycin sulfate, and amphotericin B (FUNGIZONE™) in 0.85% saline. In addition, cocktails of cytokines e.g. GM-CSF, G-CSF, SCF, IL-3, IL-6, and Epo can be included

[0088] Other culture media supplements include platelet rich plasma supplemented with heparin (2 U / ml); the basic fibroblast growth factor (bFGF) recombinant human basic fibroblast growth factor (rhubFGF), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), I nterleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-9 (IL-9), thrombopoietin (TPO) and erythropoetin (Epo).

[0089] In particular embodiments, a cell culture medium includes STEMSPAN™ Serum Free Expansion Medium (StemCell Technologies, Vancouver, British Columbia) supplemented with 10 ng / ml recombinant human lnterleukin-3 (rhlL-3), 50 ng / ml recombinant human lnterleukin-6 (rhlL-6), 50 ng / ml recombinant human Thrombopoietin (rhTPO), 50 ng / ml recombinant human Flt-3 Ligand (rhFlt-3L), 50 ng / ml and recombinant human stem cell factor (rhSCF).

[0090] (IV) Genetic Engineering Techniques. In particular embodiments, therapeutic cell populations are genetically modified to knockdown, knockout, or modify CD90 expression such that CD90-targeted therapeutics do not recognize or bind the genetically modified therapeutic cell populations. Furthermore, particular embodiments include genetically modifying therapeutic cell populations to include a therapeutic payload described elsewhere herein. Sequences to knockdown, knockout, or modify CD90 expression and / or therapeutic payload can be included on artificial expression constructs.

[0091] Artificial expression constructs disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, etal., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the artificial expression construct to the cell, so that the artificial expression construct can have its effect (e.g., knockout, knockdown, or modification of CD90 expression) and, in certain instances, preferably heritable and expressible by its cell progeny.

[0092] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g, plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids, bacterial artificial chromosomes,F053-0197PCT / 24-151 -WO-PCTviral vectors, virus-like particles (VLPs), nanoparticles, or phage. Examples of viral vectors include lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, and the like.

[0093] " Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1, and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0094] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a selfinactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. In particular embodiments, cells are genetically engineered using a lentiviral vector.

[0095] " Retroviruses" are viruses having an RNA genome. " Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0096] In particular embodiments, retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

[0097] Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science 252:431-434; Rosenfeld, etal., 1992, Cell 68: 143-155; Mastrangeli, etal., 1993, J. Clin. Invest. 91:225-234; Walsh, etal., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0098] As indicated by the foregoing discussion, there are a large number of available viral vectors suitable for use with the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packagingF053-0197PCT / 24-151 -WO-PCTcells for transducing mammalian host cells with viral particles including transgenes are described in, e.g., US 8, 119,772; Walchli, etal., 2011, PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, etal., 2003, Hum. Gene Ther. 14:1155; Frecha, etal., 2010, Mol. Ther. 18:1748; and Verhoeyen, etal., 2009, Methods Mol. Biol. 506:97.

[0099] In other embodiments, suitable vectors may include a VLP. VLPs are particles that closely resemble viruses, but do not contain viral genetic material and are therefore non-infectious. In some embodiments, VLPs include a polynucleotide encoding a transgene of interest, for example any of the gene editing components (e.g., Gas) and / or a gRNA, and, optionally, donor template polynucleotides, packaged with one or more viral structural proteins. Such particles will typically include proteins that encapsidate or package the vector genome. Suitable expression vectors may include viral expression vectors based on vesicular stomatitis virus (VSV); vaccinia virus; poliovirus; adenovirus; a retroviral vector (e.g., Murine Leukemia Virus), spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, or mammary tumor virus.

[0100] Targeted genetic engineering approaches may be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the bacteria’s "immune" response. The crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide a Cas nuclease to a region homologous to the crRNA in the target DNA called a "protospacer." The Cas nuclease cleaves the DNA to generate blunt ends at the double-strand break at sites specified by a 20-nucl eotide complementary strand sequence contained within the crRNA transcript. In some instances, the Cas nuclease requires both the crRNA and the tracrRNA for sitespecific DNA recognition and cleavage.

[0101] Guide RNA (gRNA) is one example of a targeting element. In its simplest form, gRNA provides a sequence that targets a site within a genome based on complementarity (e.g., crRNA). As explained below, however, gRNA can also include additional components. For example, in particular embodiments, gRNA can include a targeting sequence (e.g., crRNA) and a component to link the targeting sequence to a cutting element. This linking component can be tracrRNA. In particular embodiments, as described below, gRNA including crRNA and tracrRNA can be expressed as a single molecule referred to as single gRNA (sgRNA). gRNA can also be linked to a cutting element through other mechanisms such as through a nanoparticle or through expression or construction of a dual or multi-purpose molecule.

[0102] In particular embodiments, targeting elements (e.g., gRNA) can include one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). Modified backbones may include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified backbones containing a phosphorus atom may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters,F053-0197PCT / 24-151 -WO-PCTaminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', a 5' to 5' or a 2' to 2' linkage. Suitable targeting elements having inverted polarity can include a single 3' to 3' linkage at the 3'-most internucleotide linkage (i.e. a single inverted nucleoside residue in which the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms can also be included.

[0103] Targeting elements can include one or more phosphorothioate and / or heteroatom internucleoside linkages, in particular -CH2-NH--O-CH2-, --CH2-N(CH3)-O--CH2- (i.e. a methylene (methylimino) or MMI backbone), -CH2-0- N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -O-P(=O)(OH)--O-CH2-).

[0104] Targeting elements can include a morpholino backbone structure, for example, the targeting elements can include a 6-membered morpholino ring in place of a ribose ring. In some embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.

[0105] Targeting elements can include one or more substituted sugar moieties. Suitable polynucleotides can include a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO) mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to 10.

[0106] As indicated, examples of cutting elements include nucleases. CRISPR-Cas loci have more than 50 gene families and there are no strictly universal genes, indicating fast evolution and extreme diversity of loci architecture. Exemplary Gas nucleases include Casl, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cpfl, C2c3, C2c2 and C2clCsyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.

[0107] There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66). Type II Cas nucleases include Casl, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP 269215, and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470.

[0108] In particular embodiments, Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease protein orF053-0197PCT / 24-151 -WO-PCTnickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. The Cas9 enzyme, in some embodiments, includes one or more catalytic domains of a Cas9 protein derived from bacteria such as Corynebacter, Sutterella, Legionella, Treponema, Filif actor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the Cas9 is a fusion protein, e.g. the two catalytic domains are derived from different bacterial species.

[0109] As indicated previously, the CRISPR / Cas system has been engineered such that, in certain cases, crRNA and tracrRNA can be combined into one molecule called a single gRNA (sgRNA). In this engineered approach, the sgRNA guides Casto target any desired sequence (see, e.g., Jinek etal. (2012) Science 337:816-821; Jinek etal. (2013) eLife 2:e00471; Segal (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to create a double-strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR); homology-independent targeted integration (HITI)-associated microhomology-mediated end joining (MMEJ), HITI-associated non-homologous end joining (NHEJ); or complete NHEJ depending on whether a genetic construct is provided for insertion and the length of any provided homology regions (e.g., as used herein HDR occurs if a region of homology is > 75 bp and HITI occurs if a region of homology is < 75 bp).

[0110] Useful variants of the Cas9 nuclease include a single inactive catalytic domain, such as a RuvC" or HNH" enzyme or a nickase. A Cas9 nickase has only one active functional domain and, in some embodiments, cuts only one strand of the target DNA, thereby creating a single strand break or nick. In some embodiments, the mutant Cas9 nuclease having at least a D10A mutation is a Cas9 nickase. In other embodiments, the mutant Cas9 nuclease having at least a H840A mutation is a Cas9 nickase. Other examples of mutations present in a Cas9 nickase include N854A and N863A. A double-strand break is introduced using a Cas9 nickase if at least two D NA-targeting RNAs that target opposite DNA strands are used. A double-nicked induced double-strand break is repaired by NHEJ, HDR or HITI. This gene editing strategy generally favors HDR and decreases the frequency of indel mutations at off-target DNA sites. The Cas9 nuclease or nickase, in some embodiments, is codon-optimized for the target cell or target organism.

[0111] Particular embodiments can utilize Staphylococcus aureus Cas9 (SaCas9). Particular embodiments can utilize SaCas9 with mutations at one or more of the following positions: E782, N968, and / or R1015. Particular embodiments can utilize SaCas9 with mutations at one or more of the following positions: E735, E782, K929, N968, A1021, K1044 and / or R1015. In some embodiments, the variant SaCas9 protein includes one or more of the following mutations: R1015Q, R1015H, E782K, N968K, E735K, K929R, A1021T, and / or K1044N. In some embodiments, the variant SaCas9 protein includes mutations at D10A, D556A, H557A, N580A, e.g., D10A / H557A and / or D10A / D556A / H557A / N580A. In some embodiments, the variant SaCas9 protein includes one or more mutations selected from E735, E782, K929, N968, R1015, A1021, and / or K1044. In some embodiments, the SaCas9 variantsF053-0197PCT / 24-151 -WO-PCTcan include one of the following sets of mutations: E782K / N968K / R1015H (KKH variant); E782K / K929R / R1015H (KRH variant); or E782K / K929R / N968K / R1015H (KRKH variant).

[0112] A putative Class II, Type V CRISPR-Cas class exemplified by Cpf 1 has been identified Zetsche et al. (2015) Cell 163(3): 759-771. The Cpf1 nuclease particularly can provide added flexibility in target site selection by means of a short, three base pair recognition sequence (TTN), known as the protospacer-adjacent motif or PAM. CpfTs cut site is at least 18bp away from the PAM sequence. Moreover, staggered DSBs with sticky ends permit orientation-specific donor template insertion, which is advantageous in non-dividing cells.

[0113] Particular embodiments can utilize engineered Cpf 1 s. For example, US 2018 / 0030425 describes engineered Cpf1 nucleases from Lachnospiraceae bacterium ND2006 and Acidaminococcus sp. BV3L6 with altered and improved target specificity. Particular variants include Lachnospiraceae bacterium ND2006 of SEQ ID NO: 66, e.g., at least including amino acids 19-1246 of SEQ ID NO: 66, with mutations (i.e., replacement of the native amino acid with a different amino acid, e.g., alanine, glycine, or serine), at one or more of the following positions: S202, N274, N278, K290, K367, K532, K609, K915, Q962, K963, K966, K1002, and / or S1003 of SEQ ID NO: 66. Particular Cpf1 variants can also include Acidaminococcus sp. BV3L6 Cpf1 (AsCpfl) of SEQ ID NO: 67 with mutations (i.e., replacement of the native amino acid with a different amino acid, e.g., alanine, glycine, or serine (except where the native amino acid is serine)), at one or more of the following positions: N178, S186, N278, N282, R301, T315, S376, N515, K523, K524, K603, K965, Q1013, Q1014, and / or K1054 of SEQ ID NO: 67. In particular embodiments, engineered Cpf1 variants include eCfpl.

[0114] In particular embodiments, Lachnospiraceae bacterium ND2006 includes the sequence:MLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLS FINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNS FNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGE FFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEV FRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAWTEKYEDD RRKSFKKIGSFSLEQLQEYADADLSWEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAWAIMKDLLDSVKS FENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYR ATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFK KGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLY MFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELWHPANSPIANKNPDNPKKTTT LSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVWDGKGNIVEQYSLNEII NNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQWHKICELVEKYDAVIALEDLNSGFKNSRVKVEK QVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKIKYT SIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELF NKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKF053-0197PCT / 24-151 -WO-PCTNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH (SEQ ID NO: 66).

[0115] In particular embodiments, Acidaminococcus sp. BV3L6 includes the sequence:MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLF RKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMF SEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGF VTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKL FKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAWTEKYEDDRRKSFKKIGSFSLEQLQEY ADADLSWEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRD ESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKY AKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFK DSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTP NLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELWHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYE LHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVWDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKK EKERFEARQNWTSIENIKELKAGYISQWHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMV DKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVP EEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCE QSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAI GQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 67).

[0116] Other Cpf1 variants include Cpf1 homologs and orthologs of the Cpf1 polypeptides disclosed in Zetsche etal. (2015) Cell 163: 759-771 as well as the Cpf1 polypeptides disclosed in U. S. 2016 / 0208243. Other engineered Cpf1 variants are known to those of ordinary skill in the art and included within the scope of the current disclosure (see, e.g., WO / 2017 / 184768).

[0117] Additional information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, W02016205711, WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0118] Another targeted genetic engineering approach includes base editing. Methods disclosed herein include modifying CD90 expression using base editing agents (i.e., base editors) and nucleic acids encoding the same. A baseF053-0197PCT / 24-151 -WO-PCTediting system can include a base editing enzyme and / or at least one gRNA as components thereof.

[0119] Base editing refers to the selective modification of a nucleic acid sequence by converting a base or base pair within genomic DNA or cellular RNA to a different base or base pair (Rees & Liu, Nature Reviews Genetics, 19:770-788, 2018). There are two general classes of DNA base editors: (i) cytosine base editors (CBEs) that convert guanine-cytosine base pairs into thymine-adenine base pairs, and (ii) adenine base editors (ABEs) that convert adenine-thymine base pairs to guanine cytosine base pairs. In particular embodiments, components from the CRISPR system are combined with other enzymes or biologically active fragments thereof to directly install, cause, or generate mutations such as point mutations in nucleic acids, e.g, into DNA or RNA, e.g, without making, causing, or generating one or more double-stranded breaks in the mutated nucleic acid. Certain such combinations of components are known as base editors.

[0120] DNA base editors can include a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that base modify RNA.

[0121] Upon binding to its target locus in DNA, base pairing between the g RNA and target DNA strand leads to displacement of a small segment of single-stranded DNA. DNA bases within this single-stranded DNA bubble can be modified by the deaminase enzyme. In certain embodiments, to improve efficiency in eukaryotic cells, a catalytically disabled nuclease also generates a nick in the non-edited DNA strand, inducing cells to repair the non-edited strand using the edited strand as a template.

[0122] For cytosine base editors (CBEs), CRISPR-based editors can be produced by linking a cytosine deaminase with a Cas nickase, e.g., Cas9 nickase (nCas9). To provide one example, nCas9 can create a nick in target DNA by cutting a single strand, reducing the likelihood of detrimental indel formation as compared to methods that require a double-stranded break. After binding with DNA, the CBE deaminates a target cytosine (C) into a uracil (U) base. Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an original C-G pair converted to T-A or reverted to the original C-G by base excision repair mediated by uracil glycosylase. In various embodiments, expression of uracil glycosylase inhibitor (UGI), e.g., a UGI present in a payload, reduces the occurrence of the second outcome and increases the generation of T-A base pair formation.

[0123] For adenine base editors (ABEs), exemplary adenosine deaminases that can act on DNA for adenine base editing include a mutant TadA adenosine deaminases (TadA*) that accepts DNA as its substrate. E. co / / TadA typically acts as a homodimer to deaminate adenosine in transfer RNA (tRNA). TadA* deaminase catalyzes the conversion of a target ‘A’ to T (inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair can be converted to a G-C pair. As the cellular inosine excision repair is not as active as uracil excision, ABE does not require any additional inhibitor protein like UGI in CBE In some embodiments, a typical ABE can include three components including a wild-type E. coll tRNA-specific adenosine deaminase (TadA) monomer, which can play a structural role during base editing, a TadA* mutant TadA monomer that catalyzes deoxyadenosine deamination, andF053-0197PCT / 24-151 -WO-PCTa Cas nickase such as Cas9(D10A). In certain embodiments, there is a linker positioned between TadA and TadA*, and in certain embodiments there is a linker positioned between TadA* and the Cas nickase. In various embodiments, one or both linkers includes at least 6 amino acids, e.g., at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids (e.g., having a lower bound of 5, 6, 7, 8, 9, 10, or 15, amino acids and an upper bound of 20, 25, 30, 35, 40, 45, or 50 amino acids). In various embodiments, one or both linkers include 32 amino acids. In some embodiments, one or both linkers has a sequence according to (SGGS)2-XTEN-(SGGS)2, or a sequence otherwise known to those of skill in the art. In particular embodiments, the ABE includes ABE8e.

[0124] Base editors can directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products, such as insertions and deletions (indels). For example, base editors can generate less than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% indels.

[0125] DNA base editors can insert such point mutations in non-dividing cells without generating double-strand breaks. Due to the lack of double-strand breaks, base editors do not result in excess undesired editing by-products, such as insertions and deletions (indels). For example, base editors can generate fewer than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% indels as compared to technologies that do rely on double-strand breaks.

[0126] Components of most base-editing systems include (1) a targeted DNA binding protein, (2) a nucleobase deaminase enzyme, and (3) a DNA glycosylase inhibitor.

[0127] Any nuclease of the CRISPR system can be disabled and used within a base editing system. Exemplary Cas nucleases are described above.

[0128] Particular embodiments utilize a nuclease-inactive Cas9 (dCas9) as the catalytically disabled nuclease. However, any nuclease of the CRISPR system (many of which are described above) can be disabled and used within a base editing system. In particular embodiments, a Cas9 domain with high fidelity is selected wherein theCas9 domain displays decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) includes one or more mutations that decrease the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. Cas9 domains with high fidelity are known to those skilled in the art. For example, Cas9 domains with high fidelity have been described in Kleinstiver, et al., Nature 529, 490-495, 2016; and Slaymaker et al., Science 351, 84-88, 2015.

[0129] Nucleases from other gene-editing systems may also be used. For example, base-editing systems can utilize zinc finger nucleases (ZFNs) (Urnov et al., Nat Rev Genet., 11 (9): 636-46, 2010) and transcription activator like effector nucleases (TALENs) (Joung et al., Nat Rev Mol Cell Biol. 14(1):49-55, 2013) For additional information regarding DNA-binding nucleases, see US2018 / 0312825A1.

[0130] In particular embodiments, the nucleobase deaminase enzyme includes a cytidine deaminase domain or anF053-0197PCT / 24-151 -WO-PCTadenine deaminase domain.

[0131] Particular embodiments utilize a cytidine deaminase domain as the nucleobase deaminase enzyme. Particular embodiments utilize an adenine deaminase domain as the nucleobase deaminase enzyme. Further, particular embodiments utilize a uracil glycosylase inhibitor (UGI) as a glycosylase inhibitor. For example, in particular embodiments, dCas9 or a Cas9 nickase can be fused to a cytidine deaminase domain. The dCas9 or a Cas9 nickase fused to the cytidine deaminase domain can be fused to one or more UGI domains. Base editors with more than one UGI domain can generate less indels and more efficiently deaminates target nucleic acids.

[0132] In particular embodiments, a deaminase domain (cytidine and / or adenine) is fused to the N-terminus of the catalytically disabled nuclease. This is because a cytidine deaminase domain fused to the N-terminus of Cas9 can have improved base-editing efficiency when compared to other configurations. In these embodiments, a glycosylase inhibitor (e.g, UGI domain) can be fused to the C-terminus of the catalytically disabled nuclease. When multiple glycosylase inhibitors are used, each can be fused to the C-terminus of the catalytically disabled nuclease.

[0133] In particular embodiments, CBE utilizing a cytidine deaminase domain convert guanine-cytosine base pairs into thymine-adenine base pairs by deaminating the exocyclic amine of the cytosine to generate uracil Examples of cytosine deaminase enzymes include APOBEC1, APOBEC3A, APOBEC3G, CDA1, and AID. APOBEC1 particularly accepts single stranded (ss)DNA as a substrate but is incapable of acting on double stranded (ds)DNA.

[0134] Most base-editing systems also include a DNA glycosylase inhibitor that serves to override natural DNA repair mechanisms that might otherwise repair the intended base editing. In particular embodiments, the DNA glycosylase inhibitor includes an uracil glycosylase inhibitor, such as the uracil DNA glycosylase inhibitor protein (UGI) described in Wang et al. (Gene 99, 31-37, 1991).

[0135] Components of base editors can be fused directly (e.g., by direct covalent bond) or via linkers. For example, the catalytically disabled nuclease can be fused via a linker to the deaminase enzyme and / or a glycosylase inhibitor. Multiple glycosylase inhibitors can also be fused via linkers. As will be understood by one of ordinary skill in the art, linkers can be used to link any peptides or portions thereof.

[0136] Exemplary linkers include polymeric linkers (e.g., polyethylene, polyethylene glycol, polyamide, polyester); amino acid linkers; carbon-nitrogen bond amide linkers; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linkers; monomeric, dimeric, or polymeric aminoalkanoic acid linkers; aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, [3-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid) linkers; monomeric, dimeric, or polymeric aminohexanoic acid (Ahx) linkers;, carbocyclic moiety (e.g., cyclopentane, cyclohexane) linkers; aryl or heteroaryl moiety linkers; and phenyl ring linkers.

[0137] Linkers can also include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from a peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0138] In particular embodiments, linkers range from 4-100 amino acids in length. In particular embodiments, linkersF053-0197PCT / 24-151 -WO-PCTare 4 amino acids, 9 amino acids, 14 amino acids, 16 amino acids, 32 amino acids, or 100 amino acids.

[0139] For additional examples of BE complexes, including adenine deaminase base editors, see Rees & Liu Nat. Rev Genet. 19(12): 770-788, 2018. For additional information regarding base editors, see US2018 / 0312825A1, WO2018 / 165629A, Urnov etal., NatRev Genet. 11 (9):636-46, 2010; Joung eta / ., Nat Rev Mol Cell Biol. 14(1):49-55, 2013; Charpentier et a / ., Nature:, 495(7439):50-1, 2013; Seo & Kim, Nature Medicine, 24, 1493-1495, 2018, and Rees & Liu, Nature Reviews Genetics, 19, 770-78, 2018. Certain base editor constructs that can be used in various embodiments of the present disclosure are described in Zafra eta / ., Nat Biotech, 36(9):888-893, 2018, and Koblan et a / , Nat Biotech 36(9):843-846, 2018.

[0140] Expression of CD90 in therapeutic cells can also be modified using small RNAs. Small RNAs are short, noncoding RNA molecules that play a role in regulating gene expression. In particular embodiments, small RNAs are less than 200 nucleotides in length. In particular embodiments, small RNAs are less than 100 nucleotides in length. In particular embodiments, small RNAs are less than 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. In particular embodiments, small RNAs are less than 20 nucleotides in length. In various embodiments a small RNA has a length having a lower bound of 5, 10, 15, 20, 25, or 30 nucleotides and an upper bound of 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. Small RNAs include microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), tRNA-derived small RNAs (tsRNAs) small rDNA-derived RNAs (srRNAs), and small nuclear RNAs. Additional classes of small RNAs continue to be discovered.

[0141] In particular embodiments, interfering RNA molecules that are homologous to a target mRNA or to which the interfering RNA can hybridize can lead to degradation of the target mRNA molecule or reduced translation of the target mRNA, a process referred to as RNA interference (RNAi) (Carthew, Curr. Opin. Cell. Biol. 13: 244-248, 2001). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). In some instances, natural RNAi proceeds via fragments cleaved from free double-strand RNA (dsRNA) which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be manufactured, for example, to silence the expression of target genes. Exemplary RNAi molecules include small hairpin RNA (shRNA, also referred to as short hairpin RNA) and small interfering RNA (siRNA).

[0142] Without limiting the disclosure, and without being bound by theory, RNA interference in nature and / or in some embodiments is typically a two-step process. In the first step, the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) siRNA, probably by the action of Dicer, a member of the ribonuclease (RNase) III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 base pair (bp) duplexes (siRNA), each with 2-nucleotide 3' overhangs (Hutvagner & Zamore, Curr. Opin. Genet. Dev. 12: 225-232, 2002; Bernstein, Nature 409:363-366, 2001).

[0143] In a second step, an effector step, the siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC.F053-0197PCT / 24-151 -WO-PCTThe active RISC then targets the homologous transcript by base pairing interactions and typically cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA (Hutvagner & Zamore, Curr. Opin. Genet. Dev. 12: 225-232, 2002; Hammond et al., Nat. Rev. Gen. 2:110-119, 2001; Sharp, Genes. Dev. 15:485-490, 2001). Research indicates that each RISC contains a single siRNA and an RNase (Hutvagner & Zamore, Curr. Opin. Genet. Dev. 12: 225-232, 2002).

[0144] Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC (Hutvagner & Zamore, Curr. Opin. Genet. Dev. 12: 225-232, 2002; Hammond et al., Nat. Rev. Gen.2:110-119, 2001; Sharp, Genes. Dev. 15:485-490, 2001). RNAi is also described in Tuschl (Chem. Biochem. 2: 239-245, 2001); Cullen (Nat. Immunol. 3:597-599, 2002); and Brantl (Biochem. Biophys. Act. 1575:15-25, 2002).

[0145] In some embodiments, synthesis of RNAi molecules suitable for use with the present disclosure can be performed as follows. First, an mRNA sequence can be scanned downstream of the start codon of targeted transgene. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. In particular embodiments, the siRNA target sites can be selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-bi ndi ng proteins and / or translation initiation complexes may interfere with binding of the siRNA endonuclease complex (Tuschl, Chem. Biochem. 2: 239-245, 2001). It will be appreciated however, that siRNAs directed at UTRs may also be effective, as demonstrated for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) wherein siRNA directed at the 5' UTR mediated a 90% decrease in cellular GAPDH mRNA and completely abolished protein level. Second, potential target sites can be compared to an appropriate genomic database using any sequence alignment software, such as the Basic Local Alignment Search Tool (BLAST) software available from the National Center for Biotechnology Information (NCBI) server. Putative target sites which exhibit significant homology to other coding sequences can be filtered out.

[0146] Qualifying target sequences can be selected as templates for siRNA synthesis. Selected sequences can include those with low G / C content as these have been shown to be more effective in mediating gene silencing as compared to those with G / C content higher than 55%. Several target sites can be selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control can be used. Negative control siRNA can include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA may be used, provided it does not display any significant homology to other genes.

[0147] A sense strand can be designed based on the sequence of the selected portion. The antisense strand is routinely the same length as the sense strand and includes complementary nucleotides. In particular embodiments, the strands are fully complementary and blunt-ended when aligned or annealed. In other embodiments, the strands align or anneal such that 1-, 2- or 3-nucleotide overhangs are generated, i.e., the 3' end of the sense strand extendsF053-0197PCT / 24-151 -WO-PCT1, 2 or 3 nucleotides further than the 5' end of the antisense strand and / or the 3' end of the antisense strand extends 1, 2 or 3 nucleotides further than the 5' end of the sense strand. Overhangs can include nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can include deoxyribonucleotides, for example deoxythymines (dTs), or nucleotide analogs, or other suitable non-nucleotide material.

[0148] To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and 3' end of the antisense strand can be altered, e.g., lessened or reduced. In particular embodiments, the base-pair strength is less due to fewer G: C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In particular embodiments, the base pair strength is less due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. Preferably, the mismatched base pair is selected from the group including G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pair strength is less due to at least one wobble base pair, e.g., G: U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one base pair including a rare nucleotide, e.g., inosine (I). In particular embodiments, the base pair is selected from the group including an I:A, I:U and I:C. In yet another embodiment, the base pair strength is less due to at least one base pair including a modified nucleotide. In particular embodiments, the modified nucleotide is selected from, for example, 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0149] ShRNAs are single-stranded polynucleotides with a hairpin loop structure. The single-stranded polynucleotide has a loop segment linking the 3' end of one strand in the double-stranded region and the 5' end of the other strand in the double-stranded region. The double-stranded region is formed from a first sequence that is hybridizable to a target sequence, such as a polynucleotide encoding transgene, and a second sequence that is complementary to the first sequence, thus the first and second sequence form a double stranded region to which the linking sequence connects the ends of to form the hairpin loop structure. The first sequence can be hybridizable to any portion of a polynucleotide encoding transgene. The double-stranded stem domain of the shRNA can include a restriction endonuclease site.

[0150] Transcription of shRNAs is initiated at a polymerase III (Pol III) promoter and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stemloop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of 21-23 nucleotides (Brummelkamp et al., Science. 296(5567):550-553, 2002; Lee et al., Nature Biotechnol. 20(5):500-505, 2002; Miyagishi & Taira, Nature Biotechnol. 20(5):497-500, 2002; Paddison et al., Genes & Dev. 16(8): 948-958, 2002; Paul et al., Nature Biotechnol. 20 (5): 505-508, 2002; Sui, Proc. Natl. Acad. Sci. USA 99(6):5515-5520, 2002; Yu etal., Proc. Natl. Acad. Sci. USA. 99(9):6047-6052, 2002).

[0151] The stem-loop structure of shRNAs can have optional nucleotide overhangs, such as 2-bp overhangs, for example, 3' UU overhangs. While there may be variation, stems typically range from 15 to 49, 15 to 35, 19 to 35, 21 toF053-0197PCT / 24-151 -WO-PCT31 bp, or 21 to 29 bp, and the loops can range from 4 to 30 bp, for example, 4 to 23 bp. In particular embodiments, shRNA sequences include 45-65 bp; 50-60 bp; or 51, 52, 53, 54, 55, 56, 57, 58, or 59 bp. In particular embodiments, shRNA sequences include 52 or 55 bp. In particular embodiments siRNAs have 15-25 bp. In particular embodiments siRNAs have 16, 17, 18, 19, 20, 21, 22, 23, or 24 bp. In particular embodiments siRNAs have 19 bp. The skilled artisan will appreciate, however, that siRNAs having a length of less than 16 nucleotides or greater than 24 nucleotides can also function to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or Protein kinase R (PKR) response in certain mammalian cells which may be undesirable. Preferably the RNAi agents do not elicit a PKR response ( / .e., are of a sufficiently short length) However, longer RNAi agents may be useful, for example, in situations where the PKR response has been downregulated or dampened by alternative means.

[0152] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of sitespecific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells. A zinc finger is a domain of 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A well-known example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261; US 6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241,573; US 7,241,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim etal., Genome Res, 2012, 22(7): 1327-33; Urnov etal., Nature Reviews Genetics, 2010, 11:636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, etal. Genetics 161, 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, etal. The EMBO journal 4, 1609-1614 (1985).

[0153] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431; US 8,440,432; US 8,450,471; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291 -F053-0197PCT / 24-151 -WO-PCT303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0154] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0155] Particular embodiments can use transposon-based systems as gene editing agents to mediate the integration of an artificial expression construct into cells. Generally, such methods will involve introducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired genetic element that is flanked by transposon repeats. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof and encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.

[0156] Several transposon / transposase systems have been adapted for genetic insertions of heterologous DNA sequences. Examples of such transposases include sleeping beauty (" SB”, e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol1; Tol2 (e.g., derived from medaka fish); TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), Helraiser, Himarl, Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmarl, and spinON. Transposases and transposon systems are further described in U. S. Pat. Nos.6,489,458; 7,148,203; 8,227,432; and 9,228,180. The transposon / transposase system is more commonly used for inserting a gene but could be used to knockdown or knockout a gene by inserting disruptive elements.

[0157] Any genetic engineering approach that can specifically target and modify, knockout, or knockdown CD90 expression can be used. Other examples of vectors include lentiviral vectors, retroviral vectors, and other viral vectors.

[0158] (V) CD90-Targeted Therapeutics. Particular embodiments provide CD90-targeted therapeutics that bind wildtype CD90 expressed by non-therapeutic HSPC but do not bind modified CD90 expressed by therapeutic HSPC. Because genetically modified HSPCs disclosed herein have either, no, little, or altered CD90 expression, CD90-targeted therapeutics do not substantially bind to genetically modified HSPCs as compared to wildtype CD90. Therefore, a method to enrich for genetically modified therapeutic HSPCs includes administering CD90-targeted therapeutics. CD90-targeted therapeutics include any agent that binds CD90 and elicits a cytotoxic effect against the CD90-expressing cell. In particular embodiments, the CD90-targeted therapeutics includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor.

[0159] (V-a) Anti-CD90 Antibodies. Antibodies can include monoclonal antibodies (mAbs), human or humanized antibodies, antibodies with multiple binding domains, bispecific antibodies, trispecific antibodies, tetraspecificF053-0197PCT / 24-151 -WO-PCTantibodies, multi-specific antibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, antibody fusions, single chain variable fragments (scFvs), polyclonal antibodies, and fragments thereof, respectively. In particular embodiments, antibodies can include oligomers or multiplexed versions of antibodies.

[0160] Conventional naturally occurring antibody structural units include a tetramer. Each tetramer includes two pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region that is responsible for antigen recognition and epitope binding. The variable regions exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions (CDRs). The CDRs from the two chains of each pair are aligned by the framework regions, which enables binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0161] Definitive delineation of a CDR and identification of residues including the binding site of an antibody can be accomplished by solving the structure of the antibody and / or solving the structure of the antibody-epitope complex. In particular embodiments, this can be accomplished by methods such as X-ray crystallography and cryoelectron microscopy. Alternatively, CDRs are determined by comparison to known antibodies (linear sequence) and without resorting to solving a crystal structure. To determine residues involved in binding, a co-crystal structure of the Fab (antibody fragment) bound to the target can optionally be determined. Software programs and bioinformatical tools, such as ABody Builder and Paratome can also be used to determine CDR sequences. CDR coding sequence can be determined by those skilled in the art by referencing CDR amino acid sequences.

[0162] In particular embodiments, an anti-CD90 antibody includes a variable heavy chain including a CDRH1 sequence including SYWIN (SEQ ID NO: 44), a CDRH2 sequence including KIFPSDSHTNYNQKFKD (SEQ ID NO: 45) and a CDRH3 sequence including DFDTQFYAMEY (SEQ ID NO: 62); and a variable light chain including a CDRL1 sequence including RASQDISNYLN (SEQ ID NO: 63), a CDRL2 sequence including YTSRLHS (SEQ ID NO: 64), and a CDRL3 sequence including QQGNTLPRT (SEQ ID NO: 65). These CDRs are based on Kabat but CDRs for other models can be determined by methods known to those of skill in the art.

[0163] In particular embodiments, the anti-CD90 antibody includes a variable heavy chain having the sequence: QVQLLQPGAELVRPGASVRLSCKTSGYTFTSYWINWVKQRPGQGLEWIGKIFPSDSHTNYNQKFKDKATLTVDKSSS TAYMQLISPTSEDSAVYYCTRDFDTQFYAMEYWGQGTSVTVSS (SEQ ID NO: 19) and a variable light chain having the sequence: DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTFKLLIYYTSRLHSGVPSRFSGGGSGTDYSLTISNLE KEDIATYFCQQGNTLPRTFGGGTRLEVK (SEQ ID NO: 21).

[0164] Antibodies can include a carboxy-terminal portion, can include a linker connecting the variable heavy chain to the variable light chain, or can form a multi-binding protein. The carboxy-terminal portion of each chain defines a constant region, which can be responsible for effector function particularly in the heavy chain (the Fc). Examples ofF053-0197PCT / 24-151 -WO-PCTeffector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0165] As used herein, a linker can include any chemical moiety that is capable of linking two components. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose. Linkers can also include spacer regions, linkers for the purpose of linking VL and VH of antibody derived binding domains of scFvs, and junction amino acids. Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker.

[0166] For example, in particular embodiments, linkers provide flexibility and room for conformational movement between different components. Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 46), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 47), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 48), or (Gly3Ser)n(Gly4Ser)1(SEQ ID NO: 49). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 50), (Gly4Ser)3(SEQ ID NO: 51), (Gly4Ser)2(SEQ ID NO: 52), (Gly4Ser)1(SEQ ID NO: 53), (Gly3Ser)2(SEQ ID NO: 54), (Gly3Ser)1(SEQ ID NO: 55), (Gly2Ser)2(SEQ ID NO: 56) or (Gly2Ser)1, GGSGGGSGGSG (SEQ ID NO: 57), GGSGGGSGSG (SEQ ID NO: 58), or GGSGGGSG (SEQ ID NO: 59).

[0167] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, lgG1, lgG2, lgG3, and lgG4. IgM has subclasses including lgM1 and lgM2. IgA is similarly subdivided into subclasses including lgA1 and lgA2.

[0168] Linker sequences that are used to connect the VL and VH of an scFv or other forms of binding fragments are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. Linker sequences of scFv are commonly Gly-Ser linkers. In particular embodiments, the linker sequence includes the sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 51) or GSDSNAGHASAGNTS (SEQ ID NO: 60). In particular embodiments, the linker includes a Whitlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 61).

[0169] Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti-CD90 antibody or fragment thereof disclosed herein. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least, three, at least four binding domains that bind an epitope on CD90. In particular embodiments, all ofthe binding domainsof a multi-domain binding molecule bind CD90. Methods of making multi-domain binding molecules are known to those of skill in the art.

[0170] In particular embodiments, binding domains disclosed herein can be used to create bi- tri, (or more) specificF053-0197PCT / 24-151 -WO-PCTimmune cell engaging molecules (e.g., immune cell engaging antibodies). An example of a multi-specific immune cell engaging molecule includes those which bind both CD90 and an immune cell (e.g., T-cell or NK-cells) activating epitope, with the goal of bringing immune cells to CD90-expressing cells to destroy the CD90-expressing cells. See, for example, US 2008 / 0145362. Such constructs are referred to herein as immune-activating multi-specifics or I-AMS). BiTEs® (Amgen, Thousand Oaks, CA) are one form of l-AMS. Immune cells that can be targeted for localized activation by I-AMS within the current disclosure include, for example, T-cells, natural killer (NK) cells, and macrophages. Binding domains that activate these immune cells are known to those of skill in the art. For example, examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD 137), OX40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3.

[0171] (V-b) Anti-CD90 Conjugates. An antibody conjugate or anti-CD90 conjugate refers to an antibody or binding fragment thereof disclosed herein linked to another entity other than an additional binding domain. The other entity can be, for example, a toxin, a drug, or a radioisotope. In particular examples, an antibody conjugate includes an immunotoxin, an antibody-drug conjugate (ADC), or an antibody-radioisotope conjugate.

[0172] In particular embodiments, antibodies are formed as immunotoxins. Anti-CD90 immunotoxins include an anti-CD90 antibody or binding fragment thereof disclosed herein conjugated to one or more cytotoxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, Bryodin 1, bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). The toxin may be obtained from essentially any source and can be a synthetic or a natural product.

[0173] Immunotoxins with multiple (e.g., four) cytotoxins per binding domain can be prepared by partial reduction of the binding domain with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA (diethylene triamine penta-acetic acid) in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the binding domain can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker-cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting immunotoxin mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, for example, through a 0.2 m filter, and can be lyophilized if desired for storage.

[0174] In particular embodiments, immunotoxins can include anti-CD90 antibodies or binding fragments thereofF053-0197PCT / 24-151 -WO-PCTconjugated to toxins for targeted CD90+cell killing.

[0175] In particular embodiments, immunotoxins can be conjugated to anti-CD90 reagents for targeted CD90+ apoptosis.

[0176] Antibody-drug conjugates (ADC) allow for the targeted delivery of a drug moiety to a CD90-expressing cell. In particular embodiments, the drug moiety can include a cytotoxic drug or a therapeutic drug or agent.

[0177] In particular embodiments, ADC refer to targeted molecules which combine properties of both antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigen-expressing cells (Teicher, B. A. (2009) Current Cancer Drug T argets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3): 154-169; Chari, R. V. (2008) Acc. Chem. Res. 41:98-107). See also Kamath & Iyer (Pharm Res. 32(11): 3470-3479, 2015), which describes considerations for the development of ADCs.

[0178] The drug moiety of the ADC may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Cytotoxic drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone, nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0179] ADC compounds of the disclosure include those with anti-CD90 activity. In particular embodiments, the ADC compounds include an antibody conjugated, i.e., covalently attached, to the drug moiety. In particular embodiments, the antibody is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments which are also referred to as binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkerscan be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker. The ADCs selectively deliver an effective dose of a drug to cells expressing CD90 (e.g., cancer cells) whereby greater selectivity, i.e. a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0180] To prepare ADCs, linker-drug conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 2006). Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such asF053-0197PCT / 24-151 -WO-PCTdithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco's phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of a linker-drug conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-drug conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 m filter, and can be lyophilized if desired for storage.

[0181] Anti-CD90-radioisotope conjugates (or antibody-radioisotope conjugates) include a CD90 antibody or binding fragment thereof linked to a radioisotope (i.e., radioactive isotope) for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / or treatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to a subject. Therapeutic nuclear medicine is often referred to as radiation therapy or radioimmunotherapy (RIT).

[0182] Examples of radioactive isotopes that can be conjugated to antibodies or binding fragments thereof of the present disclosure include actinium-225, iodine-131, arsenic-211, iodine-131, indium-111, yttrium-90, and lutetium-177, as well as alpha-emitting radionuclides such as astatine-211 or bismuth-212 or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the disclosure.

[0183] Examples of radionuclides that are useful for radiation therapy include225Ac and227Th.225Ac is a radionuclide with the half-life of ten days. As225Ac decays the daughter isotopes221Fr,213Bi, and209Pb are formed.227Th has a halflife of 19 days and forms the daughter isotope223Ra.

[0184] Additional examples of useful radioisotopes include228Ac,111Ag,124Am,74As,211As,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,47Ca,254Cf,242Cm,51Cr,67Cu,153Dy,157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,52Fe,59Fe,251Fm,252Fm,253Fm,66Ga,72Ga,146Gd,153Gd,68Ge,170Hf,171Hf,193Hg,193mHg,160mHo,130l,131l,135l,114mln,185lr,42K,43K,76Kr,79Kr,81mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,28Mg,52Mn,90Mo,24Na,95Nb,138Nd,57Ni,66Ni,234Np,15O,1820s,189mOs,1910s,32P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,81Rb,188Re,105Rh,211Rn,103Ru,35S,44Sc,72Se,153Sm,125Sn,91Sr,173Ta,154Tb,127Te,234Th,45Ti,166Tm,230U,237U,240U,48V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, and / or97Zr. Radioisotopes can be used as a type of detectable label called a radiolabel. In particular embodiments, a radioisotope includes131I,90Y, and / or211At. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0185] (V-c) Anti-CD90 Recombinant Receptors. Anti-CD90 antibodies and fragments thereof can be utilized within recombinant receptors such as chimeric antigen receptors (CAR).

[0186] CAR include several distinct subcomponents that allow genetically modified cells (e.g., regulatory T cells) toF053-0197PCT / 24-151 -WO-PCTrecognize and kill wild-type CD90-expressing cells. The subcomponents include at least an extracellular component and an intracellular component The extracellular component includes a binding domain that specifically binds a wildtype CD90 epitope that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted epitope.

[0187] In particular embodiments, the extracellular component of a recombinant receptor includes a binding domain that binds wild-type CD90. Particular embodiments of binding domains include an anti- wild-type CD90 antibody or fragment thereof.

[0188] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, transduction markers, and tags.

[0189] Spacer regions are used to create appropriate distances and / or flexibility between sub-components of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region.

[0190] Transmembrane domains typically have a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0191] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the expressed protein (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the expressed protein (e.g., up to 15 amino acids of the intracellular components).

[0192] Intracellular effector domains activate the expressing cell when the binding domain binds the antigen. The term "effector domain" is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0193] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRα, TCRβ, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatoryF053-0197PCT / 24-151 -WO-PCTdomains selected from: CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD19, CD4, CD8a, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0194] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcRp (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3<( retain at least one, two, three, or all ITAM regions.

[0195] A co-stimulatory domain is a domain whose activation can be included for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD137), OX40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 ), NKG2C, and a ligand that specifically binds with CD83.

[0196] Methods to genetically modify cells to express recombinant receptors are well-known in the art. Recombinant receptors can additionally include tags, such as the affinity tags elsewhere herein.

[0197] In particular embodiments, the CAR includes an anti- wild-type CD90 scFv. In particular embodiments, the anti-wild-type CD90 scFv include a variable heavy chain including the sequence set forth in SEQ ID NO: 19 and a variable light chain including the sequence set forth in SEQ ID NO: 21. In particular embodiments, the variable heavy chain is connected to the variable light chain through a linker. In particular embodiments, the linker includes a Whitlow linker. In particular embodiments, the linker includes a (Gly4Ser)3 (SEQ ID NO: 51) linker. In particular embodiments, the CAR includes an anti- wild-type CD90 binding domain, a transmembrane domain, a 4-1 BB costimulatory domain, and a CD3z stimulatory domain.

[0198] In particular embodiments, the CAR includes the sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0199] (VI) Compositions and Formulations. Any of the artificial expression constructs (e.g., gRNA, shRNA) or vectors described herein in any exemplary format can be formulated alone or in combination into compositions for administration to subjects. Certain examples may include formulations. Formulations include cells genetically modified with artificial expression constructs disclosed herein within a pharmaceutically-acceptable carrier.

[0200] Salts and / or pro-drugs of the active ingredients can also be used.

[0201] A pharmaceutically-acceptable salt includes any salt that retains the activity of the active ingredient and is acceptable for pharmaceutical use. A pharmaceutically-acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.F053-0197PCT / 24-151 -WO-PCT

[0202] Suitable pharmaceutically-acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0203] Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N, N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0204] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.

[0205] Exemplary generally used pharmaceutically-acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0206] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0207] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0208] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0209] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol

[0210] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0211] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weightF053-0197PCT / 24-151 -WO-PCTbased on therapeutic weight.

[0212] The compositions disclosed herein can be formulated for administration by, for example, injection, infusion, perfusion, or lavage. The formulations and / or compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intrauterine, intraplacental, intramuscular, intravesicular, and / or subcutaneous administration.

[0213] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0214] In particular embodiments, the compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0215] In certain examples, cells are genetically modified to knockdown, knockout, or modify CD90 expression as disclosed herein. Furthermore, cells can be genetically modified to include a therapeutic payload described herein. In these embodiments, genetically modified cells can be prepared as formulations for delivery in buffers such as Hanks' solution, Ringer's solution, or physiological saline. Cells can be genetically modified using methods known in the art. Exemplary targeted genetic engineering approaches include the use of CRISPR / Cas nuclease systems, zinc finger nucleases (ZFNs), and / or transcription activator like effector nucleases (TALENs). In particular embodiments, the cells are HSPCs genetically modified to express a therapeutic payload and / or modified CD90 expression (e.g., knockdown, knockout, or modified CD90 epitope binding).

[0216] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0217] In particular embodiments, cells are in a formulation volume of a liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. Hence, the density of administered cells is typically greater than 104cells / ml, 105cells / ml, 106cells / ml, 107cells / ml, or 108cells / ml.

[0218] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically-acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically-acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, compositions and formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by U. S.F053-0197PCT / 24-151 -WO-PCTFDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0219] (VIII) Kits. Also provided herein are kits including at least one artificial expression construct (e.g., gRNA, shRNA, Cas9, base editor, therapeutic payload) or vector disclosed herein. Kits may be formed with components to practice, for example, the methods described herein. In particular embodiments, the kit includes a gene editing component (e.g., CRISPR / Cas system components, base editing components) and a guide RNA as described herein. In particular embodiments, the kit includes an interfering RNA (e.g., shRNA). In particular embodiments, the kit includes a CD90-targeted therapeutic (e.g., anti-CD90 CAR). In particular embodiments, the kit includes a therapeutic payload. In particular embodiments, the kit includes i) cells expressing a modified CD90 and / or a therapeutic payload or ii) a composition to modify cells to have modified CD90 expression and / or include a therapeutic payload. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the method described herein.

[0220] The kit according to the present disclosure may also include instructions for carrying out the method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0221] (VIII) Methods of Use. Methods disclosed herein include genetically modifying cells to modify (i.e., knockdown, knockout, or modify CD90 epitope binding) CD90 expression and / or genetically modifying cells to include a therapeutic payload. Cells can be genetically modified in vivo or ex vivo. If genetically modified ex vivo, genetically modified cells can be administered to a subject. In particular embodiments, the subject having genetically modified cells can be administered a CD90-targeted therapeutic, wherein the CD90-targeted therapeutic binds and kills cells expressing wildtype CD90.

[0222] Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions and / or formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0223] An “effective amount” is the amount of a composition or formulation necessary to result in a desired physiological change in a subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an in vitro assay, an animal model, clinical study, or clinical study relevant to the assessment of an condition’s development, progression, and / or resolution, as well as the effectsF053-0197PCT / 24-151 -WO-PCTof the condition.

[0224] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventative treatment against a condition. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a condition.

[0225] A "therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.

[0226] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0227] In particular embodiments, methods include genetically modifying HSPCs to express a therapeutic payload and genetically modifying the HSPCs to knockdown, knockout, or alter (the epitope of) CD90 expression. In particular embodiments, HSPCs can be modified to treat genetic disorders. In particular embodiments, a genetic disorder can include hemoglobinopathies, immunodeficiencies, metabolic disorders, and other disorders related to HSPCs. In particular embodiments, the genetic disorder includes sickle cell disease, thalassemia (e.g, beta thalassemia, thalassemia major), aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, SCID (combined immunodeficiency, severe combined immunodeficiency), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies, adrenoleukodystrophies, epidermolysis bullosa, severe congenital neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, leukocyte adhesion deficiency, and the like. In particular embodiments, methods disclosed herein are used to treat sickle cell disease. In particular embodiments, methods disclosed herein are used to treat thalassemia.

[0228] To treat these genetic disorders, HSPCs are genetically modified with a therapeutic payload. In particular embodiments, the therapeutic payload includes one or more of globin family genes (e.g., of γ-globin, β-globin, and / or α-globin); yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40 (e.g, soluble CD40); CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; coagulation factor genes (factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII)); CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g, FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1;F053-0197PCT / 24-151 -WO-PCTiCaspase; IDUA; IDS; interleukins (IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, IL7RA); antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; and ZAP70.

[0229] In particular embodiments, methods include genetically modifying HSPCs to have modified expression of CD90 (e.g., knockout CD90, knockdown CD90, or alter epitope binding of CD90). In this way, genetically modified HSPCs can be identified by their modified CD90 expression. Similarly, non-genetically modified HSPCs can be identified, isolated, and / or killed by their native CD90 expression.

[0230] In particular embodiments, healthy HSPCs are genetically modified to have modified expression of CD90 (e.g., knockout CD90, knockdown CD90, or alter epitope binding of CD90) in a subject with cancer (e.g, a blood cancer). In particular embodiments, administration of a CD90-targeted therapeutic can kill non-genetically modified, cancer cells within the subject. This method can be used in subject's receiving a hematopoietic cell transplantation (HCT) to treat a blood cancer. The CD90-targeted therapeutic can target and kill any residual cancer cells while the transplanted cells establish themselves.

[0231] Therapeutically effective amounts provide for identification and / or killing of non-genetically modified HSPCs. Because genetically modified therapeutic HSPCs are unaffected by a CD90-targeted therapeutic, they will essentially replace non-genetically modified HSPCs. Because the genetically modified HSPCs have a corrected or therapeutic version of a gene (i.e., a therapeutic payload), HSPCs with the corrected or therapeutic gene are dominant in the body.

[0232] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes an IC50as determined in cell culture against a particular target Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of disease, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0233] Useful doses of active ingredients within compositions range from, for example, 0.1 to 5 g / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg,F053-0197PCT / 24-151 -WO-PCT1000 mg / kg or more.

[0234] Exemplary doses of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011cells.

[0235] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol.

[0236] The compositions and / or formulations described herein can be administered on top of the current standard of care for patients, or in combination or alternation with any other compound or therapy that the healthcare provider deems beneficial for the patient. The combination and / or alternation therapy can be therapeutic, adjunctive, or palliative.

[0237] In particular embodiments, the compositions disclosed herein are administered ex vivo or in vivo. In particular embodiments, the compositions disclosed herein are administered ex vivo.

[0238] The compositions and formulations described herein can be administered by, for example, injection, inhalation, infusion, perfusion, or lavage. Routes of administration can include intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, and / or subcutaneous administration.

[0239] (IX) Exemplary Embodiments. The Exemplary Embodiments below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.1. A method of enriching for genetically modified therapeutic cells within a subject, the method including: administering hematopoietic stem and progenitor cells (HSPCs) genetically modified to express a modified CD90 to a subject or administering a composition that genetically modifies HSPCs to express a modified CD90 to a subject; and administering a CD90-targeted therapeutic to the subject,thereby enriching for genetically modified therapeutic cells within the subject.2. The method of embodiment 1, further including genetically modifying the HSPCs before the administering.3. The method of embodiments 1 or 2, wherein the expression of the modified CD90 includes knocked down expression, knocked out expression, or expression of a mutated CD904. The method of embodiment 3, wherein the mutated CD90 includes an F80 mutation.5. The method of embodiment 4, wherein the F80 mutation includes an F80P or F80S mutation.F053-0197PCT / 24-151 -WO-PCT6. The method of any of embodiments 3-5, wherein the mutated CD90 includes a D98 mutation.7. The method of embodiment 6, wherein the D98 mutation includes a D98G mutation.8. The method of any of embodiments 3-7, wherein the mutated CD90 includes an E99 mutation.9. The method of embodiment 8, wherein the E99 mutation includes an E99G mutation.10. The method of any of embodiments 3-9, wherein the mutated CD90 includes at least one mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.11. The method of any of embodiments 3-10, wherein the mutated CD90 includes at least one mutation depicted for row A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, 02, D2, E2, F2, G2, H2, A3, B3, C3, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, H4, A5, B5, C5, D5, E5, F5, G5, H5, A6, B6, C6, D6, E6, F6, G6, H6, A7, B7, C7, D7, E7, F7, G7, H7, A8, B8, 08, D8, E8, F8, G8, H8, A9, B9, 09, D9, and / or E9 in FIG. 20E.12 The method of embodiments 2-11, wherein the genetically modifying includes introducing anti-CD90 shRNA to the cells.13. The method of embodiment 12, wherein the anti-CD90 shRNA includes a sequence as set forth in SEQ ID NO: 4.14. The method of any of embodiments 2-13, wherein the genetically modifying includes introducing guide RNA (gRNA) and a gene editing component to the cells.15 The method of embodiment 14, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 5 and the gene editing component includes a nuclease.16. The method of embodiment 15, wherein the nuclease includes Cas9 or Cpf1.17. The method of embodiment 14, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and the gene editing component includes a base editor.18. The method of embodiment 17, wherein the base editor includes an adenine base editor (ABE) or a cytosine base editor (CBE).19. The method of embodiments 17 or 18, wherein the base editor includes an adenine base editor (ABE). 20. The method of embodiment 19, wherein the ABE includes ABE8e.21. The method of embodiment 14, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 6 and the gene editing component includes a base editor.22. The method of embodiment 14, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 7 and the gene editing component includes a base editor.23. The method of embodiments 21 or 22, wherein the base editor includes an adenine base editor (ABE). 24. The method of embodiment 23, wherein the ABE includes ABE8e.F053-0197PCT / 24-151 -WO-PCT25. The method of any of embodiments 1-24, wherein the CD90-targeted therapeutic includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor.26. The method of embodiment 25, wherein the anti-CD90 antibody includes a multi-domain binding molecule.27. The method of embodiment 26, wherein the multi-domain binding molecule includes at least a first binding domain that binds CD90 and a binding domain that binds an immune cell activating epitope.28. The method of any of embodiments 25-27, wherein the anti-CD90 conjugate includes an anti-CD90 antibody conjugated to a toxin, a drug, or a radioisotope.29 The method of any of embodiments 25-28, wherein the anti-CD90 recombinant receptor includes an anti-CD90 chimeric antigen receptor (CAR).30. The method of embodiment 29, wherein the anti-CD90 CAR is expressed by a cell and includes an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular component includes a binding domain that binds CD90.31. The method of embodiment 30, wherein the binding domain includes an scFv.32 The method of embodiments 30 or 31, wherein the binding domain includes a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.33. The method of embodiments 31 or 32, wherein the scFv includes a linker.34. The method of embodiment 33, wherein the linker includes a Gly-Ser linker.35. The method of embodiments 33 or 34, wherein the linker includes a Whitlow linker.36 The method of any of embodiments 30-35, wherein the intracellular component includes an effector domain including: 4-1BB (CD137), CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRα, TCRβ, TRIM, Wnt, Zap70, or a combination thereof.37. The method of any of embodiments 30-36, wherein the intracellular component includes 4-1BB and CD3δ stimulatory domains.38. The method of any of embodiments 30-37, wherein the transmembrane domain includes a transmembrane region of: an α, β or ζ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.39. The method of any of embodiments 30-38, wherein the transmembrane domain includes a CD8 transmembrane domain.40. The method of any of embodiments 25-39, wherein the anti-CD90 recombinant receptor further includes a spacer region.41 The method of embodiment 40, wherein the spacer region includes a CD8 hinge region.42. The method of any of embodiments 23-33, wherein the anti-CD90 CAR includes a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequenceF053-0197PCT / 24-151 -WO-PCTidentity thereto.43 The method of any of embodiments 1-42, wherein the genetically modified cells correct a genetic defect in the subject.44. The method of embodiment 43, wherein the genetic defect includes an immunodeficiency, a hemoglobinopathy, a lysosomal storage disease, a bone marrow failure syndrome, or a congenital anemia.45. The method of embodiments 43 or 44, wherein the genetic defect includes beta-thalassemia or sickle cell disease.46 The method of any of embodiments 1-45, wherein the genetically modified cells express a therapeutic payload.47. The method of embodiment 46, wherein the therapeutic payload includes y-globin; β-globin; α-globin; γC; ABCA3; ABCD 1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.48. A method of treating a subject in need thereof including:administering to the subject a therapeutically effective amount of HSPCs genetically modified to have altered CD90 expression; oradministering to the subject a therapeutically effective amount of a composition that genetically modifies HSPCs to have altered CD90 expression.49. The method of embodiment 48, further including:administering a therapeutically effective amount of a CD90-targeted therapeutic to the subject.F053-0197PCT / 24-151 -WO-PCT50. The method of embodiments 48 or 49, further including genetically modifying the HSPCs to have altered CD90 expression.51. The method of embodiments 49 or 50, wherein the HSPCs are autologous or allogeneic to the subject. 52. The method of embodiment 50 or 51, wherein the genetically modifying HSPCs occurs ex vivo or in vivo. 53. The method of any of embodiments 50-52, wherein the genetically modifying HSPCs occurs ex vivo and the method further includes administering the genetically modified HSPCs to the subject before administering a CD90-targeted therapeutic.54 The method of any of embodiments 48-53, wherein the subject has a hemoglobinopathy, an immune deficiency, a clotting deficiency, or other blood disorder.55. The method of any of embodiments 48-54, wherein the subject has sickle cell disease.56. The method of any of embodiments 50-55, wherein the genetically modifying corrects expression of y-globin; β-globin; α-globin; γC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sILIRI, sIL1RII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLP0; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.57. The method of any of embodiments 48-56, wherein the altered CD90 expression includes knocked down expression, knocked out expression, or mutated CD90 expression.58. The method of embodiment 57, wherein the mutated CD90 includes an F80 mutation.59 The method of embodiment 58, wherein the F80 mutation includes an F80P or F80S mutation.60. The method of any of embodiments 57-59, wherein the mutated CD90 includes a D98 mutation.61. The method of embodiment 60, wherein the D98 mutation includes a D98G mutation.F053-0197PCT / 24-151 -WO-PCT62. The method of any of embodiments 57-61, wherein the mutated CD90 includes an E99 mutation.63 The method of embodiment 62, wherein the E99 mutation includes an E99G mutation.64. The method of any of embodiments 57-63, wherein the mutated CD90 includes at least one mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.65 The method of any of embodiments 57-64, wherein the mutated CD90 includes at least one mutation depicted for row A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, C2, D2, E2, F2, G2, H2, A3, B3, C3, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, H4, A5, B5, C5, D5, E5, F5, G5, H5, A6, B6, C6, D6, E6, F6, G6, H6, A7, B7, C7, D7, E7, F7, G7, H7, A8, B8, C8, D8, E8, F8, G8, H8, A9, B9, C9, D9, and / or E9 in FIG. 20E.66. The method of any of embodiments 48-65, wherein the genetically modifying includes introducing anti-CD90 shRNA into the HSPCs.67 The method of embodiment 66, wherein the anti-CD90 shRNA includes a sequence as set forth in SEQ ID NO: 4.68. The method of any of embodiments 48-67, wherein the genetically modifying includes introducing guide RNA (gRNA) and a gene editing component into the HSPCs.69. The method of embodiment 68, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 5 and the gene editing component includes a nuclease.70 The method of embodiment 69, wherein the nuclease includes Cas9 or Cpf1.71. The method of embodiment 68, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and the gene editing component includes a base editor.72. The method of embodiment 71, wherein the base editor includes an adenine base editor (ABE).73. The method of embodiment 72, wherein the ABE includes ABE8e.74. The method of embodiment 68, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 6 and the gene editing component includes a base editor.75. The method of embodiment 68, wherein the gRNA includes a sequence as set forth in SEQ ID NO: 7 and the gene editing component includes a base editor.76. The method of embodiments 74 or 75, wherein the base editor includes an adenine base editor (ABE). 77. The method of embodiment 76, wherein the ABE includes ABE8e.78. The method of any of embodiments 48-77, wherein the CD90-targeted therapeutic includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor.79. The method of embodiment 78, wherein the anti-CD90 antibody includes a multi-domain binding molecule.80. The method of embodiment 79, wherein the multi-domain binding molecule includes at least a first bindingF053-0197PCT / 24-151 -WO-PCTdomain that binds CD90 and a binding domain that binds an immune cell activating epitope.81 The method of any of embodiments 78-80, wherein the anti-CD90 conjugate includes an anti-CD90 antibody conjugated to a toxin, a drug, or a radioisotope.82. The method of any of embodiments 78-81, wherein the anti-CD90 recombinant receptor includes an anti-CD90 chimeric antigen receptor (CAR).83. The method of embodiment 82, wherein the anti-CD90 CAR is expressed by a cell and includes an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular component includes a binding domain that binds CD90.84. The method of embodiment 83, wherein the binding domain includes an scFv.85. The method of embodiments 83 or 84, wherein the binding domain includes a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.86. The method of embodiments 84 or 85, wherein the scFv includes a linker.87 The method of embodiment 86, wherein the linker includes a Gly-Ser linker.88. The method of embodiments 86 or 87, wherein the linker includes a Whitlow linker.89. The method of any of embodiments 83-88, wherein the intracellular component includes an effector domain including: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3(, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRo, TCR|3, TRIM, Wnt, Zap70, or a combination thereof.90. The method of any of embodiments 83-89, wherein the intracellular component includes 4-1BB and CD3δ stimulatory domains91. The method of any of embodiments 83-90, wherein the transmembrane domain includes a transmembrane region of: an α, β or ζ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.92. The method of any of embodiments 83-91, wherein the transmembrane domain includes a CD8 transmembrane domain93 The method of any of embodiments 78-92, wherein the anti-CD90 recombinant receptor further includes a spacer region.94. The method of embodiment 93, wherein the spacer region includes a CD8 hinge region.95. The method of any of embodiments 67-78, wherein the anti-CD90 CAR includes a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.96 A nucleic acid that knocks down CD90 expression including SEQ ID NO: 4.97. The nucleic acid of embodiment 96, wherein the nucleic acid is a short hairpin RNA (shRNA).98. A system that knocks out CD90 expression including a guide RNA having a sequence as set forth in SEQ IDF053-0197PCT / 24-151 -WO-PCTNO: 5 and a gene editing component.99 The system of embodiment 98, wherein the gene editing component includes a nuclease.100. The system of embodiment 99, wherein the nuclease includes Cas9 or Cpf1.101. A system that knocks out CD90 expression including:a guide RNA having a sequence as set forth in SEQ ID NO: 1; anda gene editing component.102. The system of embodiment 101, wherein the gene editing component includes a base editor.103. The system of embodiment 102, wherein the base editor includes an adenine base editor.104. The system of embodiment 103, wherein the adenine base editor includes ABE8e.105. A system that mutates CD90 including SEQ ID NO: 6 and a gene editing component.106. The system of embodiment 105, wherein the gene editing component includes a base editor.107. The system of embodiment 106, wherein the base editor includes an adenine base editor.108. The system of any of embodiments 107, wherein the adenine base editor includes ABE8e.109. The system of any of embodiments 105-108, wherein an anti-CD90 antibody does not bind the mutated CD90.110. The system of embodiment 109, wherein the anti-CD90 antibody includes 5E10.111. The system of any of embodiments 105-94, wherein the system mutates CD90 at F80.112. The system of embodiment 111, wherein the F80 mutation includes an F80P or F80S mutation.113. The system of any of embodiments 105-112, wherein the system mutates CD90 at D98.114. The system of embodiment 113, wherein the D98 mutation includes a D98G mutation.115. The system of any of embodiments 105-114, wherein the system mutates CD90 at E99.116. The system of any of embodiment 115, wherein the E99 mutation includes an E99G mutation.117. The system of any of embodiments 105-116, wherein the system mutates CD90 with a mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.118. The system of any of embodiments 105-117, wherein the system mutates CD90 with a mutation depicted for row A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, C2, D2, E2, F2, G2, H2, A3, B3, C3, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, H4, A5, B5, C5, D5, E5, F5, G5, H5, A6, B6, C6, D6, E6, F6, G6, H6, A7, B7, C7, D7, E7, F7, G7, H7, A8, B8, C8, D8, E8, F8, G8, H8, A9, B9, C9, D9, and / or E9 in FIG. 20E.119. A system that mutates CD90 including SEQ ID NO: 7 and a gene editing component.120. The system of embodiment 119, wherein the gene editing component includes a base editor.121. The system of embodiment 120, wherein the base editor includes an adenine base editor.122. The system of embodiment 121, wherein the adenine base editor includes ABE8e.F053-0197PCT / 24-151 -WO-PCT123. The system of any of embodiments 119-122, wherein an anti-CD90 antibody does not bind the mutated CD90.124. The system of embodiment 123, wherein the anti-CD90 antibody includes 5E10 or FAB20671R.125. The system of any of embodiments 119-124, wherein the system mutates CD90 at F80.126. The system of embodiment 125, wherein the F80 mutation includes an F80P or F80S mutation.127. The system of any of embodiments 119-126, wherein the system mutates CD90 at D98.128. The system of embodiment 127, wherein the D98 mutation includes a D98G mutation.129. The system of any of embodiments 119-128, wherein the system mutates CD90 at E99.130. The system of any of embodiment 129, wherein the E99 mutation includes an E99G mutation.131. The system of any of embodiments 119-130, wherein the system mutates CD90 with a mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.132. The system of any of embodiments 119-131, wherein the system mutates CD90 with a mutation depicted for row A1, B1, C1, D1, E1, F1, G1, H1, A2, B2, C2, D2, E2, F2, G2, H2, A3, B3, C3, D3, E3, F3, G3, H3, A4, B4, C4, D4, E4, F4, G4, H4, A5, B5, C5, D5, E5, F5, G5, H5, A6, B6, C6, D6, E6, F6, G6, H6, A7, B7, C7, D7, E7, F7, G7, H7, A8, B8, C8, D8, E8, F8, G8, H8, A9, B9, C9, D9, and / or E9 in FIG. 20E.133. A vector including the nucleic acid or the system of any of the preceding embodiments.134. The vector of embodiment 133, further including a therapeutic payload.135. The vector of embodiment 134, wherein the therapeutic payload includes y-globin; p-globin; a-globin; yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXII I; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1;F053-0197PCT / 24-151 -WO-PCTRPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; S0D1; SOX2; STIM 1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.136. A nanoparticle encapsulating the vector of any of embodiments 133-135.137. A hematopoietic stem and progenitor cell (HSPC) genetically modified to express the vector of any of embodiments 133-135.138. The HSPC of embodiment 137, wherein a CD90-targeted therapeutic does not recognize or bind the HSPC.139. The HSPC of embodiment 138, wherein the CD90-targeted therapeutic includes an anti-CD90 antibody, an anti-CD90 conjugate, or an anti-CD90 recombinant receptor.140. The HSPC of embodiment 139, wherein the anti-CD90 antibody includes a multi-domain binding molecule.141. The HSPC of embodiment 140, wherein the multi-domain binding molecule includes at least a first binding domain that binds CD90 and a binding domain that binds an immune cell activating epitope.142. The HSPC of any of embodiments 139-141, wherein the anti-CD90 conjugate includes an anti-CD90 antibody conjugated to a toxin, a drug, or a radioisotope.143. The HSPC of any of embodiments 139-142, wherein the anti-CD90 recombinant receptor includes an anti-CD90 chimeric antigen receptor (CAR).144. The HSPC of embodiment 143, wherein the anti-CD90 CAR is expressed by a cell and includes an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular component includes a binding domain that binds CD90.145. The HSPC of embodiment 144, wherein the binding domain includes an scFv.146. The HSPC of embodiments 144 or 145, wherein the binding domain includes a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.147. The HSPC of embodiments 145 or 146, wherein the scFv includes a linker.148. The HSPC of embodiment 147, wherein the linker includes a Gly-Ser linker.149. The HSPC of embodiments 147 or 148, wherein the linker includes a Whitlow linker.150. The HSPC of any of embodiments 144-149, wherein the intracellular component includes an effector domain including: 4-1BB (CD137), CD3y, CD35, CD3E, CD3, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRα, TCRβ, TRIM, Wnt, Zap70, or a combination thereof.151. The HSPC of any of embodiments 144-150, wherein the intracellular component includes 4-1BB and CD3δ stimulatory domains.152. The HSPC of any of embodiments 144-151, wherein the transmembrane domain includes a transmembrane region of: an α, β or ζ chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.F053-0197PCT / 24-151 -WO-PCT153. The HSPC of any of embodiments 144-152, wherein the transmembrane domain includes a CD8 transmembrane domain154. The HSPC of any of embodiments 109-123, wherein the anti-CD90 recombinant receptor further includes a spacer region.155. The HSPC of embodiment 124, wherein the spacer region includes a CD8 hinge region.156. The HSPC of any of embodiments 113-125, wherein the anti-CD90 CAR includes a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.157. The HSPC of any of embodiments 137-156, wherein the HSPC is further modified to express a therapeutic payload.158. The HSPC of embodiment 157, wherein the therapeutic payload includes y-globin; β-globin; α-globin; γC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1 Ra, sILIRI, sILIRII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.159. The HSPC of any of embodiments 137-158, wherein the HSPC is autologous or allogeneic to a subject. 160. The HSPC of any of embodiments 137-159, wherein HSPC is in vivo or ex vivo.161. A composition including the vector of any of embodiments 133-135 or the nanoparticle of embodiment 136.162. A formulation including a population of cells including the HSPC of any of embodiments 137-160 and a pharmaceutically acceptable carrier.163. A kit including the nucleic acid; the system; the vector, the nanoparticle, the HSPC, the composition, and / orF053-0197PCT / 24-151 -WO-PCTthe formulation of any of the preceding embodiments 1-163.164. The kit of embodiment 133, further including a CD90-targeted therapeutic.

[0240] (X) Experimental Example. CD90 regulates developmental stage-specific hematopoiesis: implications for stem cell gene therapy and immunotherapy.

[0241] Abstract. Transplantation of genetically modified hematopoietic stem cells (HSCs) enables curative therapies for genetic disorders and malignancies. As such, expression of CD90 is canonically used for the characterization and quantification of HSCs. Here, it is reported that CD90 expression is vestigial to the self-renewal and differentiation of human and nonhuman primate adult-HSCs. Based on these findings, CD90 was utilized as an adult-HSC enrichment handle for gene therapy and cancer immunotherapy by genetic ablation of CD90. Anti-CD90 directed immunotherapy both facilitated positive selection of gene modified HSCs and demonstrated potent anti-tumor effects in a xenograft model without perturbing terminally differentiated lineages. Thus, anti-CD90 immunotherapy is applicable for targeting normal and malignant stem cells.

[0242] Main Text. Hematopoiesis occurs through a hierarchical process of differentiation beginning with multipotent self-renewing hematopoietic stem cells (HSCs) that persist throughout life and ending with transient terminally differentiated unipotent cells. This hierarchical differentiation of the hematopoietic system can be exploited in the clinic for the treatment of benign and malignant hematology. A patient's hematopoietic system can be cured by replacing diseased HSCs with healthy HSCs from an HLA-matched donor (i.e., allogeneic transplantation, allo-HSCT) (Fenske, et al., Biology of Blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation, 22:1543-1551 (2016); Gyurkocza, et al., Expert Rev. Hematol., 3:285-299 (2010)). However, allo-HSCT is limited by the need of a suitably matched donor and can also be associated with significant toxicities such as graft-versus-host disease (GVHD) (Hamilton, American Society of Hematology Education Program, 2018:228-253 (2018)). To address this, the advent of gene modification therapies has paved the way for correcting disease-driving mutations in a patient’s own HSCs via gene editing or gene therapy and subsequent re-transfusion into patients (i.e., autologous transplantation), allowing for treatment without the major toxicities associated with allo-HSCT. Therefore, efficient isolation, modification, and / or enrichment of HSCs has significant therapeutic potency by enabling the permanent replacement of pathologic hematopoiesis.

[0243] The CD34+CD45+CD38low / -CD90+ / -CD45RA-population is highly enriched for long-term (LT) repopulating HSCs with a prevalence of 1 in 100 (Majeti, et al., Cell stem cell, 1:635-645 (2007). Concomitant expression of either EPCR or CD49f increases the prevalence of LT-HSCs (Chagraoui, et al., PLoS One, 14:e0224900 (2019); Tomellini, et al., Cell Reports, 28:1063-1073 (2019)). However, the expression of CD90 (THY1) on HSC subsets strongly correlates with increased HSC engraftment kinetics and self-renewal even within these stringently defined subsets (Radtke, et al., Science Translational Medicine, 9 (2017); Radtke, et al., Blood, 142:33-43 (2023); Radtke, et al., Molecular Therapy, Methods & Clinical Development, 18:679-691 (2020)). Interestingly, CD90 is also a known cancer stem cell marker, with high antigen expression associated with metastases and chemoresistance of solid andF053-0197PCT / 24-151 -WO-PCThematologic tumors (Bowman, et al., Cancer Cell, 42:1955-1969 (2024); Tang, et al., Cancer Res., 73:2322-2332 (2013); Yamashita, et al., Hepatology, 57:1484-1497 (2013)). Thus, anti-CD90 therapeutics would be a potent anticancer strategy with broad utility. However, a limitation of such a strategy would be its detrimental impact on healthy CD90+HSCs. As such, the experiment described here aimed to evaluate whether CD90 was essential for HSC functionality to explore the potential of anti-CD90 immunotherapies for both enrichment of gene-modified HSCs and cancer stem cell immunotherapy strategies. Utilizing shRNA and base editing strategies, CD90 expression was ablated on human and nonhuman primate (NHP) HSCs and HSC-precursor cells. The impact of CD90 genetic modification on HSC functionality and ontogeny was elucidated. Also developed was an anti-CD90 chimeric antigen receptor (CAR90)-T cell platform that was evaluated for its functionality in vitro and in vivo.

[0244] Results. CD90 knockdown has no impact on HSC engraftment, differentiation, and self-renewal. CD90 expressing CD34+hematopoietic stem and progenitor cells (HSPCs) are exclusively responsible for short-term recovery and long-term reconstitution after autologous stem cell transplantation (Humbert, et al., Mol. Ther. Methods. Clin. Dev., 8:75-86 (2018); Radtke, et al., Science translational medicine, 17:eadhn2601 (2025)). However, the role and function of CD90 in hematopoiesis remains unknown. To assess whether CD90 expression is necessary for HSC functionality, shRNAs were designed and tested targeting the CD90 locus in the CD90+K562 cell line (FIG. 20D). Lentivirus-mediated delivery of the CD90-shRNA-1 displayed the highest ablation of CD90 surface expression as measured by flow-cytometry (FIG. 2A). To stably knock down CD90 in HSCs, a lentiviral vector (CD90-KD) was designed co-expressing the CD90-shRNA-1 driven by the U6 promoter and the reporter gene mCherry under Ef1a promoter regulation. Cord blood (CB)-derived CD34+HSPCs were transduced with CD90-KD or a control scramble shRNA (Control) and transplanted into NBSGW neonates as previously described (FIG. 1A) (Choo, et al., Blood Adv., 8, 916-926 (2024).

[0245] Peripheral blood (PB) chimerism was monitored via biweekly bleeds, and the frequency of mCherry+human cells was evaluated by flow cytometry. No impact of CD90-KD was observed on humanization or engraftment potential of any lineages including monocytes, granulocytes, B cells, T cells, and NK cells (FIG. 1B; FIG. 2B). To specifically evaluate the impact of transduction, mCherry expression in lineages were analzyed, revealing stable mCherry signal in mice engrafted either with control or CD90-KD HSPCs (FIG. 1B: FIG. 2C). No lymphoid or myeloid bias of mCherry expression was observed comparing CD19+ / 20+B cells with CD14+monocytes (FIG. 1B).

[0246] To evaluate whether CD90-KD has an impact on HSC and progenitor engraftment, the bone marrow (BM) of transplanted mice at necropsy 20 weeks post-transplant by flow-cytometry was assessed. No difference in the total abundance of CD34+HSPCs or CD34+CD38- multipotent progenitors (MPPs) was observed in control mice or CD90-KD mice (FIGs. 1C-1D; FIG. 2D). However, confirming CD90-KD, on average 55% reduction (P=0.0318) of CD90 expression within mScarlet+CD45+HSPCs was observed as compared to control BM (FIG. 1D) with similar trends in a second cohort using a different cord blood donor (FIG. 2D). To evaluate whether the reduction of CD90+ cells in CD90-KD mice was due to an outright loss of HSCs or the ablation of CD90 surface expression, the abundance ofF053-0197PCT / 24-151 -WO-PCTCD34+CD38~CD133+and CD34+CD38-CD117+cells were analyzed as surrogate markers for HSC immunophenotype. No difference was observed in the abundance of CD34+CD38 CD133+or CD34+CD38 CD117+cells in CD90-KD mice compared to control mice, confirming successful removal of CD90 protein rather than a loss of HSCs (FIG. 1D).

[0247] In summary, CD90-KD HSPCs show no difference in engraftment, lineage differentiation, or self-renewal compared to their wild-type counterparts despite the lack of CD90 expression. Through investigation of surrogate markers for HSC immunophenotype, it was demonstrated that CD90-KD limited CD90 expression without disturbing the total number of engrafted HSCs.

[0248] Base-editing to ablate CD90 expression does not perturb function of human HSPCs. Given known limitations of shRNA-mediated knockdown approaches, such as incomplete knockdown and need for constitutive shRNA expression in all blood cell progeny, nuclease-based editing strategies to permanently ablate CD90 expression were next evaluated. To accomplish this, an adenine base editor (ABE) was developed to knock out CD90 expression (CD90-KC) by disrupting the CD90 locus, mutating the start codon (FIG. 20D). The CRISPR RGEN Tool, BE-Designer (Li, et al., Genomics Proteomics Bioinformatics, 21:108-126 (2023)), was used to develop gRNAs that target the start codon of the CD90 allele utilizing ABE8E-NG. Two gRNAs were identified and screened for their capacity to target the CD90 start codon, with one, referred to asCD90BE-g2, showing 50% editing in HEK293T cells (FIG. 2E). The feasibility of this strategy in human CD34+ HSPCs were further evaluated. EditR analysis of genomics data showed an average of 18% editing at the CD90 start codon (A4) with bystander editing occurring at all four adenines A2, A3, A5, and A6 at varying degrees (FIG. 3B).

[0249] The maintenance of HSPC functionality after CD90 knockout was next confirmed. Adult G-CSF-mobilized peripheral blood CD34+HSPCs were treated with CD90BE-g2, transplanted into busulfan-conditioned NBSGW mice, and animals were followed for 20 weeks (FIG. 3C). As with CD90 knockdown, no impact on overall humanization or skewing of hematopoietic in CD90-KO mice relative to untreated controls was observed (FIG. 3D).

[0250] Twenty weeks post-transplant, the BM for human engraftment, lineage distribution, HSPC abundance, and editing frequency were assessed. Equal engraftment of control HSPCs or CD90-KO HSPCs in NBSGW mice was observed. On average a 31 % decrease of CD90+cells in CD90-KO mice in comparison to the controls was observed, whereas the variance for other phenotypic HSPC subsets ranged from 10-24% on average between the two groups. Importantly, CD90 editing in the BM persisted through the duration of the study. No direct correlation in between the editing efficiency of CD90 and the overall frequency of CD90+HSCs was seen.

[0251] CD90-ablated nonhuman primate HSPCs engraft and persist. The next question was to evaluate whether ABE-mediated CD90 knockout (KO) would translate to an immunocompetent nonhuman primate (NHP) model, allowing for longitudinal tracking of hematopoietic recovery and potentially revealing insidious impacts of CD90-KO. Application of previously described CD90-KO ABE approach in NHP cells demonstrated up to 80% editing in primary cells (FIG. 4A). Next, two busulfan-conditioned Rhesus macaques were transplanted with CD34+HSPCs knocked out for CD90 and simultaneously edited at the hemoglobin beta chain (HBG) promoter to induce fetal hemoglobin (HbF)F053-0197PCT / 24-151 -WO-PCTexpression (FIG. 4B) (Borot, et al., Nature Communications, 16:4899 (2025)). Engraftment and gene editing chimerism were tracked through cell blood counts (CBCs), flow cytometric assessment of the PB, and genomic sequencing of the CD90 and -175 HBG loci.

[0252] Following editing, a 40% decrease in the CD34+CD90+CD45RA- population was observed in the infusion product for both animals (FIG. 4C). Successful multiplex editing of the transplant infusion product was confirmed by Sanger sequencing showing 84% and 73% editing of CD90 at the A4 site in A23150 (NHP#1) and A24048 (NHP#2), respectively. Editing at the HBG promoter reached 83% in NHP#1 and 63% in NHP#2 (FIG. 4D). Assessment of the infusion product in colony-forming cell (CFC) assays revealed no apparent impact of editing (FIG. 5A). To confirm accurate and correct dosing of busulfan [5.5mg / kg], pharmacokinetic analysis was carried out (FIG. 4E). Both animals peaked at the expected 5.5 ng / ml with an almost identical AUC of 3093 and 3046 μMol for NHP#1 and NHP#2, respectively. Following HSCT, both animals followed typical hematopoietic recovery as assessed by time to neutrophil recovery (NHP#1: day 10, NHP#2: day 10) as well as time to platelet recovery (NHP#2: day 21) relative to previous NHPs treated with busulfan-mediated HSCT (5.0 ± 9.3d and 41.6 ± 18.0d respectively, FIG. 4F) (Murray, et al., Mol. Ther Methods. Clin Dev., 30:276-287 (2023)). Of note, NHP#1 did not experience any thrombocytopenia and was euthanized at 31 days post-transplant (DPT) due to infectious complications unrelated to hematopoietic recovery or gene-editing of CD90. Similarly, no other delays in recovery of total white blood cells (WBC), red blood cells (RBCs), or lymphocytes compared to historical busulfan-conditioned Rhesus macaque transplants were observed (FIG. 3D) (Murray, et al., Mol. Ther. Methods. Clin. Dev., 30:276-287 (2023)). Comprehensive phenotypic assessment of PB WBCs by flow cytometry demonstrated multilineage recovery (FIG. 4G). As anticipated, previously CD90-expressing granulocytes and monocytes were replaced by CD90- progeny and persisted at >64% (FIG. 4H). Concomitantly, flow-cytometric analysis of RBCs revealed permanent and stable reactivation of HbF expression (FIG. 4I). Assessment of editing of CD90 and HBG tightly corresponded with the flow cytometric data, with editing first observable three days post-transplant (DPT) and cresting at 77% and 67% editing at 30 and 14 DPT, respectively (FIG. 4J). Editing of CD90 and HBG1 / 2 in PB WBCs stabilized at 40% and 20%, respectively. Bystander editing for CD90 was found at position A9 and A2 above 1 % throughout the follow-up. Bystander editing almost exclusively co-occurred with on-target editing at A6. Next, successful editing in multipotent HSCs was confirmed by analyzing FACS-purified lineages. Editing in granulocytes, monocytes, and B cells matched the editing frequencies observed in bulk WBCs. As previously observed in historic animals undergoing autologous HSC gene therapy (Radtke, et al., Science translational medicine, 9 (2017)), editing in T cells was lower due to the delayed recovery of the adaptive immune system.

[0253] Phenotypic analysis of engrafted CD34+HSCPs in A23150 and A24048 at 1- and 3-month post-transplant, respectively, showed the expected decrease in the frequency of CD90+CD45RA- cells representing to only 1.65% and 3.65% of CD34+cells in contrast to an average of 15-20% in untreated historic controls (Radtke, et al., Science translational medicine, 9 (2017)) (FIG. 4K). Functional assessment of FACS-purified HSPCs in CFC assays confirmed erythro-myeloid differentiation potential within the expected subsets. However, substantially increased secondary CFCF053-0197PCT / 24-151 -WO-PCTpotential, a unique hallmark of CD90+HSCs, was found in the CD45RA-CD90- subset, confirming engraftment and persistence of CD90-KO HSCs (FIG. 4L). Lastly, genomic assessment of phenotypic HSPC subsets confirmed engraftment of multiplex gene-edited HSCs in the BM. Of note, CD90 editing was found in CD90+cells suggesting normal expression of CD90 in mono-allelic edited cells, whereas a strong enrichment of CD90 editing was found in the CD90~CD45RA“ subsets containing all biallelic edited HSCs (FIG. 4M).

[0254] Taken together, these data demonstrate the translatability of ABE-mediated CD90-KO in a preclinical, immunocompetent NHP model confirming that the loss of CD90 did not adversely impact the capacity for HSCs to engraft and attain multilineage fates.

[0255] Ectopic modulation of CD90 perturbs endothelial arterialization during embryonic development. In addition to its expression on adult HSCs, CD90 is highly abundant in the arterial hemogenic endothelium (HE) of Carnegie Stage 15 human dorsal aorta gonad mesonephros (AGM) tissue, from which HSCs originate during embryonic development (Hou, et al., Blood Sci., 6:e00199 (2023)). These findings have been recapitulated utilizing in vitro hematopoietic differentiation models of human pluripotent stem cells (hPSCs) (Fowler, et al., Dev. Cell, 59:1110-1131 (2024); Ng, et al., Nat. Biotechnol., 43:1274-1287 (2025); Sugimura, et al., Nature, 545:432-438 (2017); Uenishi, et al., Nature Communications, 9:1828 (2018); Calvanese, et al., Nature, 604: 534-540 (2022)). As the endothelial to hematopoietic transition (EHT) occurs, nascent HSPCs emerging from CD90 positive arterial HE lower their CD90 expression. Given the dynamic nature of CD90 during prenatal hematogenesis and vestigial role of CD90 in adult hematopoiesis, questions arose about the impact of CD90 knockdown on arterial HE formation and EHT.

[0256] Utilizing a monolayer PSC differentiation protocol that recapitulates human AGM-like hematovascular development (Palpant, et al., Nature Protocols, 12:15-31 (2017), day 5 / 6 CD34+immature endothelial cells (D5 / 6 ECs) were transduced with either scramble shRNA or CD90-KD shRNA lentivirus and differentiated towards arterial / hemogenic endothelium (AE / HE) and subsequent hematopoietic progenitors (HPs). Differentiation of transduced cells was tracked utilizing flow cytometry on day 9 (D9) of the differentiation protocol, coinciding with arterialization of the endothelial populations.

[0257] Transduction of D5 / 6 ECs with CD90-KD shRNA was highly efficient, with >90% of all cells expressing the mCherry transgene. Importantly, CD90 expression was significantly reduced in the CD90-KD condition within mCherry+cells, averaging 40%, whereas >90% of untreated and scramble-treated cells retained CD90 expression (FIG. 4B). Knockdown of CD90 led to a significantly reduced percentage of CD34+VE-Cadherin+DLL4+cells in comparison to untreated or scramble shRNA cells (FIG. 4B), with lower VE-Cadherin (VEC) expression and drastically lower deltalike ligand 4 (DLL4) expression but maintained CD34 expression. These findings suggest a role for CD90 in arterial maturation from endothelial precursors.

[0258] To better understand the mechanism by which CD90 impacts endothelial maturation, single-cell RNA-sequencing was performed on D9-differentiated cells and compared them to scramble-treated cells. Both samples primarily contributed to a dominant cluster of cells expressing endothelial-specific markers CDH5 and KDR, while veryF053-0197PCT / 24-151 -WO-PCTfew cells in either sample expressed hematopoietic-specific marker SPN (CD43) (FIGs. 4C-4E). No significant difference in expression of the pan-endothelial gene KDR was observed, whereas CD90 was specifically downregulated in the CD90-KD cells, as expected (FIGs. 4E-4F). A gene set enrichment analysis were performed on the differentially expressed genes between each sample to better understand the pathways affected by CD90 knockdown). The top enriched gene sets were related to processes involved in endothelial maturation, including vascular development, cell adhesion, and VEGFR2 (KDR) signaling. Notably, gene sets associated with known CD90-interacting pathways and also implicated in arterial HE maturation and EHT, such as integrin-mediated adhesion, RHO GTPase signaling, Focal Adhesion Kinase (FAK) activity, and VEGFA signaling, were significantly downregulated by CD90 knockdown (Sauzay, et al., Front Cell Dev. Biol., 7:66 (2019); Leyton, et al., Front Cell Dev. Biol., 7:132 (2019)).

[0259] Taken together, these data suggest that while dispensable for adult hematopoiesis, CD90 plays a role in modulating key pathways involved in HE maturation and EHT, which are essential for establishing hematopoiesis in utero. This finding provides crucial insight into the potential function of CD90 and paves the way for CD90 ablation in adult HSCs for therapeutic purposes.

[0260] In vitro validation of CD90-directed CAR-T cells. With the ability to ablate CD90 in adult HSCs without deleterious impact on engraftment or multilineage differentiation potential, a means to eliminate CD90+ cells was next devised. To this end, CD90-directed single chain variable fragments (scFvs) were installed into a 2nd generation chimeric antigen receptor (CAR) backbone with CD28 co-stimulation and a truncated EGFR (tEGFR) reporter (FIG.14A). scFvs were generated from a previously characterized human / NHP cross-reactive anti-CD90 antibody (5E10) by varying the order of the heavy chain (VH) and light chain (VL) as well as using two different linkers, G4S or Whitlow (Breckmueller, et al., Mol. Ther., 31:2901-2913 (2023). Successful transduction of T cells was confirmed by flow cytometry analysis of tEGFR, which demonstrated no significant differences between different CD90 CAR (CAR90) constructs. Secondary confirmation of CAR identity was accomplished by linker staining, demonstrating co-positivity of all constructs except CAR90.4, potentially due to confirmational inaccessibility of the linker (FIG. 14B). Cytotoxic function of CAR90 T cells was first vetted in vitro by Incucyte and ImageXpress video, which demonstrated dosedependent killing of engineered CD90+, but not CD90-, HEK293T cells relative to untransduced (UTD) T cells (FIG.14C; FIGs. 15A-15B). Secondary assessment of T-cell activation revealed that CAR90 T cells co-cultured with CD90+ cells produced the pro-inflammatory cytokines IL-2, TNFa, and IFN-y (FIG. 15C).

[0261] Having demonstrated the functionality and specificity of CAR90 in cell lines, the impact on primary HSPCs, which express CD90 at low density (1,000-3,000 molecules per cell) was determined. To this end, CAR90 T cells were cultured with HSPCs for 48h before immunophenotyping by flow-cytometry and plating into primary and secondary CFG assays. All CAR90 T-cell constructs led to production of pro-inflammatory cytokines including IFN-y, IL-2, and TNFa (FIG. 14D: FIG. 15D). In accordance with this, up-regulation of the T-cell activation-associated protein, CD25 was observed (FIG. 14E). Interestingly, co-culture of HSPCs with UTD T cells led to a partial decrease of CD90 mean fluorescence intensity (MFI); however, co-culture with CAR90 T cells led to a significant further reduction of CD90 MFIF053-0197PCT / 24-151 -WO-PCT(FIG. 14E). Utilizing CD133 as a secondary marker of HSC immunophenotype, a similar decrease of MFI in CAR90-treated conditions relative to both no T cell and UTD conditions was observed (FIG. 14E). Most importantly, while HSPCs from both UTD and CAR90 co-cultures were able to give rise to multilineage colonies in primary CFCs, CAR90 entirely ablated the most primitive HSCs exclusively capable of forming colonies in secondary CFC assays, consistent with loss of the phenotypic HSC population (FIG. 14F). Finally, in order to evaluate the utility of CAR90 to target CD90-expressing cancers, the CAR90.2 T cells were co-cultured with CD90– ML-1 or CD90+Jurkat cells. While a nonsignificant reduction of total cell viability in ML-1 co-culture was observed, CAR90 T cells led to complete killing of Jurkat cells at all effector to target (E: T) ratios tested (FIG. 14G)

[0262] CD90 editing protects modified HSPCs from CAR90T cells in vivo. To evaluate whether CD90 ablation confers protection from CAR90 T cells in vivo and can act as an enrichment handle for therapeutic purposes, adult NBSGW mice were engrafted with human HSPCs edited with CD90BE and at the HBG-175 site similar to prior studies in NHP (FIG. 16A). Eight to 10 weeks post-humanization, engrafted mice were treated with CAR90.1 (hereafter referred to as CAR90) T cells and monitored for 14 days before necropsy and collection of HSPCs for FACS analysis, CFC assays, and genomics analysis.

[0263] Successful CAR90 generation ex vivo was confirmed by flow cytometric analysis of tEGFR, demonstrating >85% tEGFR+(FIG. 16B). CAR90 T cells expanded in vivo indicated by the increased proportion of T cells (40% vs.20%, FIG. 16C), decrease of human B cells (30% vs. 60%, FIG. 16C), and concomitant CAR marking in the PB (5% vs. 0%, FIG. 16D) in CAR-treated mice relative to UTD. No adverse events were recorded in animals due to CAR T administration. At necropsy, CAR90 T cells were identifiable in the spleen and BM (2% and 0.2%, respectively, FIG.16E) Treatment with CAR90 did not significantly impact the total number of hCD45+cells in the BM. However, CAR90 did lead to significant decreases in CD90+HSCs (0.01% vs. 0.0025%), MPPs (0.1% vs. 0.05%), and total HSPCs (1% vs. 0.5%, FIG. 16F) in CAR-treated mice relative to UTD. To evaluate the maintenance of the self-renewing HSC population and the selection of CD90-modified cells, CD34+CD133+CD38−CD45RA−HSCs were plated into primary and secondary CFC assays. CAR90 did not impact colony-forming potential nor skew HSC differentiation in primary and secondary cultures (FIG. 16G). However, assessment of editing revealed enrichment of CD90 edited cells in primary culture of CAR90-treated animals compared to UTD-treated mice and a significant 2.96-fold enrichment of CD90-KO alleles in secondary culture (FIG. 16H). Finally, the question of whether enrichment of CD90 edited cells correlated with the multiplex edited HBG allele was asked. A 2.18-fold increase in base-edited HBG alleles in CAR90-treated mice compared to UTD mice was similarly observed (FIG. 16H). Thus, CD90 ablation via base editing followed by CAR90 application can be utilized as a gene-enrichment handle to increase the frequency of gene modification for therapeutic gene edits.

[0264] CD90 editing of HSCs enables a leukemia treatment strategy. With the safety of CAR90 demonstrated in vivo, next the capacity for CAR90 to rescue a human leukemia xenograft model was evaluated. To this end, adult NBSGW mice were engrafted with G-CSF-mobilized human CD90-KO HSPCs for 8-10 weeks. Mice were then retro-orbital lyF053-0197PCT / 24-151 -WO-PCTinjected with GFP+Luciferase+CD90+Jurkat cells, followed four days later by 2 x 106UTD orCAR90 T cells (FIGs. 17A-17B) Mice were then monitored biweekly for tumor growth by bioluminescent imaging (BLI) and semiweekly blood draws. As observed in the prior CAR90 treated cohort, CAR90 infusion was followed by a profound outgrowth of T cells and drop in the frequency of human B cells. Interestingly, T-cell expansion up to 40% of total hCD45+cells was previously observed, while in the cancer xenograft setting, T-cell expansion was much more marked (up to 80-90% of all hCD45+cells) with a concomitant decrease in the proportion of other lineages (B cells, myeloid cells, FIG. 17C). T-cell expansion corresponded to CAR marking in the PB, reaching up to 12% of all T cells 7 DPT (FIG. 17D). This profound T-cell outgrowth in CAR-treated mice persisted to necropsy 4 weeks later and was similarly observed in the spleen and BM (FIG. 17E). T-cell outgrowth was associated with tumor cell killing, with Jurkat outgrowth observed in the UTD condition, while CAR90-treated mice controlled the tumor starting at 8 DPT (FIGs. 17F-17G). This difference observed in BLI corresponded with a significantly increased mean survival (15 days vs. >30 days, FIG. 17G). At necropsy, no significant impact of CAR90 treatment was found compared to UTD treatment across most hematologic populations in the PB (total CD45s, B cells, and monocytes). However, similar to the prior CAR90-treated cohort a decrease of CD34+HSPCs, CD34+CD38- MPPs, CD117+, and CD133+HSPCs was observed in the BM. However, the most profound depletion was observed within the CD90+population, with an observed reduction from 0.03% to 0% of hCD45+cells (FIG. 17H).

[0265] To evaluate whether CD90-ablated HSCs were protected from CAR90, CD34+CD38|OWHSPCs were FAC-purified and plated them into CFC assays. No impact on multilineage differentiation potential nor total differentiation potential in primary culture was observed; however, a reduction in secondary CFC potential in CAR-treated animals was observed. However, assessing cells from primary and secondary CFC revealed a stark difference in the extent of editing was seen (2.36-fold increase in primary culture, 3.65-fold enrichment in secondary culture), suggesting that CD90-knockout successfully rendered HSCs resistant to CAR-mediated killing.

[0266] In summary, CAR90 is well tolerated and effectively eliminates CD90+leukemia cells in vivo. CD90 ablation of HSCs confers specific protection to the CAR90 T cells, mostly preserving hematopoietic integrity while the leukemia cells are selectively targeted.

[0267] Discussion. The current experiment characterizes the function of CD90 and its implications for the development of CD90-targeted CAR T cells for the enrichment of gene-modified HSCs and / or killing of CD90-expressing tumor cells ex vivo and in vivo. Through transduction with a CD90-directed shRNA as well as a clinically relevant base editing-mediated KO, it was demonstrated that CD90 is dispensable in adult HSCs, permitting engraftment, recovery, and complete multilineage hematopoiesis in a mouse xenograft and autologous NHP transplant models with no adverse impacts noticed. Although dispensable in adult hematopoiesis, the current studies in an iPSC differentiation model of embryonic hematopoiesis suggest that CD90 contributes to the development of arterial hemogenic endothelium (HE), which is necessary to establish the hematopoietic system prenatal ly. Simultaneously, a CD90-targeted CAR construct (CAR90) was created and comprehensively tested, demonstrating specific, CD90-F053-0197PCT / 24-151 -WO-PCTdirected CAR toxicity ex vivo. Finally, the utility of CD90-KO and CAR90 was combined in a humanized in vivo leukemia mouse model, demonstrating full clearance of tumor cells and protection as well as enrichment of gene-modified CD90-KO HSCs. These studies provide critical insight into the role of CD90 in fetal and adult hematopoiesis as well as demonstrate the utility of its targeting for treatment of malignant LSCs or the enrichment of gene-modified HSCs for gene therapy.

[0268] CD90 was first described in human hematopoiesis in 1992 (Baum, et al., Proc. Matl. Acad. Sci. USA, 89:2804-2808 (1992). Those studies showed that FACS-isolated CD34',CD90+cells were able to engraft in immunodeficient mice, demonstrating their capacity to give rise to T lymphocytes, B lymphocytes, and myeloid cells. Following the discovery and subsequent description of CD90, it was shown that CD34+CD90+cells are not only responsible for longterm hematopoiesis, but also facilitate short-term hematopoietic recovery (Radtke, et al., Blood, 142:33-43 (2023)). However, despite the use of CD90 as a primary immunophenotypic marker for HSCs, the role of CD90 in healthy hematopoiesis has not been rigorously investigated. The current findings suggest that CD90 expression on adult HSCs is not necessary to HSC function, with both the KD and KO experiments showing multilineage recovery and no negative selection pressure relative to unedited HSCs. Critically, demonstration of full hematopoietic recovery in the NHP model confirmed that ablation of CD90 does not adversely impact the multilineage output and longevity. This finding reveals that CD90, despite its near ubiquitous expression on HSCs, does not play a central role in HSC signaling or maintenance.

[0269] While CD90 has been most exhaustively evaluated in the context of adult hematopoiesis, CD90 has also been observed on fetal HSC precursors including fetal liver HSPCs, HE, and cells undergoing endothelial to hemogenic transition in the AGM (Calvanese, et al, Nature, 604:534-540 (2022); Choi, et al., Cell Reports, 2:553-567 (2012)). Additionally, given the known functional role of CD90 as an integrin-binding partner, and the critical role of integrin signaling in HSC maintenance through extracellular signaling (Leyton, et al., Front. Cell. Dev. Biol., 7:132 (2019)), it was postulated that CD90 could play a central role in developmental hematopoiesis. In the current experiments, CD90-KD perturbed the maturation of arterial HE. Single-cell analysis of CD90-KD and scramble-transduced HSPCs revealed impaired function of several pathways implicated in HE maturation and EHT. Flow-cytometric analysis of CD90-KD HSPCs revealed a loss of DLL4 and impaired overall capacity for successful transition to HE, suggesting that while CD90 is dispensable in adult hematopoiesis, it plays a critical role in mediating the development of primordial hematopoietic cells through modulating the arterialization of HE.

[0270] Though commonly used as a phenotypic marker for HSCs, CD90 has also been implicated in a number of malignancies including solid tumors such as glioblastoma multiforme (Avril, et al., Clinical cancer research: an official journal of the American Association for Cancer Research, 23:7360-7374 (2017), pancreatic adenocarcinoma (Zhu, et al., PLoS One, 9:e115507 (2014)), and hepatocellular carcinoma (Yang, et al., Cancer cell, 13:153-166 (2008)); as well as blood cancers such as T-cell acute lymphocytic leukemia and AML. Given the role of CD90 in neonatal hematogenesis and its presence on multipotent HSCs, the expression of CD90 in cancer raises its potential marker ofF053-0197PCT / 24-151 -WO-PCTcancer stem cells (CSCs). The concept of CSCs was also described to be responsible for the maintenance of AML (Bonnet, et al., Nat. Med., 3:760-737 (1997)) with CD34+CD38- CSCs being both the initiating and driving force behind malignant disease. Since then, numerous groups have not only discovered CSC populations in other cancers, but also implicated CD90 as an immunophenotypic marker of these CSCs. This finding is further supported through more rigorous characterization of AML samples through multiomic analysis, revealing a conserved hierarchy of CD90 expression on more stem-like blasts (Zhang, et al., Nat. Immunol., 25:703-715 (2024); Pei, et al., Cancer Discovery, 13:2032-2049 (2023)).

[0271] Given the newly demonstrated dispensability of CD90 in adult hematopoiesis and the established presence of CD90 in multiple cancers, the current experiment sought to evaluate the utility of CD90-ablation strategies for anticancer therapies. Multiple groups have developed strategies to edit and target hematopoietic proteins including CD45, CD117, FLT3, and CD123 (Wellhausen, et al., Sci. Transl. Med., 15:eadi1145 (2023); Petty, et al., Med., 4:749-751 (2023); Ji, et al., Cell Stem. Cell, 31:1650-1666 (2024); Casirati, et al., Nature, 621:404-414 (2023). A critical benefit of CD90 over other targets is the constrained and specific expression of CD90 on the HSC population. This is particularly important in the setting of anti-cancer therapeutic development, where strategies targeting cancer stem cells hold significant potential to limit relapse risk. To explore the utility of such an approach, several anti-CD90 CAR T-cell constructs were created and vetted their capacity in vitro. CAR90 behaved similarly to other CAR T-cell products in their capacity to proliferate and produce pro-inflammatory cytokines in response to CD90, mirroring several currently FDA-approved therapies.

[0272] Beyond the utility of targeting malignant stem cells, the expression of CD90 on healthy HSCs also benefits the use of CD90-directed targeting moieties for post-HSCT gene enrichment. The use of ABE permits the multiplex editing of CD90 as an enrichment handle together with therapeutic edits. Beyond CAR-mediated killing, the ability to protect HSCs from CD90-directed therapeutics holds broader promise. Recently, both antibody-drug conjugates (ADC) have seen an increase in the development of novel cancer therapeutics (Fu, et al., Signal Transduct. Target Then, 7:93 (2022). These antibody-based systems differ from CAR T cells in two important ways. First, antibodies are able to detect antigens with a greater sensitivity. Second, antibodies are more constrained in their ability to navigate biological barriers, such as the blood brain barrier, as compared to cell-mediated therapies (Marin, et al., Neuro Oncol., 23:2042-2053 (2021); Galea, et al., J. Exp. Med., 204:2023-2030 (2007)). This distinction could be critical in determining the best application of CD90-directed targeting for oncolytic targeting, low-toxicity conditioning, or post-transplant gene enrichment.

[0273] In conclusion, the current experiment demonstrated that the CD90 protein is dispensable in adult hematopoiesis. The current experiment further elucidated the role of CD90 by demonstrating phenotypic changes in an in vitro EHT model. Finally, the current experiment sought to functionalize CD90 KO by developing a CD90-directed CAR T cell and demonstrating its capacity to selectively kill cancer cells in vitro and in vivo while sparing edited HSCs. The current findings simultaneously provide critical new insights into the basic biology of CD90 and its function in adultF053-0197PCT / 24-151 -WO-PCTand developing HSCs while also offering a new therapeutic avenue for the specific killing of CD90+ cells, an approach that will be of high utility in post-transplant gene enrichment strategies as well as a potential anti-cancer stem cell therapy.

[0274] Materials and Methods. Study Design. This study aimed at evaluating the role of CD90 in adult and neonatal hematopoiesis. To investigate this, lentiviral shRNA constructs targeting CD90 were used to explore the impact of CD90 knockdown on HSPC engraftment potential as well as in vitro endothel i al-to-hem atopoi etic transition. The impact of CD90 knockdown on in vitro EHT was further assessed by single cell RNA sequencing. A bona fide CD90 knockout system leveraging an ABE was then developed, demonstrating CD90KO HSPC engraftment potential in an NBSGW xenograft model. Next, the impact of CD90KO was explored in the clinically relevant NHP model. Concomitantly, a CD90-directed CAR T cell was developed and demonstrated their capacity for specific cytotoxicity, activation, and pro-inflammatory cytokine production. NBSGW mice engrafted with CD90KO HSPCs were then treated with CAR90 to investigate the safety of CAR90 in vivo. Finally, CAR90 was employed in a translational ly relevant cancer model utilizing the CD90 overexpressing Jurkat cell line. All experiments were conducted in at least duplicate, with the number of replicates and statistical analyses detailed in the corresponding figure legends.

[0275] Cell Lines. K562 myelogenous leukemia cells (ATCC, CCL-243) were cultivated in RPMI-1640 (Gibco, 11875093) supplemented with 10% fetal bovine serum (FBS; Cytivia, SH3008703HI) and 1% penicillin-streptomycin (Pen / Strep; Gibco, 10378016). Jurkat T-cell leukemia cells (ATCC, TIB-152) were cultured in RPMI-1640 supplemented with 10% FBS and Penn / Strep (WOU / mL). Human embryonic kidney 293T cells (ATCC, CRL-3216) were cultivated in DMEM (Gibco, 11965092). The ML-1 (DSMZ, ACC464) follicular carcinoma cell line was cultivated in DMEM supplemented with 10% FBS and 1% Pen / Strep.

[0276] Primary human HSPCs. Primary human CD34+cells were purchased from the Co-operative Center for Excellence in Hematology (CCEH) at Fred Hutchinson Cancer Center (Fred Hutch). Collections were performed according to the Declaration of Helsinki and were approved by a local ethics committee / institutional review board of Fred Hutch (IR#3942, RG9295001). All healthy adult donors were mobilized with G-CSF. Human CD34+cells were enriched on a CliniMACS Prodigy according to manufacturer’s instructions. Human CD34+cells were cultured in StemSpan (STEMCELL Technologies, 9655) medium supplemented with Pen / Strep (100 U / mL) and 100 ng / mL of each stem cell factor (SCF, Peprotech 300-07-), thrombopoietin (TPO; PeproTech, 300-18), and Fms-related tyrosine kinase 3 ligand (FLT3-L; Peprotech, AF-300-19). Cells were cultured at 37°C, 85% relative humidity, and 5% CO2.

[0277] shRNA design. Knockdown vectors were created using the VectorBuilder.com online vector design tool (FIG.20D). Target sequences were designed by VectorBuilder.com and empirically tested by transducing K562 cells with target sequences. Five days after transduction, CD90 and mCherry expression was evaluated by flow cytometry. CD90-targeted shRNA vectors with the greatest decrease in CD90 expression determined by the mean fluorescence intensity (MFI) were used in subsequent experiments.

[0278] Base editor in vitro mRNA transcription. Similar to previous in vitro transcription of prime editor mRNAsF053-0197PCT / 24-151 -WO-PCT(Nelson, et al., Nat. Biotechnol., 40:402-410 (2022)), plasmids were cloned to encode an inactivated T7 promoter followed by a 5' untranslated region (UTR), Kozak sequence, the coding sequence of ABE8e-NRCH, and a 3' UTR. T7 promoter inactivation prevents potential transcription from circular plasmid template during mRNA generation. These components together were PCR-amplified with NEBNext polymerase (New England BioLabs, M0541S) using primers that correct T7 promoter inactivation and append a 120-nt poly(A) tail to the 3' UTR. The resulting PCR product was purified with the QIAquick PCR Purification Kit (QIAgen, 28104) and served as a template for subsequent in vitro transcription. ABE8e-NRCH mRNA was transcribed from these templates using the HiScribe T7 High-Yield RNA Synthesis Kit (New England BioLabs, E2040S) with co-transcriptional capping by CleanCap AG (TriLink Biotechnologies, N-7113) and full replacement of UTP with N1-Methylpseudouridine-5'-triphosphate (TriLink Biotechnologies, N-1081). Transcribed mRNA was precipitated in 2.5 M final concentration of lithium chloride (Invitrogen, AM9480), washed twice in 70% ethanol, then dissolved in nuclease-free water. The resulting mRNA was quantified with a NanoDrop One UV-Vis spectrophotometer (Thermo Fisher Scientific) and was aliquoted and stored at -80°C.

[0279] Base editing. ABE8e-NRCH mRNA was produced by the Liu Lab and chemically modified gRNA (2'-O-methyl analogs and 3' phosphorothioate internucleotide linkages at the first three 5' and 3' terminal RNA residues) was custom-ordered from Synthego and is listed in FIG. 20D. Briefly, purified CD34+HSPCs were electroporated using the BTX ECM830 electroporator (Harvard Apparatus) at 3×106living human / NHP cells per 2 mm cuvette for 5 ms at 250V. For the electroporation, cells were resuspended in 100 μl BTX buffer supplemented with 6 μg ABE8e-NG mRNA 0.3μM HBG-175 and / or 0.6 pM CD90 sgRNA per cuvette.

[0280] Editing efficiency determination. Allele editing frequency was determined as described by Humbert et al. (2021); Samuelson, et al., Molecular therapy, Methods & clinical development, 23:507-523 (2021). Extraction of DNA from cell pellets was performed using DNeasy Blood and Tissues kit (Qiagen, 69506) followed by PCR amplification of the CD90 and HBG loci. PCR amplicons were sequenced by Sanger sequencing and analyzed using the web-based EditR program (FIG. 20D) (Kluesner, et al., CRISPR J., 1:239-250 (2018). Indels identified via Sanger sequencing were flagged during EditR analysis when the sequence degraded within the protospacer. Samples suspected of containing indels were then Sanger-sequenced using primers from both directions and analyzed using TIDE (default settings except for max indel size set to15bp) (Brinkman, et al., Nucleic Acids Res., 42:e168 (2014).

[0281] NGs and data analysis. For NGS, genomic DNA was extracted using QIAamp DNA micro kit (Qiagen, 56304) and processed by PCR amplification using the primers described in FIG. 20D. Libraries were prepared using Illumina barcoded, 2x150 base-pair (bp), pair-end and run on the Miseq platform (Illumina). The paired-end reads were merged using Paired-End read Merger (PEAR) with default parameters (Zhang, et al., Bioinformatics, 30:614-620 (2014). A custom Python script was used for the bioinformatics analysis of the sequencing data The reads were filtered if they had more than 2 bases with low quality scores. The merged reads were aligned to the start primer and the end primer sequences allowing for 2 mismatches with no insertions and deletions (indels). The reads with good primer alignmentF053-0197PCT / 24-151 -WO-PCTwere then aligned to the wild-type locus sequence using Needle, a Needleman-Wunsch aligner from the EMBOSS Suite (Rice, et al., Trends Genet., 16:276-277 (2000). The reads were grouped based on the alignment pattern into the following: (i) reads that match the wild-type sequence (ii) reads with the CD90 / HBG base edits (iii) reads with the off-target base edits.

[0282] Flow cytometry analysis and cell sorting. Flow cytometric analysis and sorting of cell lines and human CD34+ cells were performed using the fluorochrome-conjugated antibodies. Dead cells and debris were excluded via forward light scatter (FSC) / side light scatter (SSC) gating. Flow cytometric analysis and cell sorting were performed on a FACSymphony A5, FACSCelesta, FACSAria llu, and Symphony S6 (BD Biosciences, San Jose, CA). Data were acquired using FACSDiva version 6.1.3 and newer (BD Biosciences). Data analysis was performed using FlowJo version 8 and higher (BD Biosciences).

[0283] Fetal hemoglobin measurements. HbF was measured by flow cytometry after fixation and permeabilization of unlysed blood using previously described procedures (Humbert, et al., Mol. Ther. Methods Clin. Dev., 8:75-86 (2018). Unlysed blood was fixed for 10-12 min in a 0.05% glutaraldehyde solution (Electron Microscopy Sciences, NC0268607), washed twice in PBS, permeabilized for 3-5 min in 0.01% Triton X-100 (Invitrogen, HFH10), washed 2X in PBS, and stained with PE-conjugated HbF antibody (Invitrogen, clone HBF-1, MHFH04).

[0284] Xenotransplantation of NBSGW mice. All xenograft mouse experiments were carried out under Fred Hutchinson institutional animal care & use committee (IACUC) designations 202000029 and 201900022. NSG and NBSGW neonates aged 1-3 days were intrahepatically injected with 30,000-50,000 human umbilical cord blood (UCB)-derived CD34+hematopoietic stem and progenitor cells (HSPCs, FHIRB0020199) per animal in 30 μL of injection media (filtered RPMI, 1 mM EDTA) as previously described (Traggiai, et al., Science, 304:104-107 (2004); Saito, et al., Methods Mol. Biol., 1432:309-320 (2016). Adult (8-12 week old) NBSGW were retro-orbital ly injected with 100,000 to 250,000 ex vivo gene-modified human CD34+ HSPCs (IR#3942, RG9295001) per animal in 200 pL of injection media (filtered RPMI 1640, 1 pM EDTA). Starting at 6-8 weeks after injection, human chimerism was measured as the percentage of human CD45+cells among the mouse and human CD45+in the PB. Blood analysis was performed every other week until necropsy, at which BM, PB, and spleen were collected. IVIS imaging was carried out twice weekly on the IVIS Spectrum Imaging system (Xenogen). D-Luciferin administration was carried out following the manufacturers provided SOPs for in vivo imaging in rodents.

[0285] Male and female mice were used for all studies and evenly distributed between experimental groups. Sex was recorded and separately analyzed as an independent variable. Mice were randomly assigned to experimental and control groups to preserve standard variability at study onset. All mice were ear tagged and all procedures conducted without prior knowledge of group ID, only being identified after all data collection was completed. For xenograft CAR experiments, mice were included in the study if they reached a minimum threshold of 20% human engraftment.

[0286] Group sizes were planned to be n=5 for each xenograft experiment. For each comparison, a total of up to three unique human stem cell donors were used to account for donor-to-donor variability in the quality of stem cellF053-0197PCT / 24-151 -WO-PCTproducts and the frequency of human stem cell engraftment in the murine bone marrow. Since each set of data is derived from the same human donor, the comparison is a "one-sample” comparison (i.e., the combinations have a natural pairing). If the mean difference between two conditions is 3 standard deviations from 0, this would provide 94% power to deem this difference as statistically significantly different from zero (at the one-sided significance level of.05, using a one-sample t-test).

[0287] Nonhuman primate transplants. Healthy juvenile pigtail macaques were housed at the University of Washington (UW) National Primate Research Center (WaNPRC) under conditions approved by the American Association for the Accreditation of Laboratory Animal Care. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (" The Guide”), and monkeys were randomly assigned to the study under University of Washington IACUC 3235-01. This study included at least twice-daily observation by animal technicians for basic husbandry parameters (for example, food intake, activity, stool consistency, and overall appearance), as well as daily observation by a veterinary technician and / or veterinarian. Animals were housed in cages approved by " The Guide” and in accordance with Animal Welfare Act regulations. Animals were fed twice daily and were fasted for up to 14 hours before sedation Environmental enrichment included grouping in compound, large activity, or run-through connected cages, perches, toys, food treats, and foraging activities. If a clinical abnormality was noted by WaNPRC personnel, standard WaNPRC procedures were followed to notify the veterinary staff for evaluation and determination for treatment as a clinical case. Before all procedures, animals were sedated by administration of ketamine HCI and / or telazol and supportive agents for balanced anesthesia (such as diazepam and midazolam). After sedation, animals were monitored according to WaNPRC standard protocols. WaNPRC surgical support staff are trained and experienced in the administration of anesthetics and have monitoring equipment available to assist with electronic monitoring of heart rate, respiration, and blood oxygenation; audible alarms and digital readouts; monitoring of blood pressure, temperature, etc. For minor procedures, the presence or absence of deep pain was tested by the toe-pinch reflex, and the absence of response (leg flexion) to this test indicated adequate anesthesia. In cases of general anesthesia, similar monitoring parameters were used, and anesthesia was tested by the loss of palpebral reflexes (eye blink). Analgesics (generally buprenorphine with meloxicam or buprenorphine slow release) were provided as prescribed by the clinical veterinary staff for at least 48 hours after the procedures and could be extended at the discretion of the clinical veterinarian based on clinical signs.

[0288] Autologous NHP HSC transplantation, G-CSF / AMD3100 treatment for HSC mobilization, leukapheresis, CD34 enrichment, base editing, and busulfan conditioning were conducted consistent with previously published protocols (Radtke, et al., Blood, 142:33-43 (2023); Borot, et al., Nature communications, 16:4899 (2025); Murray, et al., Mol. Ther. Methods Clin. Dev., 30:276-287 (2023). Gene-edited cell products were cryopreserved in liquid nitrogen until the day of infusion (72 hours after busulfan administration). Granulocyte colony-stimulating factor was administered daily from the day of cell infusion until the animals began to show onset of neutrophil recovery. SupportiveF053-0197PCT / 24-151 -WO-PCTcare, including antibiotics, electrolytes, fluids, and transfusions, was given as necessary, and blood counts were analyzed daily to monitor hematopoietic recovery.

[0289] CD34 enrichment. Primed NHP BM was harvested, enriched, and cultured as previously described (56, 57). Briefly, before enrichment of CD34+ cells, red blood cells were lysed in ammonium chloride lysis buffer (Fisher Chemical, A649-3), and white blood cells were incubated for 30 min with the 12.8 immuno-globulin M anti-CD34 antibody and then washed and incubated for another 30 min with MACS anti-immunoglobulin M microbeads (Miltenyi Biotec, 130-047-301). The cell suspension was run through magnetic columns to enrich for CD34+ cell fractions with a purity of 60 to 80%, which was confirmed by flow cytometry (see Flow Cytometry and FACS section).

[0290] CFC assay. For CFC assays, 200 sort-purified CD34+cells and CD34+subpopulations were seeded into 1 mL of Methocult (Stem Cell Technologies, H4434) for human HSPCs or ColonyGEL (ReachBio, 1402) for NHP HSPCs supplemented with Pen / Strep. Hematopoietic colonies were scored after 12 to 14 days. Arising colonies were identified as colony-forming unit (CFU) granulocyte (CFU-G), CFU macrophage (CFU-M), CFU granulocyte-macrophage (CFU-GM), and burst-forming unit-erythrocyte (BFU-E). Colonies consisting of erythroid and myeloid cells were scored as CFU-MIX.

[0291] Hemato-vascular differentiation of human induced pluripotent stem cells (IPSC). Human induced pluripotent stem cells (IPSCs) were differentiated to hemato-vascular lineages as previously described (Palpant, et al., Nature Protocols, 12:15-31 (2017)), with modifications as noted below. Briefly, IPSCs were maintained in mTeSR™ Plus medium (STEMCELL Technologies, Cat# 100-0276) on MatrigelO-coated plates (Corning, Cat# 354277). For differentiation, iPSCs were passaged using ReLeSR™ (STEMCELL Technologies, Cat# 05872) and seeded onto 12-well plates coated with Matrigel in cold DMEM / F12 (Thermo Fisher, Cat# 11320033) On Day -1, cells were seeded at 2 x 106cells / mL in mTeSR Plus supplemented with 1 pM CHIR99021 (Tocris, Cat# 4423). On Day 0, cells were washed with PBS (Thermo Fisher, Cat# 10010023) and cultured in RPMI 1640 (Thermo Fisher, Cat# 11875093) with the following supplements: 50 ng / mL recombinant human Activin A (PeproTech, Cat# AF-120-14E), B27 Supplement (Thermo Fisher, Cat# A1895601), and Matrigel (1X) (Corning, Cat# 354277). On day 1, the media was replaced with RPMI 1640 containing: 40 ng / mL recombinant human BMP4 (PeproTech, Cat# 120-05ET), B27 Supplement, and 1 pM CHIR99021. On day 2, the media was replaced with StemPro™-34 SFM (Thermo Fisher, Cat# 10639011) supplemented with: 10 ng / mL BMP4, 200 ng / mL recombinant human VEGF165 (PeproTech, Cat# 100-20), 5 ng / mL recombinant human bFGF (PeproTech, Cat# 100-18B), 3 pM CHIR99021, and 5 pM SB431542 (Tocris, Cat# 1614). On day 3, the media was replaced with StemPro-34 supplemented with: 10 ng / mL BMP4, 200 ng / mL VEGF165, and 5 ng / mL bFGF. On day 5, cells were dissociated using TrypLE™ Express (Thermo Fisher, Cat# 12604013) and sorted for CD34+on a CliniMACS cells using CD34 MicroBeads (Miltenyi Biotec, Cat# 130-046-702) in CliniMACS buffer (Miltenyi Biotec, Cat# 130-070-543) with IVIG (Grifols, Cat# 15019) at 2 pL per 100 pL buffer. Sorted cells were frozen at day 5 and stored in liquid nitrogen for future use. To generate a monolayer of arterialized and hemogenic endothelium (AE / HE), day 5 CD34+cells were thawed and plated on Retronectin-coated (Takara, Cat# T100B) 96-well plates at 12F053-0197PCT / 24-151 -WO-PCTx 104cells / well in StemPro-34 supplemented with: 10 ng / mL BMP4, 15 ng / mL VEGF165, and 10 ng / mL bFGF. After day 7 arterialized endothelial monolayer was exposed to StemPro-34 containing: 10 ng / mL BMP4, 15 ng / mL VEGF165, 10 ng / mL bFGF, 50 ng / mL recombinant human SCF (PeproTech, Cat# 300-07), 20 ng / mL recombinant human Flt3L (PeproTech, Cat# 300-19), 20 ng / mL recombinant human TPO (PeproTech, Cat# 300-18), and 20 ng / mL recombinant human IL-3 (PeproTech, Cat# 200-03).

[0292] shRNA knockdown of CD90 in iPSC-derived arterial hemogenic endothelium. At day 9 of culture, the arterialized hemogenic endothelial layer was dissociated with TrypLE™ Express (Gibco, 12604013), washed and stained: Cells were analyzed on a BD FACSymphony A5™ flow cytometer after cytometry data were processed with FlowJo software.

[0293] Single cell RNA sequencing. Cells for single-cell RNA sequencing were processed using the Chromium Single Cell 3' (v3) platform from 10X Genomics. Separation of single cells, library preparation, and RNA extraction were performed in accordance with the 10X Chromium Single Cell Gene Expression Solution protocol.

[0294] Single cell transcriptome analysis and quality control. Monocle3 (v.3.1.2.9) was used for downstream analysis, combining read-depth normalized data for each group of samples (Trapnell, et al., nat. Biotechnol., 32:381-386 (2014); Cao, et al / . Nature, 566:496-502 (2019). Only cells expressing detectable mCherry transcripts (transduced cells) were included for analysis. Cells with high mitochondrial genes were excluded (>5%), as were cells with low genes per cell (<1,000) and cells with low UMI per cell (UMI<10,000). Uniform Manifold Approximation (UMAP) was used for dimensionality reduction (60). The data was mapped onto the top principal components (default settings). Clustering was performed using the Leiden method implemented in Monocle3.

[0295] Cell type classification. Cell type classification was performed by supervised assignment of clusters based on expression of endothelial markers KDR and CDH5, denoting the major cluster of AE / HE, or hematopoietic-specific marker SPN, denoting the minor cluster of early emerging hematopoietic progenitors at day 9. The minor population of hematopoietic progenitors was excluded for differential gene expression, gene-set scores and Gene Ontology analysis.

[0296] Differential gene expression. A quasipoisson distribution was used to evaluate the differential expression between THY1 / CD90 knockdown and control scrambled scRNA samples with the fit_model() and coefficient table() functions in Monocle3. A q-value for multiple hypothesis testing was calculated by the Benjamini and Hochberg correction method, and q<0.05 was considered as statistically significant.

[0297] Gene ontology. The list of differentially expressed genes (q < 0.05, 0.25 < Log2(FC) < -0.25) from the comparison of CD90 shRNA knockdown and Scrambled scRHA (control) populations (EC / HEC subset) was uploaded to Metascape v.3.5 (61) for Gene Ontology (GO) analysis. Top 20 enriched terms across input gene list are shown in FIG. 20D.

[0298] Gene-set scores. Gene-set scores were calculated as the log-transformed sum of the size factor-normalized expression for each gene from the Molecular Signatures Database (gsea-msigdb.org / gsea / msigdb / index.jsp) including:F053-0197PCT / 24-151 -WO-PCTABE_VEGFA_TARGETS, GOBP_CELL_ADHESION_ MEDIATED_BY_INTEGRIN, KEGG_LEUKOCYTE_TRANSENDOTHELIAL_ MIGRATION, PID_FAK_PATHWAY, GOBP_POSITIVE_REGULATION_OF_RHO_PROTEIN_SIGNAL_TRANSDUCTION. Wilcoxon Rank Sum Test (ggupbr package v0.4.0) was used to calculate p values.

[0299] Human CAR T cell production. Plasmids for viral vector production were acquired from the center for cellular excellence in hematology (CCEH) vector core. The transfer plasmid contained a second-generation CAR backbone with a 41 BB costimulatory domain and bicistronic truncated EGFR (tEGFR) transduction reporter. oCD90 short chain variable fragments (scFvs) were designed based on the 5E10 antibody and ordered from GeneArt as plasmid DNA. aCD90 scFvs were introduced to the CAR vector by restriction enzyme digestion and ligation.

[0300] Third generation replication deficient lentivirus was produced by the CCEH vector core for all in vivo and in vitro assays. Frozen human peripheral blood mononuclear cells (PBMCs) were acquired from the CCEH. T cells were isolated using the Human T cell Isolation Kit (StemCell Technologies, 17951). Post-isolation T-cell purity and CD4: CD8 ratio was assessed by flow cytometry. T cells were cultured in R10 at a density of 1 e6 / ml_ and activated with CD3 / CD28 Dynabeads (Gibco, 11131 D) at a 3:1 ratio. 24h after activation, T cells were transduced with CAR90 lentivirus at a multiplicity of infection (MOI) of 3. Media was doubled every other day and T cells were transferred to new culture vessels as appropriate. Dynabeads were removed on day 7 of culture and assessed by FACS for CD4: CD8 ratio and CAR positivity. T cells were allowed to continue expanding until day 10 / 11. T cells were then harvested for use in vitro and in vivo.

[0301] HSPC / CAR90 co-culture assays. HSPCs were acquired from the CCEH and thawed 24h prior to assay start. HSPCs were cultured in SFEM-II media at a cellular density of 1 e6 / ml_ (details of media conditions above). 24h after thaw, HSPCs were split into treatment conditions at a density of 1 e6 / mL and mixed with T cells at a ratio of 1: 1. HSPCs were allowed to co-culture for 24, 48, or 72h with T cells, then had supernatant harvested for cytokine analysis and cells assessed by FACS for HSPC identity. CountBright counting beads (Invitrogen, C36950) were added to all samples to allow determination of total cell counts per manufacturer’s instructions.

[0302] Concomitantly, HSPCs were sorted by FACS directly into tubes containing colony forming media, targeting 100 cells per ml_. After sorting, cell / media mixture was thoroughly mixed by vortexing, then plated into 10cm plates and cultured at 37°C for 10 days. After primary culture, total colony number and ID were assessed. Cultures were melted using warm PBS, then washed twice with PBS before replating 10% of total product into secondary colony forming assays. Secondary cultures were allowed to incubate for 10 days, and were analyzed as primary colonies were.

[0303] In vitro cytotoxicity assay. HEK293T cells were engineered to overexpress the CD90 protein with a bicistronic GFP reporter. 24h before assay start, HEK293T cells were plated into a flat bottom 96-well plate at a density of 10,000 cells per well in 100 L of complete media (DMEM + 10% FBS + 1% P / S). This assay was initiated by the addition of T cells at 10:1, 5:1, 2.5:1, or 1.25:1 effector to target (E: T) ratio in 100 piL of R10. Plates were then imaged on the Incucyte microscope in the Fred Hutchison Shared Imaging Core hourly for 72h. Images were analyzed on the IncucyteF053-0197PCT / 24-151 -WO-PCTanalysis software for the number of GFP+ cells at each timepoint and normalized to total GFP+ counts at Oh.

[0304] Cytokine bead array. Supernatant from HEK293T cell assays and HSPC assays were thawed from -80C on the day of cytokine bead array (CBA). Supernatants were assessed using the BD Th1 / Th2 / Th17 CBA kit per manufacturer’s instructions (560484). Stained samples were analyzed on a BD LSRFortessa cytometer. Standard curves were generated on Microsoft Excel and used to map fluorescent intensities to cytokine concentrations.

[0305] (XI) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C. F. R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0306] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programswell known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.

[0307] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non-polar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W. H. Freeman and Company.

[0308] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); VaiF053-0197PCT / 24-151 -WO-PCT(+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-35); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5)

[0309] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity

[0310] As detailed in US 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (-0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0311] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid sidechain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistical ly-significant degree.

[0312] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0313] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. " Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suiteF053-0197PCT / 24-151 -WO-PCT(DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N. Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0314] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pig / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pig / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0315] " Binds" refers to an association of a binding domain (of, for example, a gRNA, an antibody) to its cognate binding molecule with an affinity or Ka( / .a, an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M’1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Kaof at least 107M1, at least 108M'1, at least 109M’1, at least 1010M'1, at least 1011M'1, at least 1012M'1, or at least 1013MA In particular embodiments, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M’1, up to 106M’1, up to 105MA Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction withF053-0197PCT / 24-151 -WO-PCTunits of M (e.g., 105M to 10-13M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (KOff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g, Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 51:660; and U. S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0316] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0317] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms "include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of' excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of' limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant increase in native CD90 expression, as described herein.

[0318] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, toF053-0197PCT / 24-151 -WO-PCTwithin a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1 % of the stated value.

[0319] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0320] The terms “a,” "an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0321] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0322] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0323] Furthermore, numerous references have been made to patents, printed publications, journal articles and otherF053-0197PCT / 24-151 -WO-PCTwritten text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching

[0324] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0325] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0326] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

F053-0197PCT / 24-151 -WO-PCTCLAIMSWhat is claimed is:

1. A method of enriching for genetically modified hematopoietic stem and progenitor cells (HSPCs) within a subject, the method comprising:administering HSPCs genetically modified to express a modified CD90 protein that does not bind a CD90- targeted therapeutic to the subject; andadministering the CD90-targeted therapeutic to the subject,thereby enriching for genetically modified HSPCs within the subject2. The method of claim 1, wherein the modified CD90 protein comprises a mutation at position F80, D98, or E99.

3. The method of claim 1, wherein the modified CD90 protein comprises a mutation at position F80.

4. The method of claim 3, wherein the mutation at position F80 comprises F80P or F80S.

5. The method of claim 3, wherein the HSPCs are genetically modified to express a CD90 protein having a mutation at position F80 by administering to the HSPCs a guide RNA having SEQ ID NO: 6 and an adenine base editor (ABE)6. The method of claim 1, wherein the CD90-targeted therapeutic comprises a CDRH1 having the sequence of SEQ ID NO: 44, a CDRH2 having the sequence of SEQ ID NO: 45, a CDRH3 having the sequence of SEQ ID NO: 62; a CDRL1 having the sequence of SEQ ID NO: 63, a CDRL2 having the sequence of SEQ ID NO: 64, and a CDRL3 having the sequence of SEQ ID NO: 65.

7. The method of claim 1, wherein the CD90-targeted therapeutic is a chimeric antigen receptor (CAR) that binds wild-type CD90 but does not bind the CD90 protein having a mutation at position F80.

8. The method of claim 7, wherein the CAR is expressed by an immune cell.

9. The method of claim 8, wherein the immune cell is a T cell.

10. A method of enriching for genetically modified therapeutic cells within a subject, the method comprising: administering hematopoietic stem and progenitor cells (HSPCs) genetically modified to express a modified CD90 to a subject or administering a composition that genetically modifies HSPCs to express a modified CD90 to a subject; andadministering a CD90-targeted therapeutic to the subject,thereby enriching for genetically modified therapeutic cells within the subject.

11. The method of claim 10, further comprising genetically modifying the HSPCs before the administering.

12. The method of claim 11, wherein the expression of the modified CD90 comprises knocked down expression, knocked out expression, or expression of a mutated CD90.

13. The method of claim 12, wherein the mutated CD90 includes an F80 mutation.

14. The method of claim 13, wherein the F80 mutation includes an F80P or F80S mutation.

15. The method of claim 12, wherein the mutated CD90 includes a D98 mutation.F053-0197PCT / 24-151 -WO-PCT16. The method of claim 15, wherein the D98 mutation includes a D98G mutation.

17. The method of claim 12, wherein the mutated CD90 includes an E99 mutation.

18. The method of claim 17, wherein the E99 mutation includes an E99G mutation.

19. The method of claim 12, wherein the mutated CD90 includes at least one mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63.64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81 A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.

20. The method of claim 11, wherein the genetically modifying comprises introducing anti-CD90 shRNA to the cells.

21. The method of claim 20, wherein the anti-CD90 shRNA comprises a sequence as set forth in SEQ ID NO: 4.

22. The method of claim 20, wherein the genetically modifying comprises introducing guide RNA (gRNA) and a gene editing component to the cells.

23. The method of claim 22, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 5 and the gene editing component comprises a nuclease.

24. The method of claim 23, wherein the nuclease comprises Cas9 or Cpf1.

25. The method of claim 24, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and the gene editing component comprises a base editor.

26. The method of claim 25, wherein the base editor comprises an adenine base editor (ABE) or a cytosine base editor (CBE)27. The method of claim 26, wherein the base editor comprises an adenine base editor (ABE).

28. The method of claim 26, wherein the ABE comprises ABE8e.

29. The method of claim 22, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 6 and the gene editing component comprises a base editor.

30. The method of claim 22, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 7 and the gene editing component comprises a base editor31. The method of claims 30, wherein the base editor comprises an adenine base editor (ABE).

32. The method of claim 31, wherein the ABE comprises ABE8e.

33. The method of claim 10, wherein the CD90-targeted therapeutic comprises an anti-CD90 antibody, an anti- CD90 conjugate, or an anti-CD90 recombinant receptor.

34. The method of claim 33, wherein the anti-CD90 antibody comprises a multi-domain binding molecule.

35. The method of claim 34, wherein the multi-domain binding molecule includes at least a first binding domain that binds CD90 and a binding domain that binds an immune cell activating epitope.

36. The method of claim 33, wherein the anti-CD90 conjugate comprises an anti-CD90 antibody conjugated to aF053-0197PCT / 24-151 -WO-PCTtoxin, a drug, or a radioisotope.

37. The method of claim 33, wherein the anti-CD90 recombinant receptor comprises an anti-CD90 chimeric antigen receptor (CAR).

38. The method of claim 37, wherein the anti-CD90 CAR is expressed by a cell and comprises an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular component comprises a binding domain that binds CD90.

39. The method of claim 38, wherein the binding domain comprises an scFv.

40. The method of claim 38, wherein the binding domain comprises a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.

41. The method of claim 39, wherein the scFv comprises a linker.

42. The method of claim 41, wherein the linker comprises a Gly-Ser linker.

43. The method of claim 41, wherein the linker comprises a Whitlow linker.

44. The method of claim 38, wherein the intracellular component comprises an effector domain comprising: 4-1BB (CD137), CD3y, CD35, CD3E, CD3<, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRα, TCRβ, TRIM, Wnt, Zap70, or a combination thereof.

45. The method of claim 38, wherein the intracellular component comprises 4-1BB and CD3δ stimulatory domains.

46. The method of claim 38, wherein the transmembrane domain comprises a transmembrane region of: an a, p or ( chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

47. The method of claim 38, wherein the transmembrane domain comprises a CD8 transmembrane domain.

48. The method of claim 33, wherein the anti-CD90 recombinant receptor further comprises a spacer region.

49. The method of claim 48, wherein the spacer region comprises a CD8 hinge region.

50. The method of claim 37, wherein the anti-CD90 CAR comprises a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.

51. The method of claim 10, wherein the genetically modified cells correct a genetic defect in the subject.

52. The method of claim 51, wherein the genetic defect comprises an immunodeficiency, a hemoglobinopathy, a lysosomal storage disease, a bone marrow failure syndrome, or a congenital anemia.

53. The method of claim 52, wherein the genetic defect comprises beta-thalassemia or sickle cell disease.

54. The method of claim 10, wherein the genetically modified cells express a therapeutic payload.

55. The method of claim 54, wherein the therapeutic payload comprises y-globin; p-globin; a-globin; yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5,F053-0197PCT / 24-151 -WO-PCTCD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1 ), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FaneW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; ICaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sILIRI, sILIRII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.

56. A method of treating a subject in need thereof comprising:administering to the subject a therapeutically effective amount of HSPCs genetically modified to have altered CD90 expression; oradministering to the subject a therapeutically effective amount of a composition that genetically modifies HSPCs to have altered CD90 expression.

57. The method of claim 56, further comprising:administering a therapeutically effective amount of a CD90-targeted therapeutic to the subject.

58. The method of claim 56, further comprising genetically modifying the HSPCs to have altered CD90 expression.

59. The method of claim 56, wherein the HSPCs are autologous or allogeneic to the subject.

60. The method of claim 58, wherein the genetically modifying HSPCs occurs ex vivo or in vivo.

61. The method of claim 58, wherein the genetically modifying HSPCs occurs ex vivo and the method further comprises administering the genetically modified HSPCs to the subject before administering a CD90-targeted therapeutic.

62. The method of claim 56, wherein the subject has a hemoglobinopathy, an immune deficiency, a clotting deficiency, or other blood disorder.

63. The method of claim 56, wherein the subject has sickle cell disease.

64. The method of claim 58, wherein the genetically modifying corrects expression of y-globi n; p-globin; a-globin; yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD 19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G;F053-0197PCT / 24-151 -WO-PCTantibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FaneW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sILIRI, sILIRII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.

65. The method of claim 56, wherein the altered CD90 expression comprises knocked down expression, knocked out expression, or mutated CD90 expression.

66. The method of claim 65, wherein the mutated CD90 includes an F80 mutation.

67. The method of claim 66, wherein the F80 mutation includes an F80P or F80S mutation.

68. The method of claim 65, wherein the mutated CD90 includes a D98 mutation.

69. The method of claim 68, wherein the D98 mutation includes a D98G mutation.

70. The method of claim 65, wherein the mutated CD90 includes an E99 mutation.

71. The method of claim 70, wherein the E99 mutation includes an E99G mutation.

72. The method of claim 8, wherein the genetically modifying comprises introducing anti-CD90 shRNA into the HSPCs.

73. The method of claim 72, wherein the anti-CD90 shRNA comprises a sequence as set forth in SEQ ID NO: 4 74. The method of claim 58, wherein the genetically modifying comprises introducing guide RNA (gRNA) and a gene editing component into the HSPCs.

75. The method of claim 74, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 5 and the gene editing component comprises a nuclease.

76. The method of claim 75, wherein the nuclease comprises Cas9 or Cpf1.

77. The method of claim 74, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2; and the gene editing component comprises a base editor.

78. The method of claim 77, wherein the base editor comprises an adenine base editor (ABE).F053-0197PCT / 24-151 -WO-PCT79. The method of claim 78, wherein the ABE comprises ABE8e.

80. The method of claim 74, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 6 and the gene editing component comprises a base editor.

81. The method of claim 74, wherein the gRNA comprises a sequence as set forth in SEQ ID NO: 7 and the gene editing component comprises a base editor.

82. The method of claims 81, wherein the base editor comprises an adenine base editor (ABE).

83. The method of claim 82, wherein the ABE comprises ABE8e.

84. The method of claim 57, wherein the CD90-targeted therapeutic comprises an anti-CD90 antibody, an anti- CD90 conjugate, or an anti-CD90 recombinant receptor.

85. The method of claim 84, wherein the anti-CD90 antibody comprises a multi-domain binding molecule.

86. The method of claim 85, wherein the multi-domain binding molecule includes at least a first binding domain that binds CD90 and a binding domain that binds an immune cell activating epitope.

87. The method of claim 84, wherein the anti-CD90 conjugate comprises an anti-CD90 antibody conjugated to a toxin, a drug, or a radioisotope.

88. The method of claim 84, wherein the anti-CD90 recombinant receptor comprises an anti-CD90 chimeric antigen receptor (CAR).

89. The method of claim 88, wherein the anti-CD90 CAR is expressed by a cell and comprises an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular component comprises a binding domain that binds CD90.

90. The method of claim 89, wherein the binding domain comprises an scFv.

91. The method of claim 89, wherein the binding domain comprises a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.

92. The method of claim 90, wherein the scFv comprises a linker.

93. The method of claim 92, wherein the linker comprises a Gly-Ser linker.

94. The method of claim 92, wherein the linker comprises a Whitlow linker.

95. The method of claim 89, wherein the intracellular component comprises an effector domain comprising: 4-1BB (CD137), CD3y, CD35, CD3E, CD3, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCRa, TCRf>, TRIM, Wnt, Zap70, or a combination thereof.

96. The method of claim 89, wherein the intracellular component comprises 4-1BB and CD3δ stimulatory domains.

97. The method of claim 89, wherein the transmembrane domain comprises a transmembrane region of: an a, or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.F053-0197PCT / 24-151 -WO-PCT98. The method of claim 89, wherein the transmembrane domain comprises a CD8 transmembrane domain 99. The method of claim 84, wherein the anti-CD90 recombinant receptor further comprises a spacer region.

100. The method of claim 99, wherein the spacer region comprises a CD8 hinge region.

101. The method of claim 88, wherein the anti-CD90 CAR comprises a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.

102. A nucleic acid that knocks down CD90 expression comprising SEQ ID NO: 4.

103. The nucleic acid of claim 102, wherein the nucleic acid is a short hairpin RNA (shRNA).

104. A system that knocks out CD90 expression comprising a guide RNA having a sequence as set forth in SEQ ID NO: 5 and a gene editing component.

105. The system of claim 104, wherein the gene editing component comprises a nuclease.

106. The system of claim 105, wherein the nuclease comprises Cas9 or Cpf 1.

107. A system that knocks out CD90 expression comprising:a guide RNA having a sequence as set forth in SEQ ID NO: 1; anda gene editing component.

108. The system of claim 107, wherein the gene editing component comprises a base editor.

109. The system of claim 108, wherein the base editor comprises an adenine base editor.

110. The system of claim 109, wherein the adenine base editor comprises ABE8e.

111. A system that mutates CD90 comprising SEQ ID NO: 6 or SEQ ID NO: 7 and a gene editing component.

112. The system of claim 111, wherein the gene editing component comprises a base editor.

113. The system of claim 112, wherein the base editor comprises an adenine base editor.

114. The system of claim 113, wherein the adenine base editor comprises ABE8e.

115. The system of claim 111, wherein an anti-CD90 antibody does not bind the mutated CD90.

116. The system of claim 115, wherein the anti-CD90 antibody comprises 5E10.

117. The system of claim 111, wherein the system mutates CD90 at F80.

118. The system of claim 117, wherein the F80 mutation includes an F80P or F80S mutation.

119. The system of claim 111, wherein the system mutates CD90 at D98.

120. The system of claim 119, wherein the D98 mutation includes a D98G mutation.

121. The system of claim 111, wherein the system mutates CD90 at E99.

122. The system of claim 121, wherein the E99 mutation includes an E99G mutation.

123. The system of claim 111, wherein the system mutates CD90 with a mutation including L34P; D31G; S33G; H40R; S44G; I48T; Y50H; I48V; F52L; F52P; F52S; Q49R; L54P; E51G; T55A; E57G; KK59.60RE; K60G; LF63,64PL; H61R; V67A; V69A; T66A; H72R; S76P; F80P; Y84H; T81 A; S82G; M86T; K83E; V88A; Y90H; L91P; K87R; F94P; T95A; D98G; Y102H; E99G; C104R; L106P; S109P; S112P; S116P; 1115V; V120A; V122A; N119G; L123P; N119S; and / or T121A.F053-0197PCT / 24-151 -WO-PCT124. A vector comprising encoding a system of claim 111.

125. The vector of claim 124, further comprising a therapeutic payload.

126. The vector of claim 125, wherein the therapeutic payload comprises y-globin; p-globin; a-globin; yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1 ), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; ICaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sILIRI, sILIRII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI; MYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TOR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.

127. A nanoparticle encapsulating the vector of claim 124.

128. A hematopoietic stem and progenitor cell (HSPC) genetically modified to express the vector of claim 124.

129. The HSPC of claim 128, wherein a CD90-targeted therapeutic does not recognize or bind the HSPC.

130. The HSPC of claim 129, wherein the CD90-targeted therapeutic comprises an anti-CD90 antibody, an anti- CD90 conjugate, or an anti-CD90 recombinant receptor.

131. The HSPC of claim 130, wherein the anti-CD90 antibody comprises a multi-domain binding molecule.

132. The HSPC of claim 1131, wherein the multi-domain binding molecule includes at least a first binding domain that binds CD90 and a binding domain that binds an immune cell activating epitope.

133. The HSPC of claim 130, wherein the anti-CD90 conjugate comprises an anti-CD90 antibody conjugated to a toxin, a drug, or a radioisotope.

134. The HSPC of claim 130, wherein the anti-CD90 recombinant receptor comprises an anti-CD90 chimeric antigen receptor (CAR)135. The HSPC of claim 134, wherein the anti-CD90 CAR is expressed by a cell and comprises an extracellular component linked to an intracellular component by a transmembrane domain, wherein the extracellular componentF053-0197PCT / 24-151 -WO-PCTcomprises a binding domain that binds CD90.

136. The HSPC of claim 135, wherein the binding domain comprises an scFv.

137. The HSPC of claim 135, wherein the binding domain comprises a variable heavy chain having a sequence as set forth in SEQ ID NO: 19 or a sequence having 95% sequence identity thereto; and a variable light chain having a sequence as set forth in SEQ ID NO: 21 or a sequence having 95% sequence identity thereto.

138. The HSPC of claim 136, wherein the scFv comprises a linker.

139. The HSPC of claim 138, wherein the linker comprises a Gly-Ser linker.

140. The HSPC of claim 138, wherein the linker comprises a Whitlow linker.

141. The HSPC of claim 135, wherein the intracellular component comprises an effector domain comprising: 4- 1BB (CD137), CD3y, CD35, CD3E, CD3, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, OX40, R0R2, SLAMF1, TCRa, TCRf>, TRIM, Wnt, Zap70, or a combination thereof.

142. The HSPC of claim 135, wherein the intracellular component comprises 4-1BB and CD3δ stimulatory domains.

143. The HSPC of claim 135, wherein the transmembrane domain comprises a transmembrane region of: an a, or chain of a T-cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

144. The HSPC of claim 135, wherein the transmembrane domain comprises a CD8 transmembrane domain 145. The HSPC of claim 130, wherein the anti-CD90 recombinant receptor further comprises a spacer region.

146. The HSPC of claim 145, wherein the spacer region comprises a CD8 hinge region.

147. The HSPC of claim 134, wherein the anti-CD90 CAR comprises a sequence as set forth in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or a sequence having at least 90% sequence identity thereto.

148. The HSPC of claim 128, wherein the HSPC is further modified to express a therapeutic payload.

149. The HSPC of claim 148, wherein the therapeutic payload comprises y-globin; p-globin; a-globin; yC; ABCA3; ABCD1; ABLI; ADA; AKT1; AK2; APC; APP; arylsulfatase A (ARSA); ARSB; BCL11A; BLC1; BLC6; BRCA1; BRIP1; C46; CAS9; C-CAM; CBFAI; CBL; CCR5; CD19; CDA; CD40; CD3D; CD3E; CD3Z; CD3G; antibodies to CD4, CD5, CD7, CD52; CFTR; CLN3; C-MYC; factor VIII (FVIII), FVII, von Willebrand factor (VWF), Fl, Fll, FV, FX, FXI, and FXIII; CHD7; CIITA; CORO1A; CRE; CSCR4; CSFIR; CTLA; CTS-I; CYB5R3; C9ORF72; DCC; DCLRE1B; DCLRE1C; DHFR; DKC1; DLL1; DMD; DRB1*1501 / DQB1*0602; dystrophin; EGFR; ERBA; ERBB; EBRB2; ETSI; ETS2; ETV6; Fane genes (e.g., FancA, FancB, FancC, FancDI (BRCA2), FancD2, FancE, FancF, FancG, Fancl, FancJ (BRIP1 ), FancL, FancM, FancN (PALB2), FancC (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); Fas L; FCC; FGR; FOX; FUS; FUSI; FYN; GALNS; GATA1; GLB1; GNS; GUSB; HBB; HBD; HBE1; HBG1; HBG2; HCR; HGSNAT; HOXB4; HRAS; HYAL1; ICAM-1; iCaspase; IDUA; IDS; IL4, IL10, IL12, IL13, IL1Ra, sILIRI, sILIRII, IL7RA; antibodies to IL1, IL2, IL6; JAK3; JUN; KLF4; KRAS; LCK; LIG4; LRRK2; LYN; MCC; MDM2; MGMT; MLL; MMACI;F053-0197PCT / 24-151 -WO-PCTMYB; MEN-I; MEN-II; MYC; NAGLU; NANOG; NF-1; NF-2; NHEJ1; NKX2.1; NLX2.1; NOTCH; OCT4; ORAI1; p16; p21; p27; p53; p57; p73; PALB2; PARK2; PARK7; PHOX2B; pyruvate kinase (PK); PINK1; PNP; PRKDC; PSEN1; PSEN2; PTPN22; PTPRC; RAD51C; RAG1; RAG2; ras; RFXANK; RFX5; RFXAP; RMRP; at least one of RPL3 through RPL40; RPLPO; RPLP1; RPLP2; at least one of RPS2 through RPS30; RPSA; SFTPB; SFTPC; SGSH; SLC46A1; SLX4; SNCA; SOD1; SOX2; STIM1; sTNFRI; sTNFRII; an antibody to TCR specifically present on autoreactive T cells; antibodies to TNF; TDP43; TERC; TERT; TINF2; UBQLN2; VHL; WAS; WHN; WT-I; or ZAP70.

150. The HSPC of claim 128, wherein the HSPC is autologous or allogeneic to a subject.

151. The HSPC of claim 128, wherein HSPC is in vivo or ex vivo152. A composition comprising the vector of claim 124 or the nanoparticle of claim 127.

153. A formulation comprising a population of cells comprising the HSPC of claim 128 and a pharmaceutically acceptable carrier.

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