阅读说明：本技术 一种血红蛋白病治疗有效性预测方法 (Method for predicting treatment effectiveness of hemoglobinopathy ) 是由 方日国 于玲玲 杨卉慧 于 2020-04-29 设计创作，主要内容包括：涉及一种治疗个体中的血红蛋白病的方法,包括：a)评估步骤,该评估步骤包括评估经修饰的CD34阳性造血干细胞/祖细胞的第一群体分化后产生所需水平的γ-球蛋白或胎儿血红蛋白的能力,其中第一群体的经修饰的CD34阳性HSPC来源于所述个体并经过修饰以降低BCL11A功能；并且b)治疗步骤,该治疗步骤包括向所述个体施用第二群体经修饰的CD34阳性HSPC,该经修饰的CD34阳性HSPC来源于所述个体并且经修饰降低了BCL11A功能。同时,还涉及治疗个体中的血红蛋白病的方法、选择患有血红蛋白病的个体用于以第二群体的经修饰CD34阳性HSPC进行治疗的方法和确定患有血红蛋白病的个体是否适合或不适合用来源于所述个体并经修饰降低了BCL11A功能的第二群体的经修饰CD34阳性HSPC进行治疗的方法。(Relates to a method of treating hemoglobinopathy in a subject comprising: a) an evaluation step comprising evaluating a first population of modified CD34 positive hematopoietic stem/progenitor cells for their ability to differentiate to produce a desired level of gamma-globulin or fetal hemoglobin, wherein the modified CD34 positive HSPCs of the first population are derived from the individual and modified to reduce BCL11A function; and b) a treatment step comprising administering to the individual a second population of modified CD34 positive HSPCs derived from the individual and modified to reduce BCL11A function. Also, methods of treating hemoglobinopathies in a subject, methods of selecting subjects having hemoglobinopathies for treatment with a second population of modified CD34 positive HSPCs, and methods of determining whether subjects having hemoglobinopathies are suitable or unsuitable for treatment with a second population of modified CD34 positive HSPCs derived from the subject and modified to reduce BCL11A function are also contemplated.)

A method of treating a hemoglobinopathy in a subject, comprising:

a) an evaluation step comprising evaluating a first population of modified CD34 positive hematopoietic stem/progenitor cells ("CD 34 positive HSPCs") for their ability to differentiate to produce a desired level of gamma-globulin or fetal hemoglobin (HbF), wherein the modified CD34 positive HSPCs of the first population are derived from the individual and are modified to reduce BCL11A function ("modified EV cells"); and is

b) A treatment step comprising administering to the individual a second population of modified CD34 positive HSPCs derived from the individual and modified to reduce BCL11A function ("modified TR cells").

A method of treating hemoglobinopathy in an individual comprising a treatment step comprising administering to the individual a second population of modified CD34 positive HSPCs ("modified TR cells") derived from the individual and modified to reduce BCL11A function,

wherein the individual is selected for treatment based on the functional assessment via an assessment step comprising: assessing the ability of a first population of modified CD34 positive HSPCs to differentiate to produce a desired level of gamma-globulin or fetal hemoglobin (HbF), wherein the first population of modified CD34 positive HSPCs are derived from the individual and modified to reduce BCL11A function ("modified EV cells").

A method of determining whether an individual having a hemoglobinopathy is suitable or unsuitable for treatment with a modified CD34 positive HSPC ("modified TR cell") derived from the individual and modified to reduce BCL11A function in a second population, wherein the method comprises an assessment step comprising: assessing the ability of the first population of modified CD34 positive HSPCs to differentiate to produce a desired level of gamma-globulin or fetal hemoglobin (HbF), wherein the first population of modified CD34 positive HSPCs are derived from the individual and modified to reduce BCL11A function ("modified" EV cells), wherein the individual is suitable for treatment if the first population of modified CD34 positive HSPCs produce the desired level of gamma-globulin or fetal hemoglobin (HbF); wherein the individual is not eligible for treatment with the modified TR cells if the modified CD34 positive HSPC of the first population does not produce the desired level of gamma-globulin or fetal hemoglobin (HbF).

The method of any one of claims 1-3, wherein the evaluating step comprises:

a) isolating CD 34-positive HSPCs from a blood sample of bone marrow or periphery of the individual to obtain an isolated population of CD 34-positive HSPCs ("isolated EV cells");

b) modifying the isolated EV cells to obtain a first population of modified CD34 positive HSPC cells having reduced BCL11A function ("modified EV cells"); and

c) the ability of the modified EV cells to produce the desired level of gamma-globulin or fetal hemoglobin (HbF) upon differentiation was evaluated.

The method of any one of claims 1-4, wherein the treating step comprises:

a) mobilizing CD34 positive HSPCs in the bone marrow of the subject to increase the amount of CD34 positive HSPCs in peripheral blood;

b) isolating CD 34-positive HSPCs from the peripheral blood of the individual to obtain an isolated population of CD 34-positive HSPCs ("isolated TR cells"),

c) modifying the isolated TR cells to obtain a second population of modified CD34 positive HSPCs ("modified TR cells") that have reduced BCL11A function; and

d) administering to the individual an effective amount of modified TR cells.

The method of any one of claims 1-5:

1) wherein the evaluating step comprises:

a) isolating CD 34-positive HSPCs from a bone marrow or peripheral blood sample of the individual to obtain an isolated population of CD 34-positive HSPCs ("isolated EV cells");

b) modifying the isolated EV cells to obtain a first population of modified CD34 positive HSPC cells ("modified EV cells") that have reduced BCL11A function; and

c) assessing the ability of the modified EV cells to produce a desired level of gamma-globulin or fetal hemoglobin (HbF) upon differentiation; and

2) wherein the treating step comprises:

a) mobilizing CD 34-positive HSPCs in the subject to peripheral blood;

b) isolating CD 34-positive HSPCs from the peripheral blood of the individual to obtain an isolated population of CD 34-positive HSPCs ("isolated TR cells"),

c) modifying the isolated TR cells to obtain a second population of modified CD34 positive HSPCs ("modified TR cells") that have reduced BCL11A function; and

d) administering to the individual an effective amount of modified TR cells.

The method of any one of claims 1-6, wherein the modified EV cell is modified by genetic modification.

The method of any one of claims 4-6, wherein modifying the isolated EV cells comprises genetically modifying the isolated EV cells.

The method of claim 7 or 8, wherein the isolated EV cells are genetically modified by a technique selected from the group consisting of Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and regularly interspaced short palindromic repeats (CRISPRs), RNA editing, RNA interference.

The method of any one of claims 1-9, wherein the ability of the modified EV cells to produce a desired level of γ -globulin or fetal hemoglobin (HbF) upon differentiation is assessed in an assessment step comprising: i) culturing the modified EV cell under conditions that allow differentiation to obtain a population of red blood cells; and 2) determining the level of gamma-globulin or fetal hemoglobin (HbF) produced by said red blood cells.

The method of any one of claims 1-10, wherein assessing the ability of the modified EV cells to produce a desired level of γ -globulin or fetal hemoglobin (HbF) upon differentiation in the assessing step comprises: the mRNA level of gamma globulin was determined.

The method of any one of claims 1-10, wherein assessing the ability of the modified EV cell to produce a desired level of γ -globulin or fetal hemoglobin (HbF) upon differentiation in the assessing step comprises: determining a protein level of fetal hemoglobin (HbF).

The method of any one of claims 1-12, wherein the individual has not undergone mobilization or pretreatment prior to the evaluating step.

The method of any one of claims 1-13, wherein the evaluating step is repeated at least once prior to the treating step.

The method of any one of claims 1-14, wherein

The modified TR cells are modified by genetic modification.

The method of any one of claims 5-14, wherein modifying the isolated TR cells comprises genetically modifying the isolated TR cells.

The method of claim 15 or 16, wherein the isolated TR cells are genetically modified by a technique selected from the group consisting of Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and regularly interspaced short palindromic repeats (CRISPRs), RNA editing, RNA interference.

The method of any one of claims 5-17, wherein mobilizing CD34 positive HSPCs in a treatment step comprises: treating the subject with Granulocyte Colony Stimulating Factor (GCSF) and/or plerixafor.

The method of any one of claims 1-18, wherein the treating step further comprises: pre-treating the individual prior to administering the modified TR cells.

The method of claim 19, wherein the pretreatment comprises chemotherapy, monoclonal antibody therapy, or systemic radiation.

The method of claim 20, wherein the pretreatment comprises chemotherapy.

The method of claim 21, wherein the chemotherapy comprises administering to the individual one or more chemotherapeutic agents selected from the group consisting of: busulfan, cyclophosphamide and fludarabine.

The method of any one of claims 5-22, wherein the isolated TR cells are cultured for one or more days prior to modification.

The method of any one of claims 5-23, wherein the modified TR cells are cultured for one or more days prior to administration to the individual.

The method of any one of claims 1-24, wherein the modified TR cells are stored under freezing conditions for at least 24 hours prior to administering the modified TR cells to the individual.

The method of claim 25, wherein the modified TR cells are cultured for one or more days prior to storage under freezing conditions.

[ correction 15.05.2020 according to rule 26 ] the method of any one of claims 1-26, wherein the hemoglobinopathy is selected from the group consisting of: sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, thalassemia, hemoglobin-related disorders with increased oxygen affinity, hemoglobin-related disorders with decreased oxygen affinity, unstable hemoglobinopathies, and methemoglobinemia.

The method of claim 27, wherein the hemoglobinopathy is selected from the group consisting of β -thalassemia and sickle cell anemia.

The method of claim 28, wherein the hemoglobinopathy is β 0 or β + thalassemia.

The method of any one of claims 1-29, wherein the individual is a human.

The method of any one of claims 1-30, wherein the treatment step is performed immediately following the evaluating step.