Treatment of sickle cell disease

Abstract

A method of mobilizing CD34 + cells in a subject suffering from sickle cell disease (SCD) is provided. The method comprises the following steps: a therapeutically effective amount of CXCR4 inhibitory peptide shown as SEQ ID NO: 1 and a therapeutically effective amount of natazumab are applied to a subject, and the natazumab and the CXCR4 inhibitory peptide are sequentially applied. Also provided is the use of mobilized cells for subsequent treatment of SCD in gene therapy.

Description

[0001] Related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 431,705, filed on December 11, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing Statement

[0004] The XML file named 97965SequenceListing.xml, consisting of 217088 bytes, created on December 11, 2023, and submitted simultaneously with this application, is incorporated herein by reference. Technical Field

[0005] The present invention, in some embodiments thereof, relates to the treatment of sickle cell disease. Background Art

[0006] Sickle cell disease (SCD) is one of the most common genetic disorders worldwide. Clinical manifestations of SCD typically include anemia, pain, vaso-occlusive events, and cumulative end-organ damage, leading to significant morbidity and mortality. Historically, allogeneic hematopoietic cell transplantation (HCT) has been the only curative therapy for SCD, but the high morbidity and mortality associated with HCT have limited its use (PMID: 8663884, PMID: 20007560).

[0007] Autologous hematopoietic stem cell (HSC)-based gene therapy now has therapeutic potential with reduced toxicity (PMID: 28249145). However, effective HSC-based gene therapy relies on the collection of sufficient HSCs (ideally approximately 10 × 10 per kg body weight). 6 to 15×10 6 CD34+ cells), typically collected from peripheral blood (PB) (PMID: 33956057). G-CSF and plerixafor inhibiting CXCR4 (CXCR4i) are the most widely used mobilization strategies (PMID: 19363221, PMID: 19720922). However, G-CSF has been associated with fatal vaso-occlusive events in SCD (PMID: 9734950, PMID: 11368061, PMID: 19513902). At the same time, short-acting CXCR4i and plerixafor alone cannot reliably produce optimal HSC numbers for gene therapy applications (PMID: 30282642, PMID: 29419425, PMID: 29472357). Therefore, the development of new HSC mobilization protocols to rapidly and reliably mobilize optimal CD34+ HSCs for SCD gene therapy represents an unmet need.

[0008] Additional background technology includes:

[0009] WO2018085574

[0010] Peled et al. Clin Cancer Res. 2014 Jan 15; 20(2): 469-79.

[0011] WO2020 / 148745

[0012] Doerfler J Clin Invest. 2021Apr 15;131(8):e146394. Summary of the Invention

[0013] According to one aspect of some embodiments of the present invention, there is provided a method for mobilizing CD34+ cells in a subject with sickle cell disease (SCD), the method comprising administering to the subject a therapeutically effective amount of a CXCR4 inhibitory peptide as shown in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0014] According to an aspect of some embodiments of the present invention, there is provided a combination of agents for treating sickle cell disease (SCD), the combination comprising a therapeutically effective amount of a CXCR4 inhibitory peptide as shown in SEQ ID NO:1 and a therapeutically effective amount of natalizumab, wherein natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

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

[0016] According to some embodiments of the invention, the peptide is formulated as Motixafortide.

[0017] According to some embodiments of the invention, the peptide is administered subcutaneously (SC).

[0018] According to some embodiments of the invention, the peptide is administered at a dose of 0.1 mg to 10 mg per kg body weight.

[0019] According to some embodiments of the invention, the peptide is administered at a dose of 1 mg to 2 mg per kg body weight.

[0020] According to some embodiments of the invention, the peptide is administered at a dose of 1.25 mg per kg body weight.

[0021] According to some embodiments of the invention, natalizumab is administered intravenously (iv).

[0022] According to some embodiments of the invention, natalizumab is administered at a dose of 100 mg to 1000 mg.

[0023] According to some embodiments of the invention, natalizumab is administered at a dose of 300 mg.

[0024] According to some embodiments of the invention, natalizumab is administered prior to administration of the peptide.

[0025] According to some embodiments of the invention, the peptide is administered at a time point no more than 24 hours after administration of natalizumab.

[0026] According to some embodiments of the invention, the peptide is administered 24 hours after administration of natalizumab.

[0027] According to some embodiments of the invention, the method comprises: performing apheresis to obtain CD34+ cells from the subject's peripheral blood after administering the agent.

[0028] According to an aspect of some embodiments of the present invention there is provided a population of mobilized cells obtainable according to the method described herein.

[0029] According to an aspect of some embodiments of the present invention there is provided a method of treating sickle cell disease (SCD), the method comprising:

[0030] (a) mobilizing CD34+ cells in a subject suffering from sickle cell disease (SCD) as described herein;

[0031] (b) performing phlebotomy to obtain CD34+ cells from the subject's peripheral blood; and

[0032] (c) CD34+ cells were processed to obtain red blood cells with a healthy phenotype.

[0033] According to some embodiments of the invention, the manipulation is performed by genome editing.

[0034] According to some embodiments of the invention, the healthy phenotype comprises expression of fetal hemoglobin (HbF; hemoglobin F).

[0035] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used when practicing or testing embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification including definitions shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to necessarily limit. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Some embodiments of the present invention are described herein with reference to the accompanying drawings, by way of example only. With reference now to the drawings in detail, it should be emphasized that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description taken in conjunction with the drawings makes it apparent to those skilled in the art how to practice the embodiments of the present invention.

[0037] In the attached figure:

[0038] Figure 1 Figure 1 is a schematic diagram of the study design. This study compared the therapeutic effects of motexafortide (M) with those of M+N in the study group.

[0039] Figure 2 This is a diagram depicting the lineages arising from CD34+ cells. HSC (Hematopoietic Stem Cell – a multipotent cell – can differentiate into all tissue lineages and has the ability to self-renew. MPP, a multipotent progenitor, is a cell population that has lost the self-renewal ability of HSC. LMPP, a lymphoid-biased multipotent progenitor, is actually a lymphoid progenitor with only a very low myeloid output (3%). CMP (Common Myeloid Progenitor), an oligopotent cell, can differentiate into multiple tissues / organs, but not all. MEP: Megakaryocyte / erythroid progenitor, a lineage-restricted progenitor that evolves from CMP. GMP: Granulocyte / macrophage progenitor, a lineage-restricted progenitor that evolves from CMP and LMPP. CLP: Common Lymphoid Progenitor, an oligopotent cell – can differentiate into multiple tissues / organs, but not all. DETAILED DESCRIPTION

[0040] The present invention, in some embodiments thereof, relates to the treatment of sickle cell disease (SCD).

[0041] The development of new HSC mobilization protocols to rapidly and reliably mobilize optimal CD34+ HSCs for SCD gene therapy represents an unmet need.

[0042] In conceiving embodiments of the present invention, the inventors hypothesized that combining inhibition of VLA4 by natalizumab with inhibition of CXCR4 by motexafortide would synergistically increase HSC mobilization.

[0043] Therefore, 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), the method comprising: administering to the subject a therapeutically effective amount of a CXCR4 inhibitory peptide as shown in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0044] According to one aspect of the present invention, a combination of agents is provided for treating sickle cell disease (SCD), the combination comprising a therapeutically effective amount of a CXCR4 inhibitory peptide as shown in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

[0045] As used herein, "sickle cell disease," abbreviated as SCD, refers to any form of an inherited red blood cell disorder. Red blood cells contain hemoglobin, a protein that carries oxygen. Healthy red blood cells are round, and they travel through small blood vessels to deliver oxygen to various parts of the body. In people with SCD, the hemoglobin is abnormal, which causes the red blood cells to become hard and sticky and look like a C-shaped farm implement 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 a separate embodiment. According to one specific embodiment of the present invention, beta thalassemia is also considered to be included within the term (SCD).

[0046] According to a specific embodiment, the subject is diagnosed with SCD.

[0047] As used herein, CD34+ cells refer to human hematopoietic stem cells (HSCs), which can develop into erythrocytes and serve as subjects for genetic manipulation and research (e.g., immunophenotyping, transcriptional assays such as scRNA seq and mFACS, see Figure 2 CD34+ cells can be detected using fluorescence activated cell sorting (FACS), so their presence in a sample can be assessed using this technique. Generally, CD34+ cells are present at low levels in the blood but are present in high numbers in the bone marrow.

[0048] According to a specific embodiment, the CXCR4 inhibitory peptide of the present invention is 4F-benzoyl-TN14003 (SEQ ID NO: 1), however, analogs and derivatives are also contemplated. These are structurally and functionally related to the peptides disclosed in patent applications WO 2002 / 020561 and WO 2004 / 020462, also referred to as "T-140 analogs," as described in detail below.

[0049] In various specific embodiments, the T-140 analog or derivative has the amino acid sequence represented by the following formula (I) or a salt thereof: 1 2 3 4 5 6 7 8 9 10 11 12 13 14

[0051] A1-A2-A3-Cys-Tyr-A4-A5-A6-A7-A8-A9-A10 -Cys-A 11 (I)

[0052] in:

[0053] 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-α-substituted derivative of these amino acids, or A1 is absent;

[0054] If A1 is present, A2 represents an arginine residue or a glutamic acid residue, or if A1 is absent, A2 represents an arginine residue or a glutamic acid residue or an N-α-substituted derivative of these amino acids;

[0055] A3 represents an aromatic amino acid residue;

[0056] 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;

[0057] 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;

[0058] A7 represents a proline residue, a glycine residue, an ornithine residue, a lysine residue, an alanine residue, a citrulline residue or an arginine residue;

[0059] A8 represents a tyrosine residue, a phenylalanine residue, an alanine residue, a naphthylalanine residue, a citrulline residue or a glutamic acid residue;

[0060] A 10 represents a citrulline residue, a glutamic acid residue, an arginine residue, or a lysine residue;

[0061] A 11 represents an arginine residue, a glutamic acid residue, a lysine residue or a citrulline residue, wherein the C-terminal carboxyl group may be derivatized;

[0062] Furthermore, the cysteine ​​residue at position 4 or 13 can form a disulfide bond, and the amino acid can be L-type or D-type.

[0063] Exemplary peptides according to formula (I) are peptides having an amino acid sequence as set forth in any one of SEQ ID NO: 1 to SEQ ID NO: 72, as shown in Table 1 below.

[0064] Table 1 - T-140 and currently preferred T-140 analogs

[0065]

[0066]

[0067]

[0068]

[0069] According to a specific embodiment, in each of SEQ ID NO: 1 to SEQ ID NO: 72, the two cysteine ​​residues are linked by a disulfide bond.

[0070] In another embodiment, the peptides used in the compositions and methods of the present invention consist essentially of the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the peptides used in the compositions and methods of the present invention comprise 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.

[0071] In various other embodiments, the peptide is selected from SEQ ID NO: 1 to SEQ ID NO: 72, wherein each possibility represents a separate embodiment of the present invention.

[0072] According to a specific embodiment, the peptide has the amino acid sequence shown in SEQ ID NO: 1 and is named motexafortide.

[0073] As mentioned above, the VLA-4 inhibitor (VLA4i) agent is natalizumab [abbreviated herein as (N)].

[0074] According to some embodiments, natalizumab is sold under the brand name sell.

[0075] According to some embodiments, natalizumab is sold under the brand name sell.

[0076] The above-mentioned peptides and VLA4i ("agents") can be administered to a subject per se or in the form of a pharmaceutical composition mixed with a suitable carrier or excipient. Each of the agents can be formulated as a separate preparation.

[0077] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein and 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.

[0078] The term "active ingredient" herein refers to the agent responsible for the biological effect, eg, SEQ ID NO: 1 or VLA4i.

[0079] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. Adjuvants are encompassed by these phrases.

[0080] The term "excipient" herein refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the 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.

[0081] Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, incorporated herein by reference.

[0082] Suitable routes of administration may, for example, include oral delivery, rectal delivery, transmucosal delivery (particularly nasal delivery), intestinal delivery, or parenteral delivery, parenteral delivery including intramuscular, intradermal, subcutaneous, and intramedullary injections, as well as intrathecal injections, direct intracerebroventricular injections, intracardiac injections (e.g., into the right or left ventricular cavity, into the main coronary artery), intravenous injections, intraperitoneal injections, intranasal injections, or intraocular injections.

[0083] According to a specific embodiment, the peptide of the invention or the pharmaceutical composition comprising the same is administered subcutaneously (SC).

[0084] According to a specific embodiment, the VLA4i of the present invention or a pharmaceutical composition comprising the same is administered intravenously (IV).

[0085] The pharmaceutical compositions of some embodiments of the present invention can be manufactured by processes well known in the art, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0086] Therefore, the pharmaceutical composition used according to some embodiments of the present invention can be prepared in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants), which is conducive to processing the active ingredient into a pharmaceutically usable preparation. Appropriate preparation depends on the selected route of administration.

[0087] Pharmaceutical compositions suitable for use in some embodiments of the present invention include compositions comprising an effective amount of an active ingredient to achieve the intended purpose. More specifically, depending on the embodiment, a therapeutically effective amount refers to, on the one hand, a mobilizing effective amount, and, on the other hand, an amount of the active ingredient that is effective to prevent, alleviate, or ameliorate the symptoms of a disease, such as SCD.

[0088] According to specific embodiments, the peptide of the present invention or a pharmaceutical composition comprising the same is administered in a dose range of between 0.1 mg and 10 mg per kg body weight, between 0.1 mg and 2 mg per kg body weight, between 0.1 mg and 1 mg per kg body weight, between 0.3 mg and 10 mg per kg body weight, or between 0.3 mg and 2 mg per kg body weight.

[0089] According to a specific embodiment, BL-8040 is administered at a dose of 1 mg to 2 mg per kg body weight.

[0090] According to a specific embodiment, BL-8040 is administered at a dose of 1.25 mg to 1.5 mg per kg body weight.

[0091] According to a specific embodiment, BL-8040 is administered at a dose of 1.25 mg / kg body weight.

[0092] According to a specific embodiment, BL-8040 is administered subcutaneously (SC).

[0093] According to a specific embodiment, natalizumab is administered prior to said peptide.

[0094] According to a specific embodiment, the peptide is administered no more than 24 hours after administration of said natalizumab.

[0095] According to a specific embodiment, the peptide is administered about 24 hours after administration of said natalizumab.

[0096] According to a specific embodiment, the method comprises: performing cytopheresis after administering the agent to obtain the CD34+ cells from the subject's peripheral blood.

[0097] About 4 hours and up to about 12 hours after administration of BL8040, patients underwent cytopheresis.

[0098] 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 into a container containing an anticoagulant (eg, heparin or citrate); and apheresis.

[0099] As used herein, the term "cytopheresis" refers to the process of passing an individual's peripheral blood through a device to produce a major component (e.g., HSCs) and then returning other components to the subject's blood circulation. Cytopheresis generally involves a three-step process, including: (1) drawing blood from the subject; (2) separating the blood components (e.g., based on density); and (3) returning certain components of the blood to the subject through a transfusion. Blood is generally separated into three parts: red blood cells (approximately 45% of the total blood), a "buffy coat" (less than 1% of the total blood), and plasma (approximately 55% of the total blood). Different types of cytopheresis procedures can be used depending on the blood component to be drawn.

[0100] As described above, the collected fractions can be subjected to various research or diagnostic protocols, such as transcriptional and immunophenotyping.

[0101] The collected fraction can also be processed so that the erythrocytes from which it matures have a healthy phenotype.

[0102] Thus, according to one aspect of the present invention, there is provided a method of treating sickle cell disease (SCD), the method comprising:

[0103] (a) The method of any one of claims 1 to 16, wherein CD34+ cells are mobilized in a subject with sickle cell disease (SCD);

[0104] (b) performing cytopheresis to obtain the CD34+ cells from the subject's peripheral blood; and

[0105] (c) CD34+ cells were processed to obtain red blood cells with a healthy phenotype.

[0106] Thus, the cells used are autologous to the subject.

[0107] The following is a non-limiting overview of genetic methods used to manipulate collected cells (post-cytopheresis).

[0108] These cells may be genetically manipulated or cultured first.

[0109] For example, the lentiviral transduction in CD34+HSC usually needs to be cultured in vitro under cytokine stimulation. Usually, pre-stimulation one day is performed with the serum-free culture medium including cytokines (stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L) and thrombopoietin (TPO), each 100ng / mL), followed by transduction of one day with LV, which can realize the robust implantation of CD34+ cells and effective EGFP gene labeling in xenotransplanted mice. Lower concentrations of SCF enhance the implantation of CD34+ cells, and serum albumin can be replaced by polyvinyl alcohol. Long-term cultivation can realize the implantation of CD34+ cells on fibronectin coated plates. High-density cultivation using adjuvants such as dimethyl prostaglandin E2 (PGE2) and amphiphilic drug delivery poloxamer can improve the in vitro transduction efficiency of human CD34+ cells by about 10 times. In general, the lentiviral transduction in CD34+ cell culture in vitro requires cytokine stimulation. However, minimal stimulation and short-term culture can also be used to maintain a balance between efficient gene addition and robust engraftment of CD34+ cells.

[0110] Typically, therapeutic genes such as wild-type β-globin, β- T87Q β-globin (containing an anti-sickling mutation) or γ-globin is inserted into a self-inactivating (SIN) LV. In addition, the β-globin promoter and locus control region (LCR) are inserted to control transgene expression. Because the second intron of the β-globin gene is important for its expression, traditional vectors carry the β-globin cassette in the opposite orientation relative to the vector genome to prevent transcriptional excision. Because this reverse orientation construct reduces vector titer and transduction efficiency in CD34+ cells, a forward-oriented β-globin vector has recently been developed. It addresses these issues and produces robust β-globin expression in erythroid cells.

[0111] The following are some clinical trials for SCD according to the embodiments of the present invention ( JClinInvest.2021Apr 15;131(8):e146394. Table 2).

[0112]

[0113]

[0114]

[0115] The manipulated cells can then be returned to the subject.

[0116] Each of these is contemplated herein. It is anticipated that numerous procedures related to CD34+ cells will be developed during the life of this patent application, and the scope of the term "manipulating CD34+" is intended a priori to include all such new technologies.

[0117] As used herein, the term "about" refers to ±10%.

[0118] The terms "comprises," "includes," "includes," "including," "having," and variations thereof mean "including but not limited to."

[0119] The term "consisting of" means "including and limited to."

[0120] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0121] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0122] Throughout this application, various embodiments of the present invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a hard limit to the scope of the invention. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and each numerical value within that range. For example, a description of a range from 1 to 6 should be considered to have specifically disclosed, for example, subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each number within that range, such as 1, 2, 3, 4, 5, and 6. This applies no matter how broad the range is.

[0123] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "range between a first indicated numeral and a second indicated numeral" and "from a first indicated numeral to a second indicated numeral" are used interchangeably herein and are intended to include the first indicated numeral and the second indicated numeral and all fractions and integers therebetween.

[0124] As used herein, the term "method" refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or manners, means, techniques and procedures readily developed from known manners, means, techniques and procedures.

[0125] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disorder, substantially ameliorating clinical or aesthetic symptoms of a disorder, or substantially preventing the appearance of clinical or aesthetic symptoms of a disorder.

[0126] It should be understood that certain features of the invention described in the context of separate embodiments for the sake of clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of individual embodiments for the sake of brevity may also be provided individually or in any suitable subcombination or in any other described embodiment suitable for the invention. Certain features described in the context of individual embodiments should not be considered essential features of those embodiments unless the embodiment cannot function without those elements.

[0127] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section herein find experimental support in the following examples.

[0128] Example

[0129] 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.

[0130] Treatment overview is as follows Figure 1 shown.

[0131] design

[0132] Single-arm, single-center, open-label pilot study.

[0133] Study population

[0134] N was 5 patients aged 18 to 40 years with a diagnosis of SCD (hemoglobin SS or Sβ°) who underwent automated RBC exchange via venous access with apheresis capability.

[0135] Key eligibility criteria

[0136] Able and willing to provide informed consent and meet the above study population criteria, with ECOG ≤ 1 and normal bone marrow / organ function.

[0137] Primary objective / end point

[0138] Safety and tolerability of (M) and (N) + (M) in SCD patients defined by pre-specified dose-limiting toxicities.

[0139] Secondary objectives / end points

[0140] The number of CD34+ cells mobilized by leukapheresis (CD34+ cells / kg / L) was calculated based on the total volume (tv) processed (1 blood volume (approximately 4 L to 5 L operating volume)).

[0141] The number of CD34+ cells mobilized by leukapheresis (CD34+ cells / kg / L) was calculated based on the adjusted volume (aV) for treatment (1 blood volume (approximately 4 to 5 L operating volume)).

[0142] Kinetics of CD34+ HSPC PB mobilization (cells per μl in PB).

[0143] Incidence of adverse events.

[0144] Exploratory objectives / end points

[0145] Immunophenotypic and transcriptional analysis of mobilized PB CD34+ HSCs.

[0146] Analysis of gene editing efficiency and HSC adaptability of mobilized PB CD34+HSCs in this study.

[0147] Although the present invention has been described in conjunction with specific embodiments, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. It is therefore intended to encompass all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0148] It is the applicant's intention that all publications, patents, and patent applications mentioned in this specification are incorporated herein by reference, as if each individual publication, patent, or patent application were specifically and individually indicated as being incorporated herein by reference when mentioned. In addition, the citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority documents of this application are incorporated herein by reference in their entirety.

Claims

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

2. A combination of agents for treating sickle cell disease (SCD), comprising a therapeutically effective amount of a CXCR4 inhibitory peptide as shown in SEQ ID NO: 1 and a therapeutically effective amount of natalizumab, wherein: The natalizumab and the CXCR4 inhibitory peptide are administered sequentially.

3. The method or combination according to claim 1 or 2, wherein: The natalizumab was administered intravenously (iv).

4. The method or combination according to claim 1 or 2, wherein: The peptide was formulated as motexafortide.

5. The method or combination according to claim 1 or 2, wherein: The peptides were administered subcutaneously (SC).

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

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

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

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

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

11. The method or combination of use according to claim 9, wherein: The natalizumab was administered at a dose of 300 mg.

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

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

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

15. The method or combination for use of any one of claims 1 to 14, comprising: After administering the agent, cytopheresis is performed to obtain the CD34+ cells from the subject's peripheral blood.

16. A method of treating sickle cell disease (SCD), the method 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 cytopheresis to obtain the CD34+ cells from the peripheral blood of the subject; and (c) treating the CD34+ cells to obtain red blood cells with a healthy phenotype.

17. The method according to claim 16, wherein The manipulation is performed by genome editing.

18. The method according to any one of claims 16 to 17, wherein The healthy phenotype includes the expression of fetal hemoglobin (HbF; hemoglobin F).

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