Treating sickle cell disease

Abstract

A method for mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD) is provided. The method comprises administering a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab to the subject, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially. Also provided is the use of immobilized cells in gene therapy for subsequent treatment of SCD.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 431,705, filed December 11, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing An XML file entitled 97965 Sequence Listing.xml, created on December 11, 2023, and consisting of 217,088 bytes, submitted concurrently with the filing of this application, is hereby incorporated by reference.

[0003] FIELD OF THE INVENTION The present invention, in some embodiments thereof, relates to the treatment of sickle cell disease. [Background technology]

[0004] Sickle cell disease (SCD) is one of the most prevalent genetic disorders worldwide. Clinical manifestations of SCD commonly include anemia, pain, vaso-occlusive events, and cumulative end-organ damage, which lead to significant morbidity and mortality. Historically, allogeneic hematopoietic cell transplantation (HCT) was the only curative therapy for SCD, but its use has been limited by the significant morbidity and mortality associated with HCT (PMID: 8663884, PMID: 20007560).

[0005] Autologous hematopoietic stem cell (HSC)-based gene therapy now offers the potential for cure with reduced toxicity (PMID: 28249145). However, effective HSC-based gene therapy requires the collection of sufficient HSCs (ideally approximately 10–15 × 10 ), typically from peripheral blood (PB). 6The mobilization approach relies on collecting 1000 CD34+ cells / kg (PMID: 33956057). G-CSF and CXCR4 inhibition with plerixafor (CXCR4i) are the most widely used mobilization methods (PMID: 19363221, PMID: 19720922). However, G-CSF has been associated with fatal vaso-occlusive events in SCD (PMID: 9734950, PMID: 11368061, PMID: 19513902). Meanwhile, short-acting CXCR4i with plerixafor alone does not reliably generate optimal HSC numbers for gene therapy applications (PMID: 30282642, PMID: 29419425, PMID: 29472357). Therefore, the development of novel HSC mobilization regimens to rapidly and reliably mobilize CD34+ HSCs optimal for gene therapy of SCD represents an unmet need.

[0006] Further background art includes: International Publication No. 2018085574 Peled et al. Clin Cancer Res. 2014 Jan 15;20(2):469-79. International Publication No. 2020 / 148745 Doerfler J Clin Invest. 2021 Apr 15; 131(8): e146394. Summary of the Invention

[0007] According to one aspect of some embodiments of the present invention, there is provided a method for mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0008] According to one aspect of some embodiments of the present invention, there is provided a pharmaceutical combination for use in the treatment of sickle cell disease (SCD), comprising a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0009] According to some embodiments of the invention, natalizumab is administered intravenously (iv).

[0010] According to some embodiments of the invention, the peptide is formulated as motixafortide.

[0011] According to some embodiments of the invention, the peptide is administered subcutaneously (SC).

[0012] According to some embodiments of the invention, the peptide is administered at a dose of 0.1 to 10 mg / kg body weight.

[0013] According to some embodiments of the invention, the peptide is administered at a dose of 1-2 mg / kg body weight.

[0014] According to some embodiments of the invention, the peptide is administered at a dose of 1.25 mg / kg body weight.

[0015] According to some embodiments of the invention, natalizumab is administered intravenously (iv).

[0016] According to some embodiments of the invention, natalizumab is administered in a dose of 100-1000 mg.

[0017] According to some embodiments of the invention, natalizumab is administered in a dose of 300 mg.

[0018] According to some embodiments of the invention, natalizumab is administered before the peptide.

[0019] According to some embodiments of the invention, the peptide is administered no more than 24 hours after administration of natalizumab.

[0020] According to some embodiments of the invention, the peptide is administered 24 hours after administration of natalizumab.

[0021] According to some embodiments of the invention, the method includes performing apheresis to collect CD34+ cells from the peripheral blood of the subject after administration of the agent.

[0022] According to an aspect of some embodiments of the present invention there is provided a mobilized cell population obtainable according to the methods described herein.

[0023] According to an aspect of some embodiments of the present invention there is provided a method of treating sickle cell disease (SCD), comprising: (a) mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD), as described herein; (b) performing apheresis to collect CD34+ cells from the peripheral blood of the subject; and (c) processing CD34+ cells to obtain red blood cells with a normal phenotype; A method is provided that includes:

[0024] According to some embodiments of the invention, the treatment is by genome editing.

[0025] According to some embodiments of the invention, the normal phenotype comprises expression of fetal hemoglobin (HbF, hemoglobin F).

[0026] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below.In case of conflict, the present patent specification, including definitions, will prevail.In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0027] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now made in detail to the drawings, it is emphasized that the details shown are by way of example and for purposes of illustratively discussing embodiments of the invention. In this regard, it will become apparent to those skilled in the art how embodiments of the invention may be practiced, upon reading the description in conjunction with the drawings.

[0028] The drawings are as follows: [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 is a schematic diagram of the study design. The study compares treatment with motixafortide (M) with treatment with M+N for the experimental group. [Figure 2]Figure 2 is a schematic diagram illustrating the lineages that arise from CD34+ cells. HSCs (hematopoietic stem cells), which are pluripotent cells, can self-renew and differentiate into all tissue lineages. MPP: Multipotent progenitor. A cell population that has lost the self-renewal ability of HSCs. LMPP (lymphoid-primed multipotent progenitor) cells contain almost exclusively lymphoid progenitors, with very low myeloid production (3%). CMP (common myeloid progenitor) cells are oligopotent and can differentiate into some tissues / organs, but not all. MEP: Megakaryocyte / erythroid progenitor cells, which are lineage-restricted progenitors that develop from CMPs. GMP: Granulocyte / macrophage progenitor cells, which are lineage-restricted progenitors that develop from CMPs and LMPPs. CLP: Common lymphoid progenitor. These cells are oligopotent and can differentiate into some tissues / organs, but not all. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention, in some embodiments thereof, relates to the treatment of sickle cell disease (SCD).

[0031] Developing novel HSC mobilization regimens to rapidly and reliably mobilize CD34+ HSCs optimal for gene therapy of SCD represents an unmet need.

[0032] In devising embodiments of the present invention, the inventors hypothesized that combining VLA4 inhibition with natalizumab with CXCR4 inhibition with motixafortide would synergistically increase HSC mobilization.

[0033] Thus, according to one aspect of the present invention, there is provided a method for mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0034] According to one aspect of the present invention, there is provided a pharmaceutical combination for use in the treatment of sickle cell disease (SCD), comprising a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0035] As used herein, "sickle cell disease," or SCD for short, refers to any form of this inherited red blood cell disorder. Red blood cells contain hemoglobin, a protein that carries oxygen. Healthy red blood cells are round and move through small blood vessels to deliver oxygen to all parts of the body. Individuals with SCD have abnormalities in hemoglobin that cause red blood cells to become stiff and sticky, resembling a C-shaped farm tool known as a "sickle." Specific forms of the disease include HbSS, HBSc, HbS and HbSD, HbAS, HbSE, and HbSO, each of which is contemplated herein and considered an independent embodiment. According to specific embodiments of the present invention, the disease beta-thalassemia is included in and considered under this term (SCD).

[0036] According to a specific embodiment, the subject has been diagnosed with SCD.

[0037] As used herein, CD34+ cells refer to human hematopoietic stem cells (HSCs) that can develop into red blood cells and can be subject to genetic manipulation and genetic research (e.g., immunophenotyping, transcriptional assays, e.g., scRNA sequencing and mFACS, see Figure 2). CD34+ cells can be assayed using fluorescence-activated cell sorting (FACS), and thus the presence of CD34+ cells can be evaluated in a sample using this technique. Generally, CD34+ cells are present at low levels in the blood, but are present in large amounts in the bone marrow.

[0038] According to a specific embodiment, the CXCR4 inhibitory peptide of the present invention is 4F-benzoyl-TN14003 (SEQ ID NO: 1), although analogs and derivatives are also contemplated, which are structurally and functionally related to the peptides (also known as "T-140 analogs") disclosed in patent applications WO 2002 / 020561 and WO 2004 / 020462, detailed below.

[0039] In various specific embodiments, the T-140 analog or derivative has the amino acid sequence set forth in formula (I) below, or a salt thereof:

[0040] [ka] During the ceremony, A1 is an arginine residue, a lysine residue, an ornithine residue, a citrulline residue, an alanine residue, or a glutamic acid residue, or an N-alpha substituted derivative of these amino acids, or A1 is absent; A2, when A1 is present, represents an arginine or glutamic acid residue, or A2, when A1 is absent, represents an arginine or glutamic acid residue, or an N-alpha substituted derivative of these amino acids; A3 represents an aromatic amino acid residue, A4, A5, and A9 each independently represent an arginine residue, a lysine residue, an ornithine residue, a citrulline residue, an alanine residue, or a glutamic acid residue; A6 represents a proline residue, a glycine residue, an ornithine residue, a lysine residue, an alanine residue, a citrulline residue, an arginine residue, or a glutamic acid residue; A7 represents a proline residue, a glycine residue, an ornithine residue, a lysine residue, an alanine residue, a citrulline residue, or an arginine residue; A8 represents a tyrosine residue, a phenylalanine residue, an alanine residue, a naphthylalanine residue, a citrulline residue, or a glutamic acid residue; A 10represents a citrulline residue, a glutamic acid residue, an arginine residue, or a lysine residue; A 11 represents an arginine residue, a glutamic acid residue, a lysine residue, or a citrulline residue, the C-terminal carboxyl of which may be derivatized; and The cysteine ​​residues at positions 4 or 13 can form disulfide bonds, and the amino acid can be in either the L- or D-form.

[0041] Exemplary peptides according to formula (I) are peptides having the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 72 shown in Table 1 below.

[0042] [Table 1] TIFF2025538795000003.tif253168

[0043] According to a specific embodiment, in each of SEQ ID NOs: 1-72, the two cysteine ​​residues are linked by a disulfide bond.

[0044] In another embodiment, the peptide used in the compositions and methods of the invention consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the peptide used in the compositions and methods of the invention comprises the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the peptide is at least 60%, at least 70%, or at least 80% homologous to SEQ ID NO: 1. In another embodiment, the peptide is at least 90% homologous to SEQ ID NO: 1. In another embodiment, the peptide is at least about 95% homologous to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention.

[0045] In various other embodiments, the peptide is selected from SEQ ID NOs: 1-72, with each possibility representing a separate embodiment of the present invention.

[0046] According to a specific embodiment, the peptide is named motixafortide and has the amino acid sequence set forth in SEQ ID NO:1.

[0047] As mentioned above, the VLA-4 inhibitor (VLA4i) drug is natalizumab (abbreviated herein as (N)).

[0048] According to some embodiments, natalizumab is sold under the trade name Tysabri®.

[0049] According to some embodiments, natalizumab is sold under the trade name Antegren®.

[0050] The peptides and VLA4i ("drugs") described herein above can be administered to a subject either by themselves or in a pharmaceutical composition mixed with a suitable carrier or excipient. Each drug can be formulated into a separate formulation.

[0051] As used herein, "pharmaceutical composition" refers to a preparation of one or more active ingredients, such as those described herein, with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0052] As used herein, the term "active ingredient" refers to the agent responsible for the biological effect, eg, SEQ ID NO: 1 or VLA4i.

[0053] Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound. These terms include adjuvants.

[0054] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0055] Techniques for formulation and administration of drugs may be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0056] Suitable routes of administration can include, for example, oral, rectal, transmucosal, especially nasal, intestinal, or parenteral delivery (including intramuscular, intradermal, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intracardiac (e.g., into the right or left ventricular cavity, into the common coronary artery), intravenous, intraperitoneal, intranasal, or intraocular injections).

[0057] According to a specific embodiment, the peptide of the present invention or a pharmaceutical composition comprising same is administered subcutaneously (SC).

[0058] According to a specific embodiment, the VLA4i of the present invention or a pharmaceutical composition comprising same is administered intravenously (IV).

[0059] Pharmaceutical compositions of some embodiments of the present invention may be manufactured by processes well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0060] Thus, pharmaceutical compositions for use in accordance with some embodiments of the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, that facilitate processing of the active ingredient into a pharmaceutically usable preparation. The appropriate formulation depends on the selected route of administration.

[0061] Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions containing an active ingredient in an amount effective to achieve the intended purpose. More specifically, according to specific embodiments, a therapeutically effective amount means an amount effective to mobilize, on the one hand, and an amount of active ingredient effective to prevent, reduce, or alleviate the symptoms of a disorder, such as SCD, on the other hand.

[0062] According to a specific embodiment, the peptide of the present invention or a pharmaceutical composition comprising the same is administered at a dose ranging from 0.1 to 10 mg / kg body weight, 0.1 to 2 mg / kg body weight, 0.1 to 1 mg / kg body weight, 0.3 to 10 mg / kg body weight, or 0.3 to 2 mg / kg body weight.

[0063] According to a specific embodiment, BL-8040 is administered at a dose of 1-2 mg / kg body weight.

[0064] According to a specific embodiment, BL-8040 is administered at a dose of 1.25 to 1.5 mg / kg body weight.

[0065] According to a specific embodiment, BL-8040 is administered at a dose of 1.25 mg / kg body weight.

[0066] According to a specific embodiment, BL-8040 is administered subcutaneously (SC).

[0067] According to a specific embodiment, natalizumab is administered before the peptide.

[0068] According to a specific embodiment, the peptide is administered no more than 24 hours after administration of natalizumab.

[0069] According to a specific embodiment, the peptide is administered about 24 hours after administration of natalizumab.

[0070] According to a specific embodiment, the method includes performing apheresis to collect CD34+ cells from the peripheral blood of the subject after administration of the agent.

[0071] Approximately 4 hours to up to approximately 12 hours after administration of BL8040, patients undergo apheresis.

[0072] Methods for collecting peripheral blood are well known in the art and include, but are not limited to, drawing up to 500 ml of whole blood from a subject and collecting it in a container containing an anticoagulant (e.g., heparin or citrate), and apheresis.

[0073] As used herein, the term "apheresis" refers to a procedure in which an individual's peripheral blood is passed through a device to obtain major components (e.g., HSCs) and return other components to the subject's circulation. Apheresis is generally a three-step process that involves (1) withdrawing blood from a subject, (2) separating blood components (e.g., based on density), and (3) returning certain components of the blood to the subject via transfusion. Blood is typically separated into three fractions: red blood cells (approximately 45% of total blood), "buffy coat" (less than 1% of total blood), and plasma (approximately 55% of total blood). Various types of apheresis procedures can be used depending on the blood components being removed.

[0074] As previously described herein, the collected fractions are subjected to various research or diagnostic protocols, such as transcription and immunophenotyping.

[0075] The collected fraction can also be subjected to a treatment capable of conferring a normal phenotype to erythrocytes maturing from the red blood cells.

[0076] Thus, according to one aspect of the present invention, there is provided a method of treating sickle cell disease (SCD), comprising: (a) mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD) according to any one of claims 1 to 16; (b) performing apheresis to collect CD34+ cells from the peripheral blood of the subject; and (c) processing CD34+ cells to obtain red blood cells with a normal phenotype; A method is provided that includes:

[0077] Thus, the cells used are autologous to the subject.

[0078] Below is a non-limiting summary of genetic approaches used to manipulate collected cells (post-apheresis).

[0079] The cells may be subjected to genetic manipulation or may be initially cultured.

[0080] For example, lentiviral transduction of CD34+ HSCs typically requires ex vivo culture with cytokine stimulation. Typically, prestimulation with serum-free culture medium containing cytokines (stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L), and thrombopoietin (TPO) at 100 ng / mL each) for 1 day, followed by lentiviral transduction for 1 day, allows robust engraftment and efficient EGFP transfection in CD34+ cells in xenografted mice. Lower concentrations of SCF enhance CD34+ cell engraftment. Serum albumin can also be replaced with polyvinyl alcohol. Long-term culture on fibronectin-coated plates allows CD34+ cell engraftment. High-density culture with supplements, such as dimethyl-prostaglandin E2 (PGE2) and amphiphilic drug-delivery poloxamers, can improve in vitro transduction efficiency of human CD34+ cells by approximately 10-fold. Overall, cytokine stimulation is required for lentiviral transduction of ex vivo CD34+ cell cultures. However, minimal stimulation and short-term culture can also be used to maintain a balance between efficient gene transfer and robust CD34+ cell engraftment.

[0081] Wild-type β-globin, β T87QTherapeutic genes, such as β-globin (including anti-sickling mutations) or γ-globin, are typically inserted into self-inactivating (SIN) lentiviruses. Furthermore, a β-globin promoter and locus control region (LCR) are inserted to control transgene expression. Because the second intron of the β-globin gene is critical for its expression, conventional vectors carry the β-globin cassette in the reverse orientation relative to the vector genome to prevent transcriptional excision. Because this inverted construct reduces vector titer and transduction efficiency in CD34+ cells, a forward β-globin vector was recently developed. This forward β-globin vector addresses these issues and results in robust β-globin expression in erythroid cells.

[0082] The following are some clinical trials for SCD, which are embodiments of the present invention: J Clin Invest. 2021 Apr 15; 131(8): e146394 Table 2).

[0083] [Table 2]

[0084] The engineered cells can then be transfused back into the subject.

[0085] Each of these is contemplated herein. It is anticipated that many related CD34+ cell manipulations will be developed during the life of the patent resulting from this application, and it is intended that all such new technologies be included a priori within the scope of the term "manipulating CD34+."

[0086] As used herein, the term "about" refers to ±10%.

[0087] The terms "comprises," "comprising," "includes," "including," "having," and their conjugations mean "including but not limited to."

[0088] The term "consisting of" means "including and limited to."

[0089] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or moieties, but only if the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0090] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0091] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as each individual numerical value within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed each individual numerical value within that range, e.g., 1, 2, 3, 4, 5, and 6, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0092] Whenever a range of values ​​is given herein, the range is meant to include any recited number (fractional or integer) within the range given. The phrases "range between" a first recited number and a second recited number and "range from" a first recited number to a second recited number are used interchangeably herein and are meant to include the first recited number and the second recited number, and all fractional and integer numbers therebetween.

[0093] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, any manners, means, techniques, and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or readily developed by such practitioners from known manners, means, techniques, and procedures.

[0094] As used herein, the term "treating" includes inhibiting, substantially inhibiting, slowing, or reversing the progression of a condition, substantially alleviating clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0095] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately, or in any suitable subcombination, or as suitable in any otherwise described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0096] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0097] Reference is now made to the following examples, which together with the above descriptions, illustrate some embodiments of the invention in a non-limiting fashion.

[0098] The procedure is outlined in Figure 1.

[0099] design Single-arm, single-center, open-label pilot study.

[0100] Study population N are five patients aged 18–40 years diagnosed with SCD (hemoglobin SS or Sβ°) undergoing automated RBC exchange via apheresis-capable venous access.

[0101] Main eligibility criteria Able and willing to provide consent, meet the study population criteria above, have ECOG ≤ 1, and have adequate bone marrow / organ function.

[0102] Primary Objective / Evaluation Item Safety and tolerability of (M) and (N)+(M) in patients with SCD, as defined by pre-specified dose-limiting toxicities.

[0103] Secondary Objectives / Endpoints Number of CD34+ cells mobilized via leukapheresis (CD34+ cells / kg / L) relative to total volume treated (tv) (1 blood volume (approximately 4-5 L treatment)).

[0104] Number of CD34+ cells mobilized via leukapheresis (CD34+ cells / kg / L) relative to the adjusted volume (aV) treated (1 blood volume (approximately 4-5 L treatment)).

[0105] Kinetics of peripheral blood mobilization of CD34+ HSPCs (cells per microliter of peripheral blood).

[0106] Incidence of adverse events.

[0107] Exploratory Objectives / Endpoints Immunophenotypic and transcriptional profiling of peripheral blood CD34+ HSCs mobilized in the study.

[0108] Gene editing efficiency and HSC fitness profiling of peripheral blood CD34+ HSCs mobilized in the study.

[0109] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0110] It is the intention of the applicant(s) that all publications, patents, and patent applications mentioned herein be incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein at the time of reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent section headings are used, they should not be construed as necessarily limiting. Additionally, any priority documents of this application are incorporated herein by reference in their entirety.

Claims

1. A method for mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

2. A pharmaceutical combination for use in the treatment of sickle cell disease (SCD), comprising a therapeutically effective amount of a CXCR4 inhibitory peptide set forth in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein the natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

3. 3. The method or combination for use of claim 1 or 2, wherein the natalizumab is administered intravenously (i.v.).

4. 3. The method or combination for use of claim 1 or 2, wherein the peptide is formulated as motixafortide.

5. 3. The method or combination for use of claim 1 or 2, wherein the peptide is administered subcutaneously (S.C.).

6. 6. The method or combination for use according to claim 5, wherein the peptide is administered at a dose of 0.1 to 10 mg / kg body weight.

7. 6. The method or combination for use according to claim 5, wherein the peptide is administered at a dose of 1 to 2 mg / kg body weight.

8. 6. The method or combination for use according to claim 5, wherein the peptide is administered at a dose of 1.25 mg / kg body weight.

9. The method or combination for use of any one of claims 1 to 6, wherein the natalizumab is administered intravenously (iv).

10. 10. The method or combination for use of claim 9, wherein said natalizumab is administered at a dose of 100-1000 mg.

11. 10. The method or combination for use of claim 9, wherein the natalizumab is administered at a dose of 300 mg.

12. The method or combination for use of any one of claims 1 to 11, wherein said natalizumab is administered before said peptide.

13. The method or combination for use of any one of claims 1 to 11, wherein the peptide is administered no more than 24 hours after administration of the natalizumab.

14. The method or combination for use of any one of claims 1 to 11, wherein the peptide is administered 24 hours after the administration of natalizumab.

15. 15. The method or combination of any one of claims 1 to 14, comprising performing apheresis to recover the CD34+ cells from the peripheral blood of the subject after administration of the agent.

16. 1. A method for treating sickle cell disease (SCD), comprising: (a) mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD) according to any one of claims 1 to 15; (b) performing apheresis to recover the CD34+ cells from the peripheral blood of the subject; and (c) treating the CD34+ cells to obtain red blood cells with a normal phenotype; A method comprising:

17. 17. The method of claim 16, wherein the treatment is by genome editing.

18. The method of any one of claims 16 to 17, wherein the normal phenotype comprises expression of fetal hemoglobin (HbF, hemoglobin F).

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