Engineered hematopoietic cells and methods of use thereof

Abstract

The present disclosure relates to an engineered hematopoietic cell comprising a very late antigen-4 (VLA-4) variant and uses thereof in treating an inherited genetic disorder or an acquired disorder.

Description

[0001] Docket No. 11624-006W01

[0002] ENGINEERED HEMATOPOIETIC CELLS

[0003] AND METHODS OF USE THEREOF

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims benefit of U.S. Provisional Application No. 63 / 685,779, filed August 22, 2024, incorporated herein by reference in its entirety.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on August 20, 2025, is entitled “11624-006W01_ST26.xml”, and is 111,755 bytes in size.

[0008] FIELD

[0009] The present disclosure relates to an engineered hematopoietic cell comprising a very late antigen-4 (VLA-4) variant and uses thereof.

[0010] BACKGROUND

[0011] Transplantation of hematopoietic cells can treat a variety of inherited and acquired diseases, but currently requires treatment of the patient with preparative chemotherapy to allow the transplanted hematopoietic cells to engraft. This preparative chemotherapy which consists of high- dose alkylating chemotherapy leads to short-term toxicities like pain, nausea, inflammation, mucositis, alopecia, cytopenia, and organ injury as well as long-term toxicities like infertility and secondary malignancies, increasing the complexity of providing the therapy. This toxic conditioning regimen, used to deplete the bone marrow and make space for transplanted HSPCs, and insufficient engraftment of edited hematopoietic stem and progenitor cells (HSPCs) are major limitations of transplantation of hematopoietic stem cells as a treatment.

[0012] What is needed are new compositions and / or methods to improve hematopoietic cell retention, including engraftment, to treat a variety of inherited and acquired diseases. Docket No. 11624-006W01

[0013] SUMMARY

[0014] Disclosed herein is an engineered hematopoietic cells and methods of use thereof. The inventors have identified a method of making a mutation in the ITGA4 (alpha 4 or a4) subunit of the VLA-4 receptor on hematopoietic cells, wherein this approach allows hematopoietic retention in the bone marrow of a subject in the absence of radiation therapy or chemotherapy.

[0015] Accordingly, in one aspect, disclosed herein is an engineered hematopoietic cell comprising a VLA-4 receptor variant, wherein the VLA-4 receptor variant comprises a mutation in an ITGA4 subunit, and wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

[0016] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand.

[0017] In some embodiments, the mutation is located between amino acids R146 to N259 of the ITGA4 subunit.

[0018] In some embodiments, the mutation comprises an amino acid mutation.

[0019] In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I,

[0020] K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T,

[0021] A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D,

[0022] H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F,

[0023] K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is L206P. In some embodiments, the amino acid mutation is L206S. In some embodiments, the amino acid mutation is L206V. In some embodiments, the amino acid mutation is L206W. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K256S.

[0024] In some embodiments, the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some embodiments, the silent mutation comprises a Y151Y mutation, wherein the Y151Y mutation is encoded by a 453T>C mutation.

[0025] In some embodiments the VLA-4 receptor variant binds to VC AM- 1 expressed on bone marrow stromal cells. Docket No. 11624-006W01

[0026] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab.

[0027] In one aspect disclosed herein, is a method of treating an inherited genetic disorder in a subject. In some embodiments, the method comprises isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder.

[0028] In some embodiments, the method further comprises administering natalizumab to the subject.

[0029] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand.

[0030] In some embodiments, the mutation is located between amino acids R146 to N259 of the ITGA4 subunit.

[0031] In some embodiments, the mutation comprises an amino acid mutation.

[0032] In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I,

[0033] K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T,

[0034] A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D,

[0035] H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F,

[0036] K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is L206P. In some embodiments, the amino acid mutation is L206S. In some embodiments, the amino acid mutation is L206V. In some embodiments, the amino acid mutation is L206W. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K256S.

[0037] In some embodiments, the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some Docket No. 11624-006W01 embodiments, the silent mutation comprises a Y151Y mutation, wherein the Y151Y mutation is encoded by a 453T>C mutation.

[0038] In some embodiments the VLA-4 receptor variant binds to VC AM- 1 expressed on bone marrow stromal cells.

[0039] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab.

[0040] In some embodiments, natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

[0041] In some embodiments the inherited genetic disorder is selected from the group consisting of sickle cell anemia, thalassemia, a bone marrow failure disorder, an inherited leukocyte disorder, an inherited anemia, an inherited error of immunity disorder, a metabolic disorder, a skeletal disorder, an inherited thrombocytopenia, and an immunodeficiency. In some embodiments, the bone marrow failure disorder is selected from a group consisting of Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, severe congenital neutropenia, Diamond-Blackfan anemia, GATA2 deficiency, SAMD9 / SAMD9L disorders, MECOM syndromes, and congenital amegakaryocytic thrombocytopenia. In some embodiments, the inherited leukocyte disorder is chronic granulomatous disease. In some embodiments, the inherited anemia is a hemoglobinopathy, a membranopathy, an enzymopathy, or a congenital dyserythropoietic anemia. In some embodiments, the inherited error of immunity disorder includes but is not limited to severe congenital immunodeficiency. In some embodiments, the metabolic disorder is a leukodystrophy or a lysosomal storage disorder. In some embodiments, the skeletal disorder includes but is not limited to infantile osteopetrosis. In some embodiments, the inherited genetic disorder is sickle cell anemia.

[0042] In some embodiments, the genetic mutation causing the inherited genetic disorder is an E6V mutation in a beta-globin gene (HBB).

[0043] In one aspect disclosed herein, is a method of treating an acquired disorder in a subject. In some embodiments, the method comprises isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor Docket No. 11624-006W01 using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

[0044] In some embodiments, the method further comprises administering natalizumab to the subject.

[0045] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule-1 (VCAM-1) ligand.

[0046] In some embodiments, the mutation is located between amino acids R146 to N259 of the ITGA4 subunit.

[0047] In some embodiments, the mutation comprises an amino acid mutation.

[0048] In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I,

[0049] K20IL, K201M, K201N, K20IQ, K201R, K201S, K20IT, K201V, K201Y, Y202S, Y202T,

[0050] A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D,

[0051] H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F,

[0052] K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is L206P. In some embodiments, the amino acid mutation is L206S. In some embodiments, the amino acid mutation is L206V. In some embodiments, the amino acid mutation is L206W. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K201S.

[0053] In some embodiments, the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some embodiments, the silent mutation comprises a Y151 Y mutation, wherein the Y 151 Y mutation is encoded by a 453T>C mutation.

[0054] In some embodiments the VLA-4 receptor variant binds to VCAM-1 expressed on bone marrow stromal cells.

[0055] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab.

[0056] In some embodiments, natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the Docket No. 11624-006W01 subject. In some embodiments, natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

[0057] In some embodiments, the acquired disorder is selected from the group consisting of HIV / AIDS, a malignancy, and an autoimmune disease.

[0058] Also disclosed is method of treating an inherited or acquired genetic disorder in a subject in vivo, comprising: engineering a hematopoietic cell in vivo, wherein said engineering comprises: delivering a gene modification vector to produce a natalizumab resistance variant, wherein said variant comprises a mutation; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and administering to the subject natalizumab to select for gene modified hematopoietic cells.

[0059] BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0061] FIG. 1 shows engineering HSCs for gene therapy without chemotherapy. In the unperturbed state, HSPCs predominantly home to and are retained within the bone marrow (BM). Mobilization antagonizes HSC receptors that retain HSCs in the BM and temporarily relocates HSCs to the peripheral blood (PB). Gene modified cells that are natalizumab resistant (and disease ameliorated) could selectively engraft the BM in the presence of natalizumab.

[0062] FIG. 2 shows prime editing design for 1TGA4 Q152NNK and Q152K. Target location as marked on the main transcript of ITGA4 (dashed box), in this annotation exon 3 and 4. Further zoom to sequence marked by black solid box, presenting PBS (primer binding site), RT (reverse transcription) template, spacer and nicking guide RNA (gRNA) target sequences to install edits at amino acid Y151 and QI 52 locations (amino acid numbering starting here after signal peptide). This edit produces a silent mutation at Y151 and a Q-to-any amino acid (upper row) or Q-to-K (lower row) missense mutation at Q152. Shorter versions of PBS and RT template sequence give rise to (e)pegRNA versions 1-6 as shown in Tables 1 and 3.

[0063] FIGS. 3A-3C show prime editing all amino acids for Q152 in ITGA4. FIG. 3A shows per the design as shown in Figure 2, a pegRNA oligo pool inserting NNK codons (N: A, G, C, T; K: G, T) was used in place of AAA at position 152, inserting 32 codons encoding all 20 amino acids and a stop codon. As shown here, between 18-30% overall editing was achieved in three replicates. FIG. 3B shows natalizumab resistance of all possible amino acids at QI 52 was tested through VCAM-1 binding assay. Shown here is the log 2 foldchange difference of amino acid presence (averaged codons encoding each amino acid) in sorted populations high (per ligand binding assay, Docket No. 11624-006W01 natalizumab resistance, VCAM-1+) versus low (natalizumab sensitive, VCAM-1-), nominating natalizumab resistance through insertion of positively charged (H, K, R) and aromatic residues (F, Y, W). FIG. 3C shows flow cytometry results of the ligand binding assay after prime editing (PE) of ITGA4 Q152Y in Jurkat cells. Using prime editing, the Q152Y mutation was installed in Jurkat cells and VCAM-1 binding (by Alexa-647 fluorescence) was determined in absence and presence of natalizumab. After Q152Y editing, VCAM-1 binding partially persists in presence of natalizumab (100 nM VCAM-1, 2 nM natalizumab).

[0064] FIG. 4 shows representative flow cytometry results showing natalizumab resistance after prime editing of ITGA4 Q152K in lurkat cells. Using prime editing, the ITGA4 Q152K mutation was installed in a subset of Jurkat cells and VCAM-1 binding (by Alexa-647 fluorescence) was determined in absence and presence of natalizumab (100 nM VCAM-1, 2 nM natalizumab). Binding of VCAM-1 persists in 18% of cells, corresponding to observed editing frequency for ITGA4 (v5 + nick) of 18% allelic editing.

[0065] FIG. 5 shows prime editing of ITGA4 Q152K in Jurkat cells confers natalizumab resistance. Using prime editing, a VLA-4 (ITGA4) mutation was installed in a subset of Jurkat cells and VCAM-1 binding was determined in absence and presence of natalizumab (NAT). In contrast to B2M-edited cells (control), ITGA4 editing (vl, v5, v6) yielded a natalizumab-resistant cell population, in which VCAM-1 binding persists in -10-20% of cells, corresponding to observed allelic editing rates.

[0066] FIG. 6 shows prime editing of ITGA4 Q152K in Jurkat cells confers quantitative natalizumab resistance. The fraction of natalizumab-resistant cells corresponded to the bulk editing efficiency of 7 different experiments (R2=0.99), indicating that the designed mutation abolishes natalizumab binding (ITGA4_\ \ + nick, ITGA4_ 5 + nick, ITGA4_\6 + nick, control), as shown in Table 2 and Figure 2 (100 nM VCAM-1 and 1 or 2 nM natalizumab). Given that these Jurkat cells carry 3 ITGA4 alleles, these results suggest that mere editing of a single ITGA4 allele is sufficient to confer natalizumab resistance.

[0067] FIG. 7 shows flow cytometry results demonstrate dominant natalizumab resistance in prime edited ITGA4 Q152K Jurkat single cell derived clones. Using prime editing, the ITGA4 Q152K mutation was installed in a subset of Jurkat cells which have 3 copies of the ITGA4 gene. Clonal cell populations were generated from the prime edited cells, resulting in clone A and B, both having a single allele ITGA4 Q152K and two alleles ITGA4 Q152Q (wild type, “+”). Both these clones show dominant and complete resistance to natalizumab of a single ITGA4 Q152K allele, with comparable frequency and mean fluorescence intensity of VCAM-1 binding (100 nM) in presence and absence of natalizumab (2 nM). Docket No. 11624-006W01

[0068] FIG. 8 shows ITGA4 Q152K, K201I, and K256S mutants confer natalizumab resistance. Transient expression of ITGA4 Q152K, K201I, and K256S, achieved by electroporating ITGA4 deficient Jurkat cells with plasmids carrying ITGA4 Q152K, K201I and K256S expression cassettes, confers resistance to natalizumab. VCAM-1+ binding shown as percentage cells stained positive for VLA-4 in wild type (WT) and ITGA4 knock out Jurkat cells in presence and absence of natalizumab.

[0069] FIG. 9 shows representative flow cytometry results of natalizumab resistance acquired after prime editing (PE) of ITGA4 Q152K in CD34+ HSPCs. Using prime editing, the ITGA4 Q152K mutation was installed in CD34+ HSPCs and VCAM-1 binding (by Alexa-647 fluorescence, 150 nM) was determined in absence and presence of natalizumab (3 nM). After editing (ITGA4 v5 + nick), VCAM-1 binding persists in presence of natalizumab at a frequency (32.8%) similar to the observed allelic editing frequency (28%).

[0070] FIG. 10 shows prime editing of ITGA4 Q152K in CD34+ HSPCs confers natalizumab resistance. Using prime editing, a VLA-4 (ITGA4) mutation was installed in CD34+ HSPCs and VCAM-1 binding (100 nM) was determined in absence and presence of natalizumab (NAT, 2 nM). In contrast to mock-edited or wild-type (WT) cells, VLA-4 edited cells demonstrated a natalizumab-resistant cell population, in which VCAM-1 binding persists in presence of natalizumab, with resistant cell frequency closely approximating allelic editing frequency.

[0071] FIG. 11 shows prime editing of ITGA4 Q152K in CD34+ HSPCs confers quantitative natalizumab resistance. The fraction of natalizumab-resistant CD34+ HSPCs corresponded to the bulk editing efficiency of 6 experiments (R2=0.97), indicating that the ITGA4 Q152K mutation abolishes natalizumab-dependent inhibition of VCAM-1 binding (JTGA4 vl + / - nick, 1TGA4 v5 + / - nick, Mock, WT), as shown in Table 4 and Figures 9 and 10 (100 nM VCAM-1 and 2 nM natalizumab).

[0072] FIGS. 12A-12C show co-selection for natalizumab resistance and sickle cell disease correction after multiplex prime editing. FIG. 12A shows that sickle cell disease (HbSS) patient CD34+ HSPCs were unedited or multiplex prime edited (PE) at ITGA4 to introduce a natalizumab- resistance mutation (Q152K) and at HBB to correct the Glu6Val mutation (E6V>E). Unedited:PE HSPCs mixed at 10:1 ratio were sorted based on VCAM-1 binding in presence of natalizumab prior to in vitro erythroid differentiation culture and hemoglobin HPLC. FIG. 12B shows nextgeneration sequencing of genomic DNA shows enrichment for the edited alleles (Q152K for ITGA4 and E6V>E for HBB) based on selection by natalizumab resistant VCAM-1 binding. FIG. 12C shows gene corrected HSPCs with corrected hemoglobin expression potential were efficiently selected by natalizumab resistance. Docket No. 11624-006W01

[0073] FIG. 13A-B shows saturating mutagenesis of the natalizumab epitope in subunit alpha-4 (ITGA4) of VLA4. FIG 13 A shows protein structure (PDB ID 4IRZ) showing natalizumab interaction with alpha-4 subunit of VLA4. Schematic showing the 4 libraries comprising the saturating mutagenesis cassette (libraries 2-4 cover the nominated natalizumab epitope, in black, library 1 an additional proximate loop, in dark gray). FIG 13B shows frequency of VCAM1+ cells with and without natalizumab, showing between 1-4% resistant cells per library.

[0074] FIG. 14A-D shows in vivo selection of ITGA4 edited HSPCs. FIG. 14A shows two groups of mice (n=4) were injected with a mix of PE (mNeonGreen(mNG)):control cells (mTagBFP(mtBFP)) (1 :3 or 1 :5). One group was pretreated 3 hours before cell injection with subcutaneous injection of 600 ug of natalizumab and continued receiving biweekly injections of 170 ug natalizumab until experiment end point. The control group (PBS) received the same number of injections with PBS. After initial sacrifice (primary, 1st), a fraction of the collected bone marrow cells were transplanted into new mice (secondary transplant (2nd)). FIG 14B shows chimerism in the bone marrow (BM) of the mice after primary and secondary transplant. FIG 14C shows flow cytometry reporter of transduced (TDX) hCD45+ cells in the bone marrow, in both primary and secondary transplants, showing selection of mNG (Q152K prime edited) HSCs in the natalizumab treated mice. FIG 14D shows allele frequency in the BM of the primary transplanted mice as determined by next generation or Sanger sequencing, showing selection of Q152K edited hematopoietic cells.

[0075] FIG. 15 describes that toxicity of conditioning regimens reduces the accessibility of ex vivo HSC gene therapies and that this can be ameliorated by providing a gene modification to allow pharmacologic selection of gene modified HSCs at the expense of nonmodified HSCs.

[0076] FIG. 16 shows VCAM-1 binding in Jurkat cells.

[0077] FIG. 17 shows natalizumab inhibition of VCAM-1 binding at 30nM and lOOnM. Natalizumab has an IC50 of 0.9-1.2 nM.

[0078] FIG. 18 shows the schematic of double prime editing and differentiation of sickle cell disease (SCD) HSPCs.

[0079] DETAILED DESCRIPTION

[0080] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure Docket No. 11624-006W01 belongs. The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0082] The following definitions are provided for the full understanding of terms used in this specification.

[0083] Terminology

[0084] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0085] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0086] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as Docket No. 11624-006W01 between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0087] “Administration” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra- arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and the administration by another.

[0088] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3- Aminoadipic acid, Hydroxy lysine, [3-alanine, p- Aminopropionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3 -Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0089] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of" when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure. Docket No. 11624-006W01

[0090] As used herein, “codon” refers to the genetic code used by living cells to translate information encoded by genetic material (DNA or mRNA sequences of nucleotide triplets) into protein. This term also refers to the genetic code that specifies which amino acids will be added next during protein synthesis.

[0091] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0092] The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.

[0093] As used herein, “diagnose”, “diagnosed”, “diagnosing”, and any grammatical variations thereof as used herein, refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms.

[0094] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0095] As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.

[0096] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA occurs. Docket No. 11624-006W01

[0097] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0098] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0099] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0100] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et Docket No. 11624-006W01 al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0101] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0102] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or Docket No. 11624-006W01 any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.

[0103] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0104] As used herein, a “mutation” refers to changing the structure of a gene. A mutation is caused by the alteration of single nucleotides in DNA, or the deletion, insertion, or rearrangement of larger sections of genes. A mutation can lead to the expression of a protein that has been changed physically or functionally leading to lethality, non-lethal dysfunction effects, or no effects.

[0105] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0106] As used herein, the term “preventing” a disease, a disorder, or unwanted physiological event in a subject refers to the prevention of a disease, a disorder, or unwanted physiological event or prevention of a symptom of a disease, a disorder, or unwanted physiological event

[0107] The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0108] The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.

[0109] The term "nucleobase" refers to the part of a nucleotide that bears the Watson / Crick basepairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

[0110] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0111] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides. Docket No. 11624-006W01

[0112] The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers.

[0113] The term “reduced”, “reduce”, “reduction”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level so long as the decrease is statistically significant.

[0114] As used throughout, by a "subject" (or a “host”) is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.

[0115] “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0116] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, Docket No. 11624-006W01 analogs, and the like. When the term “therapeutic agent” is used, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0117] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, and improvement or remediation of damage.

[0118] The term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position. In relation to substitutions, ‘immediately adjacent’ may be to the N-side (‘upstream’) or C-side (‘downstream’) of the amino acid occupying a position (‘the named amino acid’). Therefore, for an amino acid named / numbered ‘X,’ the insertion may be at position ‘X+l ’ (‘downstream’) or at position ‘X-l’ (‘upstream’).

[0119] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least

[0120] Docket No. 11624-006W01

[0121] 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide. A variant polypeptide may have substantially the same functional activity as a reference polypeptide.

[0122] “Hematopoietic cells” include, but are not limited to, hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, and basophils.

[0123] Disclosed herein are the components to be used to prepare the disclosed compositions as to be used in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. If a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0124] Engineered Hematopoietic Cell Compositions

[0125] Disclosed herein is an engineered hematopoietic cell and its use thereof in treating inherited genetic disorders and acquired disorders. Hematopoietic cells that express VLA-4 include, but are not limited to, hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, and basophils.

[0126] Accordingly, in one aspect, disclosed herein is an engineered hematopoietic cell comprising a VLA-4 receptor variant, wherein the VLA-4 receptor variant comprises a mutation in an ITGA4 subunit, and wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding. Docket No. 11624-006W01

[0127] The VLA-4 receptor or the very late antigen-4 is an integrin dimer. It is composed of CD49d (alpha 4, a4) and CD29 (beta 1, pi) and in some embodiments the VLA-4 receptor is called Integrin oc4 1. The alpha 4 (a4) subunit is 155 kDa, and the beta 1 subunit is 150 kDa. VLA- 4 is expressed on the cell surfaces of cells, progenitor cells, T and B cells, monocytes, natural killer cells, eosinophils, but not neutrophils. It functions to promote an inflammatory response by the immune system by assisting in the movement of leukocytes to tissue that requires inflammation. It is a key player in cell adhesion.

[0128] In some embodiments, the mutation in the ITGA4 subunit is present in the natalizumab binding epitope. In some embodiments, the mutation in the natalizumab binding epitope is located between amino acids R146 to N259 of the ITGA4 subunit. In some embodiments, the mutation in the ITGA4 subunit is located between amino acids R146 to N259 of SEQ ID NO: 19.

[0129] In some embodiments, the mutation comprises an amino acid mutation. In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152H. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is Q152R. In some embodiments, the amino acid mutation is Q152Y. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201Q. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is L206P. In some embodiments, the amino acid mutation is L206S. In some embodiments, the amino acid mutation is L206V. In some embodiments, the amino acid mutation is L206W. In some embodiments, the amino acid mutation is K256N. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K256S.

[0130] In some embodiments, the amino acid mutation is selected from a Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y,

[0131] V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H,

[0132] K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S,

[0133] Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, Docket No. 11624-006W01

[0134] H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M mutation in SEQ ID NO: 19.

[0135] In some embodiments, the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some embodiments, the silent mutation comprises a Y151Y mutation (in the amino acid sequence SEQ ID NO: 19), wherein the 453rd nucleotide is changed from a thymine (T) to a cytosine (C) (in the nucleic acid sequence SEQ ID NO:18).

[0136] In some embodiments, the mutations are made using gene editing. In some embodiments, the gene editing is prime editing. Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. In some embodiments, the pegRNA comprises SEQ ID NOS: 1-11, 12-14, 24-38, or 39-62. In some embodiments, the pegRNA comprise the replacement of thymidine residues with uridine residues.

[0137] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand. In some embodiments the VLA-4 receptor variant binds to VCAM- 1 expressed on bone marrow stromal cells.

[0138] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab. Natalizumab is a humanized monoclonal antibody against the cell adhesion molecule a4-integrin.

[0139] In some embodiments, disclosed herein is a VLA-4 receptor variant comprising a mutation in an ITGA4 subunit, and wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

[0140] Methods of Treating an Inherited Genetic Disorder

[0141] In one aspect disclosed herein, is a method of treating an inherited genetic disorder in a subject, comprising: isolating a hematopoietic cell from a blood sample from the subject; Docket No. 11624-006W01 engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and administering to the subject the engineered hematopoietic cell.

[0142] In one aspect disclosed herein, is a method of treating an inherited genetic disorder in a subject. In some embodiments, the method comprises isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo or in vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder.

[0143] In some embodiments, the gene editing is in vivo and comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using an integrating or non-integrating viral vector, a viral like particle, a direct mRNA protein or a cell membrane delivery.

[0144] In one aspect disclosed herein, is a method of treating an inherited genetic disorder in a subject, comprising: isolating a hematopoietic cell from a blood sample from an allogeneic donor; engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and administering to the subject the engineered hematopoietic cell.

[0145] In some embodiments, the method of treating an inherited genetic disorder in a subject comprises isolating a hematopoietic cell from a blood sample from an allogeneic donor; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 Docket No. 11624-006W01 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder.

[0146] In some embodiments, the hematopoietic cell from a blood sample is an autologous sample (from the same subject being treated). In some embodiments, the hematopoietic cell from a blood sample is from a person other than the subject being treated (from an allogeneic donor).

[0147] In some embodiments, the method further comprises administering natalizumab to the subject.

[0148] In some embodiments, the mutation in the ITGA4 subunit is present in the natalizumab binding epitope. In some embodiments, the mutation in the natalizumab binding epitope is located between amino acids R146 to N259 of the ITGA4 subunit. In some embodiments, the mutation in the ITGA4 subunit is located between amino acids R146 to N259 of SEQ ID NO: 19.

[0149] In some embodiments, the mutation comprises an amino acid mutation. In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152H. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is Q152R. In some embodiments, the amino acid mutation is Q152Y. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201Q. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is L206P. In some embodiments, the amino acid mutation is L206S. In some embodiments, the amino acid mutation is L206V. In some embodiments, the amino acid mutation is L206W. In some embodiments, the amino acid mutation is K256N. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K256S.

[0150] In some embodiments, the amino acid mutation is selected from a Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, Docket No. 11624-006W01

[0151] H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M mutation in SEQ ID NO: 19.

[0152] In some embodiments, the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some embodiments, the silent mutation comprises a Y151Y mutation (in the amino acid sequence SEQ ID NO: 19), wherein the 453rd nucleotide is changed from a thymine (T) to a cytosine (C) (in the nucleic acid sequence SEQ ID NO:18).

[0153] In some embodiments, the mutations are made using gene editing. In some embodiments, the gene editing is prime editing. Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. In some embodiments, the pegRNA comprises SEQ ID NOS: 1-11, 12-14, 24-38, or 39-62. In some embodiments, the pegRNA comprise the replacement of thymidine residues with uridine residues.

[0154] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand. In some embodiments the VLA-4 receptor variant binds to VCAM- 1 expressed on bone marrow stromal cells.

[0155] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab. Natalizumab is a humanized monoclonal antibody against the cell adhesion molecule a4-integrin.

[0156] In some embodiments, natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

[0157] In some embodiments, natalizumab is administered to the subject at multiple times and / or multiple doses. In some embodiments, natalizumab is administered to the subject at several different timepoints. Docket No. 11624-006W01

[0158] In some embodiments the inherited genetic disorder is selected from the group consisting of sickle cell anemia, thalassemia, a bone marrow failure disorder, an inherited leukocyte disorder, an inherited anemia, an inherited error of immunity disorder, a metabolic disorder, a skeletal disorder, an inherited thrombocytopenia, and an immunodeficiency.

[0159] In some embodiments, the bone marrow failure disorder is selected from a group consisting of Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, severe congenital neutropenia, Diamond-Blackfan anemia, GATA2 deficiency, SAMD9 / SAMD9L disorders, MECOM syndromes, and congenital amegakaryocytic thrombocytopenia.

[0160] In some embodiments, the inherited leukocyte disorder is chronic granulomatous disease. In some embodiments, the inherited anemia is a hemoglobinopathy, a membranopathy, an enzymopathy, or a congenital dyserythropoietic anemia.

[0161] In some embodiments, the inherited error of immunity disorder includes but is not limited to severe congenital immunodeficiency.

[0162] In some embodiments, the metabolic disorder is a leukodystrophy or a lysosomal storage disorder.

[0163] In some embodiments, the skeletal disorder includes but is not limited to infantile osteopetrosis.

[0164] In some embodiments, the inherited genetic disorder is sickle cell anemia. In some embodiments, the genetic mutation causing the inherited genetic disorder is an E6V mutation in a beta-globin gene (HBB).

[0165] In some embodiments, the engineered hematopoietic cell is administered to the subject hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 hours, daily once, twice or three times, weekly, for up to 1, 2, 3, 4 week(s), monthly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 month(s), 1, 2, or 3 years and wherein the subject is a human.

[0166] In some embodiments, natalizumab is administered to the subject hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 hours, daily once, twice or three times, weekly, for up to 1, 2, 3, 4 week(s), monthly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 month(s), 1 , 2, or 3 years and wherein the subject is a human.

[0167] In some embodiments, the method suppresses symptoms of the inherited genetic disorder in a subject relative to an untreated subject. In some embodiments, the method suppresses the symptoms of the inherited genetic disorder by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more in a subject relative to an untreated subject.

[0168] In one aspect disclosed herein, is a method of treating an inherited genetic disorder in a subject, comprising: Docket No. 11624-006W01 isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and administering to the subject the engineered hematopoietic cell.

[0169] In some embodiments, the VLA-4 receptor variant (wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding) can be edited ex vivo. In some embodiments, the VLA-4 receptor variant (wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding) can be edited in vivo. In some embodiments, the VLA-4 receptor variant is produced, for example, by integrating or non-integrating viral vector delivery, viral like particle delivery, direct mRNA delivery for protein expression, cell membrane delivery, or other similar mechanisms.

[0170] In some embodiments, the VLA-4 receptor variant can be produced through an appropriate gene modification or gene editing method or through direct mRNA delivery. In some embodiments, the VLA-4 receptor variant can be produced through prime editing, base editing, CRISPR gene editing (for example CRISPR / Cas9 editing), nuclease-driven homology directed repair (HDR), DNA-dependent DNA polymerase mediated editing, integrase or recombinase-mediated gene editing, lentiviral-mediated or retroviral-mediated gene transfer, adeno-associated virus (AAV)- mediated gene transfer, or mRNA delivery, to achieve a similar outcome (for example, using VLA- 4 mutants to confer natalizumab resistance and enabling pharmacological selection of hematopoietic cells).

[0171] Methods of Treating an Acquired Disorder

[0172] In one aspect disclosed herein, is a method of treating an acquired disorder in a subject, comprising: isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and administering to the subject the engineered hematopoietic cell. Docket No. 11624-006W01

[0173] In one aspect disclosed herein, is a method of treating an acquired disorder in a subject. In some embodiments, the method comprises isolating a hematopoietic cell from a blood sample from the subject; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

[0174] In one aspect disclosed herein, is a method of treating an acquired disorder in a subject, comprising: isolating a hematopoietic cell from a blood sample from an allogeneic donor; engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and administering to the subject the engineered hematopoietic cell.

[0175] In some embodiments, the method of treating an acquired disorder in a subject comprises isolating a hematopoietic cell from a blood sample from an allogeneic donor; engineering the hematopoietic cell to obtain an engineered hematopoietic cell and administering to the subject the engineered hematopoietic cell. In some embodiments, engineering the hematopoietic cell comprises introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

[0176] In some embodiments, the hematopoietic cell from a blood sample is an autologous sample (from the same subject being treated). In some embodiments, the hematopoietic cell from a blood sample is from a person other than the subject being treated (from an allogeneic donor).

[0177] In some embodiments, the method further comprises administering natalizumab to the subject.

[0178] In some embodiments, the mutation in the ITGA4 subunit is present in the natalizumab binding epitope. In some embodiments, the mutation in the natalizumab binding epitope is located between amino acids R146 to N259 of the ITGA4 subunit. In some embodiments, the mutation in the ITGA4 subunit is located between amino acids R146 to N259 of SEQ ID NO: 19.

[0179] In some embodiments, the mutation comprises an amino acid mutation. In some embodiments, the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, Docket No. 11624-006W01

[0180] F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L,

[0181] K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F,

[0182] A204H, A204K, A204W, A204Y, E206P, E206S, E206V, E206W, E206Y, H226D, H226K,

[0183] H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, E258F, E258P, E258W, or I260M, or a combination thereof. In some embodiments, the amino acid mutation is Q152H. In some embodiments, the amino acid mutation is Q152K. In some embodiments, the amino acid mutation is Q152R. In some embodiments, the amino acid mutation is Q152Y. In some embodiments, the amino acid mutation is K201I. In some embodiments, the amino acid mutation is K201Q. In some embodiments, the amino acid mutation is K201R. In some embodiments, the amino acid mutation is E206P. In some embodiments, the amino acid mutation is E206S. In some embodiments, the amino acid mutation is E206V. In some embodiments, the amino acid mutation is E206W. In some embodiments, the amino acid mutation is K256N. In some embodiments, the amino acid mutation is K256R. In some embodiments, the amino acid mutation is K256S.

[0184] In some embodiments, the amino acid mutation is selected from a Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H,

[0185] K201I, K201E, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S,

[0186] Y202T, A204F, A204H, A204K, A204W, A204Y, E206P, E206S, E206V, E206W, E206Y,

[0187] H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E,

[0188] K256F, K256G, K256I, K256E, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, E258F, E258P, E258W, or I260M mutation in SEQ ID NO: 19.

[0189] In some embodiments, the VEA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation. In some embodiments, the silent mutation comprises a Y151 Y mutation (in the amino acid sequence SEQ ID NO:19), wherein the 453rd nucleotide is changed from a thymine (T) to a cytosine (C) (in the nucleic acid sequence SEQ ID NO: 18).

[0190] In some embodiments, the genetic mutation causing the acquired disorder is corrected.

[0191] In some embodiments, the mutations are made using gene editing. In some embodiments, the gene editing is prime editing. Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses Docket No. 11624-006W01 a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. In some embodiments, the pegRNA comprises SEQ ID NOS: 1-11, 12-14, 24-38, or 39-62. In some embodiments, the pegRNA comprise the replacement of thymidine residues with uridine residues.

[0192] In some embodiments, the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand. In some embodiments the VLA-4 receptor variant binds to VCAM- 1 expressed on bone marrow stromal cells.

[0193] In some embodiments, the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in the presence of natalizumab.

[0194] Natalizumab is a humanized monoclonal antibody against the cell adhesion molecule a4- integrin.

[0195] In some embodiments, natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the subject. In some embodiments, natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

[0196] In some embodiments, natalizumab is administered to the subject at multiple times and / or multiple doses. In some embodiments, natalizumab is administered to the subject at several different timepoints.

[0197] In some embodiments, the acquired disorder is selected from the group consisting of HIV / AIDS, a malignancy, and an autoimmune disease. In some embodiments, the method further comprises instructing a genetic change for treating the acquired disorder.

[0198] In some embodiments, the engineered hematopoietic cell is administered to the subject hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 hours, daily once, twice or three times, weekly, for up to 1, 2, 3, 4 week(s), monthly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 month(s), 1, 2, or 3 years and wherein the subject is a human.

[0199] In some embodiments, natalizumab is administered to the subject hourly, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23 hours, daily once, twice or three times, weekly, for up to 1, 2, 3, 4 week(s), monthly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 month(s), 1, 2, or 3 years and wherein the subject is a human. Docket No. 11624-006W01

[0200] In some embodiments, the method suppresses symptoms of the inherited genetic disorder in a subject relative to an untreated subject. In some embodiments, the method suppresses the symptoms of the inherited genetic disorder by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more in a subject relative to an untreated subject.

[0201] In some embodiments, VLA-4 receptor variant (wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding) can be edited ex vivo. In some embodiments, VLA-4 receptor variant (wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding) can be edited in vivo. In some embodiments, the VLA-4 receptor variant is produced, for example, by integrating or non-integrating viral vector delivery, viral like particle delivery, direct mRNA delivery for protein expression, cell membrane delivery, or other similar mechanisms.

[0202] Disclosed is method of treating an inherited or acquired genetic disorder in a subject in vivo, comprising: engineering a hematopoietic cell in vivo, wherein said engineering comprises: delivering a gene modification vector to produce a natalizumab resistance variant, wherein said variant comprises a mutation; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and administering to the subject natalizumab to select for gene modified hematopoietic cells.

[0203] EXAMPLES

[0204] The following examples are set forth below to illustrate the compositions, cells, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0205] Example 1: Pharmacological selection for gene modified hematopoietic cells

[0206] Disclosed herein is an approach to enable hematopoietic retention that would reduce the toxicity and simplify the delivery of the therapy by bypassing the need for preparative chemotherapy. The approach relies on manipulating the response of hematopoietic and progenitor cells (HSPCs) to reversible adhesive interactions that allow these cells to home from the peripheral blood (PB) to the bone marrow (BM) and to be retained in the BM. A major receptor expressed on HSPCs that serves this adhesive function is the integrin receptor VLA-4 (alpha-4 / beta- 1 (a4|31 ) heterodimer) which binds to its ligand VCAM-1. VLA-4 antagonists such as the FDA-approved Docket No. 11624-006W01 agent natalizumab result in the mobilization of HSPCs from the BM to the PB and block the homing of HSPCs from the PB to the BM.

[0207] Shown here are variants of VLA-4 that can be engineered and can render the receptor resistant to the antagonistic effects of natalizumab. HSPCs engineered to express these variants by gene editing retain similar VCAM-1 binding potential in the presence of natalizumab as do unmanipulated HSPCs in the absence of natalizumab. This strategy enables selective hematopoietic retention, such as for autologous gene therapy, without requiring chemotherapy, which greatly reduces the toxicity.

[0208] Gene transfer and gene editing of hematopoietic stem and progenitor cells (HSPCs) is a promising treatment for inherited and acquired diseases, including primary immunodeficiencies, hemoglobinopathies, metabolic diseases, HIV / AIDS, cancer, and bone marrow failure syndromes. The use of autologous HSPCs for gene therapy and transplantation has removed previous limitations of allogeneic HSPC transplantation, such as the risk of graft-versus-host disease, limited donor availability, and the need for post-transplantation immunosuppression treatment. The general procedure for autologous HSPC transplantation and gene therapy consists of i) mobilization of HSPCs from the bone marrow (BM) to the peripheral blood (PB), ii) collection through apheresis, iii) genetic correction ex vivo or in vivo, iv) myeloablative conditioning therapy to deplete bone marrow HSPCs, and v) infusion and engraftment (or retention) of gene modified HSPCs. Disclosed herein is a therapeutic strategy to eliminate the need for toxic conditioning regimens and improve retention / engraftment of edited HSPCs for broader and safer use of these therapies.

[0209] HSPC therapies start with the collection of HSPCs, which are mobilized from patient’s bone marrow by drugs such as plerixafor or natalizumab. These drugs block the interaction of receptors on HSPCs with their ligands in the bone marrow niche. One of the major factors mediating HSPC retention to the bone marrow is integrin very late antigen 4 (VLA-4 or a4| 1 ), a heterodimer constituted of an alpha-4 subunit (a4) (encoded by ITGA4) and a beta-1 subunit (Pl ) (encoded by ITGB1), binding to its primary ligand vascular cell adhesion molecule-1 (VCAM-1).

[0210] Saturation mutagenesis of the natalizumab epitope of VLA4 subunit alpha-4 (ITGA4) was performed to identify VLA4 variants that maintain binding of VC AMI but no longer bind to its inhibitor natalizumab. Gene editing technologies were used to install these mutations at the endogenous ITGA4 locus of Jurkat cells and healthy donor CD34+ HSPCs and it has been demonstrated that these mutations no longer respond to natalizumab, but retain VCAM1 binding and receptor function. Docket No. 11624-006W01

[0211] The premise is these natalizumab-resistant HSPCs can be pharmacologically selected in vivo based on their ability to home, engraft and be retained in the patient’s bone marrow in the presence of natalizumab (Figure 1). In contrast, unmodified HSPCs vacate the bone marrow and allow for increased engraftment of the edited HSPCs. Patients are re-dosed with natalizumab until edited cells exceed a therapeutic threshold. Thus, natalizumab-resistant HSPCs avoid the need for chemotherapeutic conditioning regimens typically required to make space for therapeutic HSPCs in the bone marrow by enabling pharmacological selection in vivo for gene modified hematopoietic cells.

[0212] Through multiple sequence alignment and structural analysis, multiple residues were nominated in the natalizumab epitope of VLA-4 that are important for antibody binding: K256, K201 and Q152. Mutations were investigated at amino acid position Q152, including Q152K in ITGA4 (encoding the alpha-4 subunit (a4) of VLA-4), through prime editing in Jurkat cells and CD34+ HSPCs (Table 1 , Figures 2 and 3). Ligand binding assays show these mutations dominantly confer resistance to natalizumab and retain binding of VCAM-1, the primary ligand of VLA-4 (Tables 2-4, Figures 4-11). Using sickle cell disease (SCD) patient CD34+ HSPCs, shown herein is that multiplex genetic modification of both VLA-4 to confer natalizumab resistance and prime editing to correct the SCD mutation enables selection of SCD mutation-corrected HSPCs by retained VCAM-1 binding capacity in presence of natalizumab (Figure 12). Additionally, disclosed herein is that VCAM-1 binding is restored and natalizumab resistance is conferred in ITGA4 deficient Jurkat cells through transient expression of a 1TGA4 construct carrying the Q152K mutation.

[0213] Methods:

[0214] Jurkat cells: Using PEmax-La a single amino acid change Q152K (CAA to AAA) was introduced to eliminate natalizumab binding and a silent mutation Y151Y (TAT to TAC) to improve prime editing efficiency (epegRNA with protospacer of 19 nt, and 3’ extension sequences including PBS and RTT sequences between 20 and 28 nt; shown in Table 1 and Figure 2). 5xl05Jurkat cells were electroporated with 500 ng PEmax plasmid, 250 ng epegRNA plasmid, and + / - 100 ng nicking guide plasmid, using SE Lonza kit, Jurkat E6.1 program, pulse CL-120. As a control, a B2M targeting pegRNA was used. The cells were recovered in 1 ml pre- warmed media, and grew over 8 days, after which genomic DNA was collected to evaluate editing with Sanger sequencing. After 15 days a ligand binding assay was performed.

[0215] For the binding assay, cells were incubated for 30 minutes with 1 or 2 nM natalizumab (Human Integrin alpha-4 beta-1 (a4pi) research grade natalizumab biosimilar antibody, R&D Docket No. 11624-006W01 systems, MAB10603) at room temperature, followed by incubation with 30 or 100 nM VCAM-1 (Recombinant human VCAM-1 / CD106 FC Chimera, C-term 6-His tag, R&D systems, 862-VC), 30 minutes at room temperature (added directly to cells with natalizumab, no washing in between, natalizumab is still present). The cells were washed, after which bound VCAM-1 was stained with Alexa-Fluor His-tag secondary antibody (30 minutes at room temperature; His Tag Alexa Fluor 647-conjugated Antibody, R&D systems, IC0501R-100UG).

[0216] HSPCs (CD34+ cells): After 24 hours of culture with cytokines, 5xl05HSPCs were electroporated with 2000 ng PEmax-La mRNA, 200 pmol synthetic 1TGA4-QA 52K_v5 pegRNA, and 100 pmol PE3b nick sgRNA using pulse code DS-130. As a control 5xlO5HSPCs were electroporated with 2000 ng PEmax-La mRNA and 200 pmol B2M targeting pegRNA using pulse code DS-130. HSPCs were cultured for 72 hours, after which genomic DNA was harvested to evaluate editing and a VCAM-1 binding assay was performed as described above.

[0217] ITGA4 saturation mutagenesis using lentiviral screening: Saturation libraries were designed to replace all residues in the ITGA4 natalizumab epitope with all 20 amino acids, each as an individual variant. Oligo pools (Twist Biosciences) for saturation libraries were flanked with primer binding sites for PCR amplification and Type IIS restriction sites for cloning (New England Biolabs). Libraries were assembled by Golden Gate Cloning at the respective library positions into a lentiviral backbone in frame with an IRES-PURO cassette under an SFFV promoter. Library quality was assessed by Sanger sequencing of single colonies. Resulting plasmid libraries were transformed into electrocompetent Endura DUOs cells (Bioresearch Technologies) providing >1000x library coverage (colonies per library member).

[0218] The resulting plasmid libraries were transfected into HEK293T cells with packaging plasmid psPAX2 (Addgene #12260) and envelope plasmid VSV-G (Addgene #8454) to produce lentivirus. The virus-containing supernatant was collected 48 h after transfection, clarified by filtration (0.45 pm) and concentrated by ultracentrifugation. Lentivirus was then titrated in Jurkat cells to determine the amount of lentivirus resulting in a multiplicity of approximately 0.2-0.3 which minimizes the possibility of multiple VLA4 variants in individual cells. For each library, three independent lentiviral transductions were prepared in parallel and maintained independently throughout the screen. 24 hours after transduction, Jurkat cells were selected by supplementing media with puromycin to a final concentration of 1 pg / mL and expanded for ~ 1 week. For each replicate, ~3 million cells were first stained for viability, then incubated with natalizumab for 30 Docket No. 11624-006W01 min (at 50 or 20 nM), then 100 or 150 nM VCAM1 were added, and cells were incubated for another 30 min at room temperature. The cells were washed, after which bound VC AMI was stained with Alexa-Fluor His-tag secondary antibody (30 minutes at room temperature; His Tag Alexa Fluor 647-conjugated Antibody, R&D systems, IC0501R-100UG). The cells were then sorted in 3 populations based on VCAM1-AF647 fluorescence: VCAM1 high, medium and low cells using an BD FACS Aria cell sorter (BD Biosciences). After sorting cells, DNA was extracted using QuickExtract and the library cassette was PCR-amplified first with a primer set specific to the lentiviral cassette. Then additional PCR steps added Illumina adaptors and barcodes. Amplicon libraries were sequenced using Novaseq to >1500-fold coverage (Figure 13).

[0219] Enrichment analysis of amino acid substitutions: FASTQ sequencing files from the different experimental conditions were processed to generate count matrices reporting the number of reads corresponding to each amino acid substitution. Comparisons were performed between the presort sample and two test groups: high and medium fractions. Each group included three biological replicates. For each amino acid substitution, we modeled the observed read counts using logistic regression for binomial data, where the number of reads corresponding to a substitution was modeled against the total number of reads (coverage) for that replicate. Specifically, paired variables representing the number of reads for a given substitution and total reads were used as the response and binomial denominator, respectively. A regression analysis was conducted: Presort vs High+Medium. We used a likelihood ratio test (LRT) to assess the statistical significance of enrichment for each substitution in the test group relative to Presort. P-values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate (FDR) method (adjusted p- value), and a threshold of FDR < 0.05 was used to define statistical significance. To improve robustness and reduce the number of potential false positives, we also computed the coefficient of variation (CV) of the normalized substitution frequency across replicates in the test group. This was defined as the standard deviation divided by the mean of the substitution-to-coverage ratio across the three replicates (CV test ratio). Amino acid substitutions were considered significantly enriched if they met adjusted p-value (adjpvalue) < 0.05, CV test ratio < 2 and enrichment coefficient (enrichment) > 0.8 (corresponding to a > 1.7-fold increase relative to Presort). The substitutions meeting these criteria are reported in Table 5. The analysis was performed in R, using the data, table package for data handling and fastglm for efficient model estimation.

[0220] In vivo selection of ITGA4 edited HSPCs: Competitive xenograft experiments were performed with ITGA4 Q152K prime edited and unedited human HSPCs from two donors. HSPCs were thawed and transduced with either a lentiviral mNeonGreen (mNG) or mTagBFP (mtBFP) Docket No. 11624-006W01 fluorescent marker. After 24 hours of prestimulation with cytokines, the mNG transduced cells were electroporated with 2000 ng PEmax-La mRNA, 150 or 200 pmol synthetic 1TGA4- Q152K_v5 pegRNA, and 50 or 100 pmol PE3b nick sgRNA. The mtBFP transduced cells were either electroporated without any RNA, or received a AAVS1 pegRNA (safe locus control edit). After an additional 48 hours of culture, these cells were mixed in a 1:3 or 1 :5 ratio (Q152K PE:control), and injected into mice. We divided the mice into two groups; one received biweekly subcutaneous injections of 170 mcg of natalizumab, the other biweekly control injections with PBS. Peripheral blood was drawn every 4 weeks, and after 20 to 22 weeks the mice were euthanized. Cells were collected from the bone marrow for flow cytometry analysis, genotyping and secondary transplants. Secondary transplanted mice were not treated and sacrificed after 12- 16 weeks after cell injection. Again, bone marrow cells were collected for flow cytometry analysis and genotyping. In the absence of natalizumab, a neutral impact of the prime edit (variant Q152K) on engraftment function was observed. In the presence of natalizumab, profound selection for engraftment of receptor modified HSPCs was observed, based on reporter fluorescent protein expression and gene edited allele frequency (Figure 14). These results establish proof-of-concept that natalizumab may be repurposed for hematopoietic selection.

[0221] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0222] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0223] Docket No. 11624-006W01

[0224] TABLES

[0225] Table 1. Prime editing guide RNAs (pegRNAs) and engineered pegRNAs (epegRNAs) for ITGA4 Q152 prime editing by plasmid expression via the U6 promoter, target location as shown in Figure 2. Sequences show the spacer, in italics the scaffold sequence, in bold the reverse transcription template (RTT) and primer binding site (PBS), bold underlined the target edit, and underlined the tevopreQi structural motif (in the epegRNAs). (e)pegRNA sequences are shown as DNA versions, with U’s converted to T’s and with M for A and C. Docket No. 11624-006W01

[0226] Table 2. Prime editing (PE) in Jurkat cells, quantification of allelic editing frequency using BEAT webtool (Xu, Liu, and Han 2019, PMID: 31328964) from Sanger sequence data. Editing is reported at the cognate target locus, so at ITGA4 for ITGA4 targeting epegRNAs and B2M for B2M targeting pegRNA. Docket No. 11624-006W01

[0227] Table 3. Sequences for synthetic pegRNA ITGA4 Q152K, as shown in Figures 2, 9-12 and Table 4. Sequence shows the spacer, in italics the scaffold sequence, in bold the reverse transcription template (RTT) and primer binding site (PBS), bold underlined the target edit, and finally the polyU tail. 2’-O-Methyl modified bases are highlighted with a lowercase ‘’m”, and phosphorothioate linkages are highlighted with an asterisk in the synthetic pegRNA sequences.

[0228] Table 4. Prime editing (PE) in CD34+ HSPCs, quantification of allelic editing frequency using BEAT webtool from Sanger sequence data. Editing is reported at the cognate target locus, so at ITGA4 for ITGA4 targeting pegRNAs.

[0229] Table 5. ITGA4 variants, identified in ITGA4 site-saturation screening, that bind VCAM1 in presence of natalizumab. The amino acid change (variant) and the ITGA4 residue number are listed along with enrichment score (Enrichment), statistical significance value (Adjusted p-value), and coefficient of variance test ratio (CV test ratio). Enrichment is calculated as log2 fold change comparing VCAM1 positive (sorted) to total (unsorted) population in the presence of natalizumab. Adjusted p-value is calculated as an FDR corrected permutationbased p-value. Variants with positive enrichment (Enrichment > 0.8), statistical significance (Adjusted p-value < 0.05), and low variance across replicates (CV test ratio < 2) are shown. Adjusted p- values less than IxlO"16are rounded to 0. Docket No. 11624-006W01

[0230] Variant Enrichment Adjusted p- value CV test ratio

[0231] Q72E 0.87727677 0 0.65398704

[0232] L73I 0.92394967 0 0.86246786

[0233] N77S 0.82279383 0 1.26719995

[0234] G82C 0.80259644 0 0.92689765

[0235] T84I 1.07876983 0 1.34288998

[0236] C85E 0.99676426 0 1.28592218

[0237] Q152K 4.2010159 0 0.77363231

[0238] Q152N 3.93250266 0 1.23300554

[0239] Q152I 2.74939203 0 0.93777174

[0240] Q152R 1.74761836 0 1.12202066

[0241] Q152C 1.64529687 0 1.01899092

[0242] Q152V 1.18131529 0 1.10481358

[0243] Q152Y 0.90419506 0 1.18301515

[0244] Q152P 0.8580151 0 1.85356202

[0245] V155D 1.34488254 0 0.8812489

[0246] K157R 1.11764132 0 1.92186345

[0247] F162W 1.02330006 0 1.13960301

[0248] M180S 1.98061771 0 1.5649531

[0249] K201V 1.56389533 0 0.31451245

[0250] K201T 1.42455837 0 0.12030489

[0251] K201E 1.36390812 0 0.15393806

[0252] K201G 1.31333063 0 0.17772076

[0253] K201H 1.21465675 0 0.20780789

[0254] K201A 1.21195985 0 0.15096686

[0255] K201Q 1.20055882 0 0.21803966

[0256] K201N 1.13901369 0 0.12165879

[0257] K201D 1.10632104 0 0.18410155

[0258] K201I 1.08427895 0 0.14768601

[0259] K201Y 1.08293051 0 0.19961022

[0260] K201F 1.07953215 0 0.28296982

[0261] K201S 1.0458393 0 0.33958598

[0262] K201M 1.03428287 0 0.11298764 Docket No. 11624-006W01

[0263] K201C 1.00615713 0 0.19779703

[0264] K201L 0.87885913 0 0.24108189

[0265] K201R 0.84782324 0 0.20315743

[0266] Y202T 1.28590385 0 0.08321999

[0267] Y202S 1.11761448 0 0.26689234

[0268] A204K 1.40288852 0 0.33126162

[0269] A204Y 1.3830474 0 0.15293464

[0270] A204F 1.30760686 0 0.11564006

[0271] A204H 1.23119389 0 0.20665042

[0272] A204W 1.09517797 0 0.10807938

[0273] L206Y 1.1334255 0 0.21579977

[0274] H226D 1.29910406 0 0.94880411

[0275] H226Y 1.16510364 0 0.40797743

[0276] H226R 0.88284983 0 1.07310078

[0277] H226K 0.87495361 0 0.83608368

[0278] E234V 0.96776335 0 0.64239134

[0279] H242C 0.8841198 0 0.59814144

[0280] I250K 0.84976057 0 0.57815726

[0281] K256D 2.5071344 0 0.40779333

[0282] K256E 2.40770159 0 0.30713036

[0283] K256W 2.154082 0 0.35098115

[0284] K256P 1.96387316 0 0.26819571

[0285] K256F 1.65915575 0 0.27975263

[0286] K256C 1.57134775 0 0.28163283

[0287] K256I 1.56801682 0 0.34194097

[0288] K256G 1.37044157 0 0.48948183

[0289] K256Y 1.3573225 0 0.33077357

[0290] K256S 1.0979253 0 0.32482466

[0291] K256L 1.01520833 0 0.34991162

[0292] K256V 0.93862074 0 0.19491255

[0293] K256Q 0.91601798 0 0.32796778

[0294] K256A 0.87482725 0 0.42638635

[0295] E257W 1.31945305 0 0.63075284 Docket No. 11624-006W01

[0296] E257C 1.10846461 0 0.45507246

[0297] E257F 1.03638573 0 0.51603528

[0298] E257D 0.98689914 0 1.32782294

[0299] E257K 0.94024728 0 0.4722625

[0300] E257Y 0.89187199 0 0.47453591

[0301] L258F 1.01673958 0 0.47253708

[0302] L258W 0.99788343 0 0.48379537

[0303] L258P 0.88641739 0 0.65067607

[0304] I260M 0.94541941 0 0.77893534

[0305] Table 6. Prime editing guide RNAs (pegRNAs) for ITGA4 L206, A204 and Y202 prime editing. Sequences show the spacer, in italics the scaffold sequence, in bold the reverse transcription template (RTT) and primer binding site (PBS), and bold underlined the target edit. pegRNA sequences are shown as DNA versions, with U’s converted to T’s and with M for A and C. Additional silent mutations are added in the RTT to optimize editing efficiency. Docket No. 11624-006W01

[0306] Table 7. Base editing guide RNAs (sgRNA) for plasmid-based ITGA4 base editing. sgRNAs are expressed from a U6 promotor cassette. Sequences show the spacer underlined; the target base(s) in bold underlined. Terminator T stretch at the 3’ end is not shown. sgRNA sequences are shown as DNA versions, with U’s converted to T’s. All targeting human ITGA4 gene. Docket No. 11624-006W01 Docket No. 11624-006W01

[0307] SEQUENCES

[0308] SEQ ID NO. 1:

[0309] ITGA4_Q152_pegRNA_protospacer

[0310] GAATAGCTCCGTGTTATCA

[0311] SEQ ID NO. 2:

[0312] ITGA4_Q152K_epegRNA_vl

[0313] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

[0314] TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCCTTACCTTTGTAACACG

[0315] GAGCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0316] SEQ ID NO. 3:

[0317] ITGA4_Q152K_epegRNA_v2

[0318] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

[0319] TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCCTTACCTTTGTAACACG

[0320] GAGCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0321] SEQ ID NO. 4:

[0322] ITGA4_Q152K_epegRNA_v3

[0323] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

[0324] TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTACCTTTGTAACACGGAG

[0325] CTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0326] SEQ ID NO. 5:

[0327] ITGA4 Q152K epegRNA v4

[0328] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

[0329] TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTACCTTTGTAACACGGAG

[0330] CCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0331] SEQ ID NO. 6:

[0332] ITGA4_Q152K_epegRNA_v5 Docket No. 11624-006W01

[0333] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATGCCTTACCTTTGTAA CACGGAGCTCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0334] SEQ ID NO. 7:

[0335] ITGA4_Q152K_epegRNA_v6

[0336] GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATGCCTTACCTTTGTAA CACGGAGCCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA

[0337] SEQ ID NO. 8:

[0338] ITGA4_Q152NNK_pegRNA (same spacer, RTT and PBS sequence as in epegRNA_v5 with the exception of the target edit) GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATGCCTTACCMNNGTAA CACGGAGCT

[0339] SEQ ID NO. 9:

[0340] ITGA4_Q152Y_pegRNA (same spacer, RTT and PBS sequence as in epegRNA_v5 with the exception of the target edit) GAATAGCTCCGTGTTATCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCATGCCTTACCATAGTAA CACGGAGCT

[0341] SEQ ID NO. 10:

[0342] ITGA4 PE3b Nick sgRNA (19 nucleotide spacer with a G mismatch at PAM distal position 20 [shown in lowercase] for U6 promoter expression), for use with ITGA4_Q152K_epegRNAs_vl-v6 gATGCCTTACCTTTGTAACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTA GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0343] SEQ ID NO. 11:

[0344] ITGA4_Q152NNK_PE3_Nick_sgRNA, for use with ITGA4_Q152NNK and Q152Y pegRNAs Docket No. 11624-006W01

[0345] GTTCTGTTCGTAAATCAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG

[0346] TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

[0347] SEQ ID NO. 12:

[0348] ITGA4_Q152K _pegRNA_vl (2’-O-Methyl modified bases are highlighted with a lowercase ‘’m”, and phosphorothioate linkages are highlighted with an asterisk4’*” in the synthetic pegRNA sequences) mA*mG*mA*AUAGCUCCGUGUUAUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGCCUUACC UUUGUAACACGGAGCUUU*mU*mU*mUU

[0349] SEQ ID NO. 13:

[0350] ITGA4_Q152K _pegRNA_v5 (2’-O-Methyl modified bases are highlighted with a lowercase4’m”, and phosphorothioate linkages are highlighted with an asterisk4’*” in the synthetic pegRNA sequences) mA*mG*mA*AUAGCUCCGUGUUAUCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUCAUGCCU UACCUUUGUAACACGGAGCUUU*mU*mU*mUU

[0351] SEQ ID NO. 14:

[0352] ITGA4_Q152K_PE3b_Nick_sgRNA (2’-O-Methyl modified bases are marked with a lowercase4’m”, and phosphorothioate linkages are highlighted with an asterisk4’*” in the synthetic pegRNA sequences) mC*mA*mU*GCCUUACCUUUGUAACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmU*mU*mU *U

[0353] SEQ ID NO. 15: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0354] Reference genome hg38: chr2:181, 457, 655-181, 535, 527. Full cDNA sequence (exons only):

[0355] Signal peptide (amino acid -33 to 0), Natalizumab epitope (amino acid 146 upto including 259), Codon positions of mutations at Q152, K201, K256 not shown (original nonmutant codon sequences shown).

[0356] ATGGCTTGGGAAGCGAGGCGCGAACCCGGCCCCCGAAGGGCCGCCGTCCG Docket No. 11624-006W01

[0357] ACAACGTGGACACTGAGAGCGCGCTGCTTTACCAGGGCCCCCACAACACG

[0358] CTGTTCGGCTACTCGGTCGTGCTGCACAGCCACGGGGCGAACCGATGGCTC

[0359] CTAGTGGGTGCGCCCACTGCCAACTGGCTCGCCAACGCTTCAGTGATCAAT

[0360] CCCGGGGCGATTTACAGATGCAGGATCGGAAAGAATCCCGGCCAGACGTG

[0361] CGAACAGCTCCAGCTGGGTAGCCCTAATGGAGAACCTTGTGGAAAGACTT

[0362] GTTTGGAAGAGAGAGACAATCAGTGGTTGGGGGTCACACTTTCCAGACAG

[0363] CCAGGAGAAAATGGATCCATCGTGACTTGTGGGCATAGATGGAAAAATAT

[0364] ATTTTACATAAAGAATGAAAATAAGCTCCCCACTGGTGGTTGCTATGGAGT

[0365] GCCCCCTGATTTACGAACAGAACTGAGTAAAAGAATAGCTCCGTGTTATC

[0366] AAGATTATGTGAAAAAATTTGGAGAAAATTTTGCATCATGTCAAGCTGGA

[0367] ATATCCAGTTTTTACACAAAGGATTTAATTGTGATGGGGGCCCCAGGATCA

[0368] TCTTACTGGACTGGCTCTCTTTTTGTCTACAATATAACTACAAATAAATAC

[0369] AAGGCTTTTTTAGACAAACAAAATCAAGTAAAATTTGGAAGTTATTTAGG

[0370] ATATTCAGTCGGAGCTGGTCATTTTCGGAGCCAGCATACTACCGAAGTAGT

[0371] CGGAGGAGCTCCTCAACATGAGCAGATTGGTAAGGCATATATATTCAGCA

[0372] TTGATGAAAAAGAACTAAATATCTTACATGAAATGAAAGGTAAAAAGCTT

[0373] GGATCGTACTTTGGAGCTTCTGTCTGTGCTGTGGACCTCAATGCAGATGGC

[0374] TTCTCAGATCTGCTCGTGGGAGCACCCATGCAGAGCACCATCAGAGAGGA

[0375] AGGAAGAGTGTTTGTGTACATCAACTCTGGCTCGGGAGCAGTAATGAATG

[0376] CAATGGAAACAAACCTCGTTGGAAGTGACAAATATGCTGCAAGATTTGGG

[0377] GAATCTATAGTTAATCTTGGCGACATTGACAATGATGGCTTTGAAGATGTT

[0378] GCTATCGGAGCTCCACAAGAAGATGACTTGCAAGGTGCTATTTATATTTAC

[0379] AATGGCCGTGCAGATGGGATCTCGTCAACCTTCTCACAGAGAATTGAAGG

[0380] ACTTCAGATCAGCAAATCGTTAAGTATGTTTGGACAGTCTATATCAGGACA

[0381] AATTGATGCAGATAATAATGGCTATGTAGATGTAGCAGTTGGTGCTTTTCG

[0382] GTCTGATTCTGCTGTCTTGCTAAGGACAAGACCTGTAGTAATTGTTGACGC

[0383] TTCTTTAAGCCACCCTGAGTCAGTAAATAGAACGAAATTTGACTGTGTTGA

[0384] AAATGGATGGCCTTCTGTGTGCATAGATCTAACACTTTGTTTCTCATATAA

[0385] GGGCAAGGAAGTTCCAGGTTACATTGTTTTGTTTTATAACATGAGTTTGGA

[0386] TGTGAACAGAAAGGCAGAGTCTCCACCAAGATTCTATTTCTCTTCTAATGG

[0387] AACTTCTGACGTGATTACAGGAAGCATACAGGTGTCCAGCAGAGAAGCTA

[0388] ACTGTAGAACACATCAAGCATTTATGCGGAAAGATGTGCGGGACATCCTC

[0389] ACCCCAATTCAGATTGAAGCTGCTTACCACCTTGGTCCTCATGTCATCAGT Docket No. 11624-006W01

[0390] GAAAGAAAAAGACATAATGAAAAAAACAATAAACTTTGCAAGGTTTTGTG CCCATGAAAATTGTTCTGCTGATTTACAGGTTTCTGCAAAGATTGGGTTTT TGAAGCCCCATGAAAATAAAACATATCTTGCTGTTGGGAGTATGAAGACA TTGATGTTGAATGTGTCCTTGTTTAATGCTGGAGATGATGCATATGAAACG ACTCTACATGTCAAACTACCCGTGGGTCTTTATTTCATTAAGATTTTAGAG CTGGAAGAGAAGCAAATAAACTGTGAAGTCACAGATAACTCTGGCGTGGT ACAACTTGACTGCAGTATTGGCTATATATATGTAGATCATCTCTCAAGGAT AGATATTAGCTTTCTCCTGGATGTGAGCTCACTCAGCAGAGCGGAAGAGG ACCTCAGTATCACAGTGCATGCTACCTGTGAAAATGAAGAGGAAATGGAC AATCTAAAGCACAGCAGAGTGACTGTAGCAATACCTTTAAAATATGAGGT TAAGCTGACTGTTCATGGGTTTGTAAACCCAACTTCATTTGTGTATGGATC AAATGATGAAAATGAGCCTGAAACGTGCATGGTGGAGAAAATGAACTTAA CTTTCCATGTTATCAACACTGGCAATAGTATGGCTCCCAATGTTAGTGTGG AAATAATGGTACCAAATTCTTTTAGCCCCCAAACTGATAAGCTGTTCAACA TTTTGGATGTCCAGACTACTACTGGAGAATGCCACTTTGAAAATTATCAAA GAGTGTGTGCATTAGAGCAGCAAAAGAGTGCAATGCAGACCTTGAAAGGC ATAGTCCGGTTCTTGTCCAAGACTGATAAGAGGCTATTGTACTGCATAAAA GCTGATCCACATTGTTTAAATTTCTTGTGTAATTTTGGGAAAATGGAAAGT GGAAAAGAAGCCAGTGTTCATATCCAACTGGAAGGCCGGCCATCCATTTT AGAAATGGATGAGACTTCAGCACTCAAGTTTGAAATAAGAGCAACAGGTT TTCCAGAGCCAAATCCAAGAGTAATTGAACTAAACAAGGATGAGAATGTT GCGCATGTTCTACTGGAAGGACTACATCATCAAAGACCCAAACGTTATTTC ACCATAGTGATTATTTCAAGTAGCTTGCTACTTGGACTTATTGTACTTCTAT TGATCTCATATGTTATGTGGAAGGCTGGCTTCTTTAAAAGACAATACAAAT CTATCCTACAAGAAGAAAACAGAAGAGACAGTTGGAGTTATATCAACAGT AAAAGCAATGATGATTAA

[0391] SEQ ID NO. 16: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0392] Reference genome hg38: chr2:181, 457, 655-181, 535, 527. Full protein (amino acid) sequence: Signal peptide (amino acid -33 to 0), Natalizumab epitope (amino acid 146 upto including 259), Mutation positions Q152, K201, K256 not indicated (original nonmutant AA sequences shown).

[0393] MAWEARREPGPRRAAVRETVM LLLC L GVPT GRP YNVDTE S ALLY QG Docket No.11624-006W01

[0394] GKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGI VTCGHRWKNIFYIKNENKLPTGGC YG VPPDLRTELSKRIAPC YQD YV KKFGENFASC Q AGIS S FYTKDLIVMG APGS S YWTGSLFV YNITTNKY KAFLDKQNQVKFGS YLGYS VGAGHFRSQHTTEVVGGAPQHEQIGKA YIFS IDE KELNILHEM KGKKLGS YFG AS VC A VDLN ADGFS DLL V GAP MQS TIREEGR VF V YINS GS G A VMN AMETNL VGS DKY A ARFGES I VNL GDIDND GFEDV AIGAPQEDDL Q GAI YIYN GRAD GIS S TFS QRIEGLQIS KS LS MFGQS IS GQID ADNNG Y VD V A VG AFRS DS A VLLRTRP V VI VD A SLSHPES VNRTKFDC VENGWPS VCIDLTLCFS YKGKEVPGYIVLFYN MSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMR KD VRDILTPIQIE A A YHLGPH VIS KRSTEEFPPLQPILQQKKEKDIMKK TINFARFCAHENCS AD LQ V S AKIGFL KPHENKT YL A VG S M KTLMLN V SLFN AGDD A YETTLH V KLP VGL YFIKILELEEKQINC E VTDNS G V VQL DCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDN LKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKM NLTFH VINTGNS M APN VS VEIM VPNS FSPQTD KLFNILD VQTTTGECH FENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNF LCNFGKMESGKEAS VHIQLEGRPSILEMDETS ALKFEIR ATGFPEPNP RVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLIS Y VMWKAGFFKRQYKSILQEENRRDS WS YINSKSNDD*

[0395] SEQ ID NO.17: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0396] Reference genome hg38: chr2:181, 457, 655-181, 535, 527. Full protein (amino acid) sequence: Signal peptide (amino acid -33 to 0), Natalizumab epitope (amino acid 146 up to including 259), Natalizumab resistant mutations Q152K, K201I, K256S indicated.

[0397] MAWEARREPGPRRAAVRETVMLLLCLGVPTGRPYN VDTESALLYQG PHNTLFGYS VVLHSHGANRWLLVG APT ANWLAN AS VINPGAIYRCRI GKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENG SIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPC YKDY VKKFGENF AS C Q AGIS SFYTKDLIVMGAPGSS YWTGSLFV YNITTNIY KAFLDKQNQVKFGS YLGYS VGAGHFRSQHTTEVVGGAPQHEQIGKA YIFS ID E S ELNILHEM KGKKLGS YFG A SVC A VDLN ADGFS DLL VGAP MQS TIREEGR VFV YINS GS GA VMN AMETNL VGS DKY A ARFGES I VNL Docket No.11624-006W01

[0398] KSLSMFGQSISGQIDADNNGYVDVAVGAFRSDS A VLLRTRP V VIVD A SLSHPES VNRTKFDC VENGWPS VCIDLTLCFS YKGKE VPG YIVLFYN MS LD VNRK AESPPRF YFS S NGTSD VITGSIQ VS S RE ANC RTHQ AFMR KDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKK TINFARFCAHENCS AD LQ VS A KIGFL KPHENKT YL A VGS M KTLMLN V SLFN AGDD A YETTLH VKLP VGL YFIKILELEEKQINC E VTDNS G V VQL DCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDN LKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETCMVEKM NLTFH VINTGNS M APN VS VETM VPNS FSPQTD KLFNILD VQTTTGEC H FENYQR VC ALEQQKS AMQTLKGI VRFLS KTDKRLL YC IK ADPHCLNF LCNFGKMESGKEASVHIQLEGRPSILEMDETS ALKFEIR ATGFPEPNP RVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLIS Y VMWKAGFFKRQYKSILQEENRRDS WS YINSKSNDD*

[0399] SEQ ID NO.18: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0400] Reference genome hg38: chr2:181, 457, 655-181, 535, 527. Full cDNA sequence (exons only) (without signal peptide sequence):

[0401] Natalizumab epitope (amino acid 146 up to including 259), Codon positions of mutations at Q152, K201, K256 not indicated (original nonmutant codon sequences shown).

[0402] AACGTGGACACTGAGAGCGCGCTGCTTTACCAGGGCCCCCACAACACGCT GTTCGGCTACTCGGTCGTGCTGCACAGCCACGGGGCGAACCGATGGCTCCT AGTGGGTGCGCCCACTGCCAACTGGCTCGCCAACGCTTCAGTGATCAATCC CGGGGCGATTTACAGATGCAGGATCGGAAAGAATCCCGGCCAGACGTGCG AACAGCTCCAGCTGGGTAGCCCTAATGGAGAACCTTGTGGAAAGACTTGT TTGGAAGAGAGAGACAATCAGTGGTTGGGGGTCACACTTTCCAGACAGCC AGGAGAAAATGGATCCATCGTGACTTGTGGGCATAGATGGAAAAATATAT TTTACATAAAGAATGAAAATAAGCTCCCCACTGGTGGTTGCTATGGAGTGC CCCCTGATTTACGAACAGAACTGAGTAAAAGAATAGCTCCGTGTTATCAA GATTATGTGAAAAAATTTGGAGAAAATTTTGCATCATGTCAAGCTGGAAT ATCCAGTTTTTACACAAAGGATTTAATTGTGATGGGGGCCCCAGGATCATC TTACTGGACTGGCTCTCTTTTTGTCTACAATATAACTACAAATAAATACAA GGCTTTTTTAGACAAACAAAATCAAGTAAAATTTGGAAGTTATTTAGGATA TTCAGTCGGAGCTGGTCATTTTCGGAGCCAGCATACTACCGAAGTAGTCGG Docket No. 11624-006W01

[0403] ATGAAAAAGAACTAAATATCTTACATGAAATGAAAGGTAAAAAGCTTGGA

[0404] TCGTACTTTGGAGCTTCTGTCTGTGCTGTGGACCTCAATGCAGATGGCTTC

[0405] TCAGATCTGCTCGTGGGAGCACCCATGCAGAGCACCATCAGAGAGGAAGG

[0406] AAGAGTGTTTGTGTACATCAACTCTGGCTCGGGAGCAGTAATGAATGCAA

[0407] TGGAAACAAACCTCGTTGGAAGTGACAAATATGCTGCAAGATTTGGGGAA

[0408] TCTATAGTTAATCTTGGCGACATTGACAATGATGGCTTTGAAGATGTTGCT

[0409] ATCGGAGCTCCACAAGAAGATGACTTGCAAGGTGCTATTTATATTTACAAT

[0410] GGCCGTGCAGATGGGATCTCGTCAACCTTCTCACAGAGAATTGAAGGACT

[0411] TCAGATCAGCAAATCGTTAAGTATGTTTGGACAGTCTATATCAGGACAAAT

[0412] TGATGCAGATAATAATGGCTATGTAGATGTAGCAGTTGGTGCTTTTCGGTC

[0413] TGATTCTGCTGTCTTGCTAAGGACAAGACCTGTAGTAATTGTTGACGCTTC

[0414] TTTAAGCCACCCTGAGTCAGTAAATAGAACGAAATTTGACTGTGTTGAAA

[0415] ATGGATGGCCTTCTGTGTGCATAGATCTAACACTTTGTTTCTCATATAAGG

[0416] GCAAGGAAGTTCCAGGTTACATTGTTTTGTTTTATAACATGAGTTTGGATG

[0417] TGAACAGAAAGGCAGAGTCTCCACCAAGATTCTATTTCTCTTCTAATGGAA

[0418] CTTCTGACGTGATTACAGGAAGCATACAGGTGTCCAGCAGAGAAGCTAAC

[0419] TGTAGAACACATCAAGCATTTATGCGGAAAGATGTGCGGGACATCCTCAC

[0420] CCCAATTCAGATTGAAGCTGCTTACCACCTTGGTCCTCATGTCATCAGTAA

[0421] ACGAAGTACAGAGGAATTCCCACCACTTCAGCCAATTCTTCAGCAGAAGA

[0422] AAGAAAAAGACATAATGAAAAAAACAATAAACTTTGCAAGGTTTTGTGCC

[0423] CATGAAAATTGTTCTGCTGATTTACAGGTTTCTGCAAAGATTGGGTTTTTG

[0424] AAGCCCCATGAAAATAAAACATATCTTGCTGTTGGGAGTATGAAGACATT

[0425] GATGTTGAATGTGTCCTTGTTTAATGCTGGAGATGATGCATATGAAACGAC

[0426] TCTACATGTCAAACTACCCGTGGGTCTTTATTTCATTAAGATTTTAGAGCT

[0427] GGAAGAGAAGCAAATAAACTGTGAAGTCACAGATAACTCTGGCGTGGTAC

[0428] AACTTGACTGCAGTATTGGCTATATATATGTAGATCATCTCTCAAGGATAG

[0429] ATATTAGCTTTCTCCTGGATGTGAGCTCACTCAGCAGAGCGGAAGAGGAC

[0430] CTCAGTATCACAGTGCATGCTACCTGTGAAAATGAAGAGGAAATGGACAA

[0431] TCTAAAGCACAGCAGAGTGACTGTAGCAATACCTTTAAAATATGAGGTTA

[0432] AGCTGACTGTTCATGGGTTTGTAAACCCAACTTCATTTGTGTATGGATCAA

[0433] ATGATGAAAATGAGCCTGAAACGTGCATGGTGGAGAAAATGAACTTAACT

[0434] TTCCATGTTATCAACACTGGCAATAGTATGGCTCCCAATGTTAGTGTGGAA

[0435] ATAATGGTACCAAATTCTTTTAGCCCCCAAACTGATAAGCTGTTCAACATT Docket No.11624-006W01

[0436] GTGTGTGCATTAGAGCAGCAAAAGAGTGCAATGCAGACCTTGAAAGGCAT AGTCCGGTTCTTGTCCAAGACTGATAAGAGGCTATTGTACTGCATAAAAGC TGATCCACATTGTTTAAATTTCTTGTGTAATTTTGGGAAAATGGAAAGTGG AAAAGAAGCCAGTGTTCATATCCAACTGGAAGGCCGGCCATCCATTTTAG AAATGGATGAGACTTCAGCACTCAAGTTTGAAATAAGAGCAACAGGTTTT CCAGAGCCAAATCCAAGAGTAATTGAACTAAACAAGGATGAGAATGTTGC GCATGTTCTACTGGAAGGACTACATCATCAAAGACCCAAACGTTATTTCAC CATAGTGATTATTTCAAGTAGCTTGCTACTTGGACTTATTGTACTTCTATTG ATCTCATATGTTATGTGGAAGGCTGGCTTCTTTAAAAGACAATACAAATCT ATCCTACAAGAAGAAAACAGAAGAGACAGTTGGAGTTATATCAACAGTAA AAGCAATGATGATTAA

[0437] SEQ ID NO.19: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0438] Reference genome hg38: chr2:181, 457, 655-181, 535, 527. Full protein (amino acid) sequence (without signal peptide sequence):

[0439] Natalizumab epitope (amino acid 146 upto including 259), Mutation positions Q152, K201, K256 not indicated (original nonmutant AA sequences shown).

[0440] YNVDTES ALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLA NASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQ WLGVTLSRQPGENGIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLR TELS KRI APC YQD Y VKKFGENFASC Q AGIS S F YTKDLI VMG APGS S Y WTGSLFVYNITTNKYKAFLDKQNQ VKFGS YLGYS VGAGHFRSQHTT EVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGS YFGASVCAV DLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLV GSD K Y A ARFGES I VNLGDIDNDGFED V AIG APQEDDLQG Al YI YNGR AD G1S STFS QRIEGLQIS KS LS MFGQS IS GQID ADNN G Y V D V A V G AFR SDS AVLLRTRPVVIVDASLSHPES VNRTKFDC VENGWPS VCIDLTLCF SYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQV SSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPP LQPILQQKKEKDIMKKTINFARFC AHENCS ADLQVS AKIGFLKPHEN KT YL A VGS M KTLMLN VS LFN AGDD A YETTLH V KLP VGL YFIKILELE EKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEED

[0441] LSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFV YGSNDENEPETCMVEKMNLTFH VINT GNS MAP N VS VEIM VPNS FS PQ Docket No.11624-006W01

[0442] TDKLFNILD VQTTTGECHFEN YQR VC ALEQQKS AM QTLKGI VRFLS K TDKRLL YCIK ADPHCLNFLCNFGKMES G KE AS VHIQLEGRPS ILEMD ETS ALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFT IVIISSSLLLGLIVLLLIS YVM WKAGFFKRQ YKSILQEENRRDS WS YIN SKSNDD*

[0443] SEQ ID NO.20: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0444] Reference genome hg38: chr2: 181,457,655-181,535,527. Full protein (amino acid) sequence (without signal peptide sequence): Natalizumab epitope (amino acid 146 upto including 259), Natalizumab resistant mutations Q152K, K201I, K256S shown.

[0445] YNVDTES ALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLA NASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQ WLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDL RTELS KRI APC YKD Y VKKFGENF AS C Q AGIS S F YTKDLI VM G APGS S Y WTGSLFVYNITTNIYKAFLDKQNQVKFGS YLGYSVGAGHFRSQHTTE V VGG AP QHEQIGKA YIFS IDES ELNILHEM KGKKLGS YFG AS VCAVD LN AD GFS DLL V G APM QS TIREEGR VF V YINS GS G A VMN AMETNL VG SDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRA DGIS S TFS QRIEGLQIS KS LS MFGQS IS GQID ADNNG Y VD V A VG AFRS DSAVLLRTRPVVIVDASLSHPES VNRTKFDC VENGWPSVCIDLTLCFS YKGKE VP G YI VLF YNM S LD VNRKAESPPRF YFS S NGTSD VITGSIQ VS SREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPL QPILQQKKEKDIMKKTINFARFC AHENCS ADLQVS AKIGFLKPHENK TYLA VGS MKTLMLN VS LFN AGDD A YETTLH VKLP VGL YFIKILELEE KQINCE VTDNS G V VQLDC S IG YI Y VDHLSRIDIS FLLD VS S LS R AEEDL SITVHATCENEEEMDNLKHSRVTVA1PLKYEVKLTVHGFVNPTSFVY GS NDENEPETCM VEKMNLTFH VINTGNSM APN VS VEIM VPNS FS P QT DKLFNILD VQTTTGECHFEN YQR VC ALEQQKS AM QTLKGI VRFLS KT DKRLL YCIK ADPHCLNFLCNFGKMES G KE AS VHIQLEGRPS ILEM DE TSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTI VIIS S SLLLGLI VLLLIS YVM WKAGFFKRQ YKSILQEENRRDS WSYINS KSNDD* Docket No.11624-006W01

[0446] SEQ ID NO.21: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0447] Reference genome hg38: chr2:181, 457, 655-181, 535, 527.

[0448] Natalizumab epitope (amino acid 146 up to including 259), Codon positions of mutations at Q152, K201, K256 not indicated (original nonmutant codon sequences shown).

[0449] AGAATAGCTCCGTGTTATCAAGATTATGTGAAAAAATTTGGAGAAAATTTT

[0450] GCATCATGTCAAGCTGGAATATCCAGTTTTTACACAAAGGATTTAATTGTG

[0451] ATGGGGGCCCCAGGATCATCTTACTGGACTGGCTCTCTTTTTGTCTACAAT

[0452] ATAACTACAAATAAATACAAGGCTTTTTTAGACAAACAAAATCAAGTAAA ATTTGGAAGTTATTTAGGATATTCAGTCGGAGCTGGTCATTTTCGGAGCCA GCATACTACCGAAGTAGTCGGAGGAGCTCCTCAACATGAGCAGATTGGTA

[0453] AGGCATATATATTCAGCATTGATGAAAAAGAACTAAAT

[0454] SEQ ID NO.22: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0455] Reference genome hg38: chr2:181, 457, 655-181, 535, 527.

[0456] Natalizumab epitope (amino acid 146 up to including 259), Mutation positions Q152, K201, K256 not indicated (original nonmutant AA sequences shown).

[0457] RIAPC YQDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSS YWTGSL FV YNITTNKYK AFLD KQNQ VKFGS YLG YS VG AGHFRS QHTTE V VGG APQHEQIGKAYIFSIDEKELN

[0458] SEQ ID NO.23: Locus of ITGA4, ENSG00000115232; ENST00000397033.7

[0459] Reference genome hg38: chr2:181, 457, 655-181, 535, 527.

[0460] Natalizumab epitope (amino acid 146 up to including 259), Natalizumab resistant mutations Q152K, K201I, K256S shown.

[0461] RIAPC YKDYVKKFGENFASCQAGISSFYTKDLIVMGAPGSS YWTGSL FV YN1TTN I Y K AFLD KQN Q V KFGS YLGYS V G AGHFRS QHTTE V VGGA

[0462] PQHEQIGKAYIFSIDESELN Docket No. 11624-006W01

[0463] SEQ ID NO: 24: ITGA4_L206-Y202_pegRNA_protospacer

[0464] TATAACTACAAATAAATACA

[0465] SEQ ID NO: 25: ITGA4_L206Y_pegRNA

[0466] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0467] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTGTTTGTCGT

[0468] AGAACGCTTTATATTTATTTGTAGTTAT

[0469] SEQ ID NO: 26: ITGA4_Y202S_pegRNA_vl

[0470] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0471] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAAAAGCCTTA

[0472] GATTTATTTGTAGTTAT

[0473] SEQ ID NO: 27: ITGA4_Y202S_pegRNA_v2

[0474] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0475] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAAAAGCCTTG

[0476] GATTTATTTGTAGTTAT

[0477] SEQ ID NO: 28: ITGA4_Y202S_pegRNA_v3

[0478] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0479] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAAAAGCCTTTG

[0480] ATTTATTTGTAGTTAT

[0481] SEQ ID NO: 29: ITGA4_Y202S_pegRNA_v4

[0482] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0483] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAAAAGCCTTC

[0484] GATTTATTTGTAGTTAT

[0485] SEQ ID NO: 30: ITGA4_A204Y_pegRNA_vl

[0486] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0487] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAATA

[0488] TTTATATTTATTTGTAGTTAT Docket No. 11624-006W01

[0489] SEQ ID NO: 31: ITGA4_A204Y_pegRNA_v2

[0490] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0491] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAGTA

[0492] TTTATATTTATTTGTAGTTAT

[0493] SEQ ID NO: 32: ITGA4_A204F_pegRNA_vl

[0494] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0495] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAAAA

[0496] TTTATATTTATTTGTAGTTAT

[0497] SEQ ID NO: 33: ITGA4_A204F_pegRNA_v2

[0498] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0499] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAGA

[0500] ATTTATATTTATTTGTAGTTAT

[0501] SEQ ID NO: 34: ITGA4_A204H_pegRNA_vl

[0502] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0503] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAATG

[0504] TTTATATTTATTTGTAGTTAT

[0505] SEQ ID NO: 35: ITGA4_A204H_pegRNA_v2

[0506] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0507] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAAGT

[0508] GTTTATATTTATTTGTAGTTAT

[0509] SEQ ID NO: 36: ITGA4 A204W pegRNA

[0510] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0511] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAACCA

[0512] TTTATATTTATTTGTAGTTAT

[0513] SEQ ID NO: 37: ITGA4_A204K_pegRNA_vl

[0514] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0515] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAACTT

[0516] TTTATATTTATTTGTAGTTAT Docket No. 11624-006W01

[0517] SEQ ID NO: 38: ITGA4_A204K_pegRNA_v2

[0518] TATAACTACAAATAAATACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

[0519] CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGTCTAAAAATTT

[0520] TTTATATTTATTTGTAGTTAT

[0521] SEQ ID NO: 39: ITGA4_K201_vl CTACAAATAAATACAAGGCT SEQ ID NO: 40: ITGA4_K201_v2 TACAAATAAATACAAGGCT SEQ ID NO: 41: ITGA4_K201_v3 ACAAATAAATACAAGGCTTT SEQ ID NO: 42: ITGA4_K201_v4 CAAATAAATACAAGGCTTTT SEQ ID NO: 43: ITGA4_K201_v5 AAATAAATACAAGGCTTTTT SEQ ID NO: 44: ITGA4_K201_v6 AATAAATACAAGGCTTTTTT SEQ ID NO: 45: ITGA4_K201_v7 ATAAATACAAGGCTTTTTTA SEQ ID NO: 46: ITGA4_K201_v8 TAAATACAAGGCTTTTTTAG

[0522] SEQ ID NO: 47: ITGA4_K256_vl TTGATGAAAAAGAACTAAAT SEQ ID NO: 48: ITGA4_K256_v2 TGATGAAAAAGAACTAAATA SEQ ID NO: 49: ITGA4_K256_v3 GATGAAAAAGAACTAAATAT SEQ ID NO: 50: ITGA4_K256_v4 ATGAAAAAGAACTAAATATC SEQ ID NO: 51: ITGA4_K256_v5 TGAAAAAGAACTAAATATCT SEQ ID NO: 52: ITGA4_K256_v6 GAAAAAGAACTAAATATCTT SEQ ID NO: 53: ITGA4_K256_v7 AAAAAGAACTAAATATCTTA SEQ ID NO: 54: ITGA4_K256_v8 AAAAGAACTAAATATCTTAC

[0523] SEQ ID NO: 55: ITGA4_L206_vl TGTTTGTCTAAAAAAGCCTT SEQ ID NO: 56: ITGA4 L206 v2 GTTTGTCTAAAAAAGCCTTG SEQ ID NO: 57: ITGA4_L206_v3 TTTGTCTAAAAAAGCCTTGT SEQ ID NO: 58: ITGA4_L206_v4 TTGTCTAAAAAAGCCTTGTA SEQ ID NO: 59: ITGA4_L206_v5 TGTCTAAAAAAGCCTTGTAT SEQ ID NO: 60: ITGA4_L206_v6 GTCTAAAAAAGCCTTGTATT SEQ ID NO: 61: ITGA4_L206_v7 TCTAAAAAAGCCTTGTATTT SEQ ID NO: 62: ITGA4_L206_v8 CTAAAAAAGCCTTGTATTTA

Claims

Docket No. 11624-006W01CLAIMSWhat is claimed is:

1. An engineered hematopoietic cell comprising a VLA-4 receptor variant, wherein the VLA-4 receptor variant comprises a mutation in an ITGA4 subunit, and wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding.

2. The engineered hematopoietic cell of claim 1 , wherein the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule-1 (VCAM-1) ligand.

3. The engineered hematopoietic cell of claim 1 or 2, wherein the mutation is located between amino acids R 146 to N259 of the ITGA4 subunit.

4. The engineered hematopoietic cell of any one of claims 1-3, wherein the mutation comprises an amino acid mutation.

5. The engineered hematopoietic cell of claim 4, wherein the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C,K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q,K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K,A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R,H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof.

6. The engineered hematopoietic cell of any one of claims 4-5, wherein the amino acid mutation is Q 152K.

7. The engineered hematopoietic cell of any one of claims 1-6, wherein the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation.Docket No. 11624-006W018. The engineered hematopoietic cell of claim 7, wherein the silent mutation comprises a Y151Y mutation, wherein the Y151Y mutation is encoded by a 453T>C mutation.

9. The engineered hematopoietic cell of any one of claims 1-8, wherein the VLA-4 receptor variant binds to VCAM-1 expressed on bone marrow stromal cells.

10. The engineered hematopoietic cell of any one of claims 1-9, wherein the engineered hematopoietic cell has an increased ability for selective retention in bone marrow in presence of natalizumab.

11. The engineered hematopoietic cell of any one of claims 1-10, wherein the hematopoietic cell comprises hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, or basophils.

12. A method of treating an inherited genetic disorder in a subject, comprising: a) isolating a hematopoietic cell from a blood sample from the subject; b) engineering the hematopoietic cell, comprising: introducing a mutation in an TTG A4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and c) administering to the subject the engineered hematopoietic cell.

13. The method of claim 12, further administering natalizumab to the subject.

14. The method of any one of claims 12-13, wherein the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand.

15. The method of any one of claims 12-14, wherein the mutation is located between amino acids R 146 to N259 of the ITGA4 subunit.Docket No. 11624-006W0116. The method of any one of claims 12-15, wherein the mutation comprises an amino acid mutation.

17. The method of claim 16, wherein the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combinatin thereof.

18. The method of any one of claims 16-17, wherein the amino acid mutation is Q152K.

19. The method of any one of claims 12-18, wherein the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation.

20. The method of claim 19, wherein the silent mutation comprises a Y151Y mutation, wherein the Y151 Y mutation is encoded by a 453T>C mutation.

21. The method of any one of claims 12-20, wherein the VLA-4 receptor variant binds to VCAM-1 expressed on bone marrow stromal cells.

22. The method of any one of claims 12-21, wherein the engineered hematopoietic cell has an increased ability for selective retention in bone marrow of the subject, in presence of natalizumab.

23. The method of any one of claims 13-22, wherein natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject.

24. The method of any one of claims 13-23, wherein natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the subject.Docket No. 11624-006W0125. The method of any one of claims 13-24, wherein natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

26. The method of any one of claims 12-25, wherein the inherited genetic disorder is selected from the group consisting of sickle cell anemia, thalassemia, a bone marrow failure disorder, an inherited leukocyte disorder, an inherited anemia, an inherited error of immunity disorder, a metabolic disorder, a skeletal disorder, an inherited thrombocytopenia, and an immunodeficiency.

27. The method of claim 26, wherein the bone marrow failure disorder is selected from the group consisting of Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, severe congenital neutropenia, Diamond-Blackfan anemia, GATA2 deficiency, SAMD9 / SAMD9L disorders, MECOM syndromes, and congenital amegakaryocytic thrombocytopenia.

28. The method of any one of claims 26-27, wherein the inherited leukocyte disorder is chronic granulomatous disease.

29. The method of any one of claims 26-28, wherein the inherited anemia is selected from the group consisting of a hemoglobinopathy, a membranopathy, an enzymopathy, and a congenital dyserythropoietic anemia.

30. The method of any one of claims 26-29, wherein the inherited error of immunity disorder is severe congenital immunodeficiency.

31. The method of any one of claims 26-30, wherein the metabolic disorder is selected from a group consisting of a leukodystrophy and a lysosomal storage disorder.

32. The method of any one of claims 26-31 , wherein skeletal disorder is infantile osteopetrosis.

33. The method of any one of claims 12-32, wherein the inherited genetic disorder is sickle cell anemia.

34. The method of any one of claims 12-33, wherein the genetic mutation causing the inherited genetic disorder is an E6V mutation in a beta-globin gene (HBB).Docket No. 11624-006W0135. The method of any one of claims 12-34, wherein the gene editing to produce a VLA-4 receptor variant comprises using a pegRNA, wherein the pegRNA comprises SEQ ID NO: 1-11, 12-14, 24-38, or 39-62.

36. The method of any one of claims 12-35, wherein the hematopoietic cell comprises hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, or basophils.

37. A method of treating an acquired disorder in a subject, comprising: a) isolating a hematopoietic cell from a blood sample from the subject; b) engineering the hematopoietic cell, comprising: introducing a mutation in an ITGA4 subunit of a VLA-4 receptor using ex vivo gene editing to produce a VLA-4 receptor variant, wherein the mutation in the ITGA4 subunit confers resistance to natalizumab binding; and c) administering to the subject the engineered hematopoietic cell.

38. The method of claim 37, further administering natalizumab to the subject.

39. The method of any one of claims 37-38, wherein the VLA-4 receptor variant binds to a Vascular Cell Adhesion Molecule- 1 (VCAM-1) ligand.

40. The method of any one of claims 37-39, wherein the mutation is located between amino acids R 146 to N259 of the ITGA4 subunit.

41. The method of any one of claims 37-40, wherein the mutation comprises an amino acid mutation.

42. The method of claim 41, wherein the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A,Docket No. 11624-006W01K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof.

43. The method of any one of claims 41-42, wherein the amino acid mutation is Q152K.

44. The method of any one of claims 37-43, wherein the VLA-4 receptor variant further comprises one or more additional mutations, wherein the one or more additional mutations is a silent mutation.

45. The method of claim 44, wherein the silent mutation comprises a Y 151 Y mutation, wherein the Y151 Y mutation is encoded by a 453T>C mutation.

46. The method of any one of claims 37-45, wherein the VLA-4 receptor variant binds to VCAM-1 expressed on bone marrow stromal cells.

47. The method of any one of claims 38-46, wherein the engineered hematopoietic cell has an increased ability for selective retention in bone marrow of the subject, in presence of natalizumab.

48. The method of any one of claims 38-47, wherein natalizumab is administered to the subject before administration of the engineered hematopoietic cell to the subject.

49. The method of any one of claims 38-48, wherein natalizumab is administered to the subject during administration of the engineered hematopoietic cell to the subject.

50. The method of any one of claims 38-49, wherein natalizumab is administered to the subject after administration of the engineered hematopoietic cell to the subject.

51. The method of any one of claims 37-50, wherein the acquired disorder is selected from the group consisting of HIV / AIDS, a malignancy, and an autoimmune disease.

52. The method of any one of claims 37-51, wherein the gene editing to produce a VLA-4 receptor variant comprises using a pegRNA, wherein the pegRNA comprises SEQ ID NO: 1-11, 12-14, 24-38, or 39-62.Docket No. 11624-006W0153. The method of any one of claims 37-52, wherein the hematopoietic cell comprises hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, or basophils.

54. A method of treating an inherited or acquired genetic disorder in a subject in vivo, comprising: a) engineering a hematopoietic cell in vivo, wherein said engineering comprises: i) delivering a gene modification vector to produce a natalizumab resistance variant, wherein said variant comprises a mutation; ii) correcting a genetic mutation causing the inherited genetic disorder to obtain an engineered hematopoietic cell; and b) administering to the subject natalizumab to select for gene modified hematopoietic cells.

55. The method of claim 54, wherein the mutation comprises an amino acid mutation.

56. The method of claim 55, wherein the amino acid mutation is selected from Q72E, L73I, N77S, G82C, T84I, C85E, Q152C, Q152H, Q152I, Q152K, Q152N, Q152P, Q152R, Q152V, Q152Y, V155D, K157R, F162W, M180S, K201A, K201C, K201D, K201E, K201F, K201G, K201H, K201I, K201L, K201M, K201N, K201Q, K201R, K201S, K201T, K201V, K201Y, Y202S, Y202T, A204F, A204H, A204K, A204W, A204Y, L206P, L206S, L206V, L206W, L206Y, H226D, H226K, H226R, H226Y, E234V, H242C, I250K, K256A, K256C, K256D, K256E, K256F, K256G, K256I, K256L, K256N, K256Q, K256R, K256S, K256V, K256W, K256Y, E257C, E257D, E257F, E257K, E257W, E257Y, L258F, L258P, L258W, or I260M, or a combination thereof.

57. The method of any one of claims 54-56, wherein the hematopoietic cell comprises hematopoietic stem cells, multipotent progenitor cells, lymphocytes (including B cells, T cells, and natural killer cells), monocytes, macrophages, dendritic cells, eosinophils, or basophils.

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