Conceptio › Archive › Google Patents

Google Patentsopen access

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and … — Vertex Pharmaceuticals Incorporated (US20250041349A1)

Vertex Pharmaceuticals Incorporated · Google Patents

Google Patents · Patents · License: Open Access

patent, google patents, intellectual property, US20250041349A1, Vertex Pharmaceuticals Incorporated, Ewelina Morawa, en, 2025

ABSTRACT

Abstract

Provided herein, in some embodiments, are methods and compositions for treatment of subjects with Î²-thalassemia and subjects with severe sickle cell disease using autologous CRISPR-Cas9 modified CD34+ human hematopoietic stem and progenitor cells.

Description

RELATED APPLICATIONS

The application is a national stage filing under 35 U.S.C. Â§ 371 of international application number PCT/US2018/063973, filed Dec. 5, 2018, and claims the benefit of priority under 35 U.S.C. Â§ 119 (e) of U.S. provisional application No. 62/594,689, filed Dec. 5, 2017, U.S. provisional application No. 62/664,023, filed Apr. 27, 2018, U.S. provisional application No. 62/671,770, filed May 15, 2018, U.S. provisional application No. 62/734,431, filed Sep. 21, 2018, and U.S. provisional application No. 62/734,543, filed Sep. 21, 2018; the contents of each of which are incorporated by reference herein in their entirety.

BACKGROUND

Hemoglobin (Hb), is a tetramer formed of four globin peptides, each tightly associated with a heme group that contains an atom of iron. During gestation, the predominant form of hemoglobin is fetal hemoglobin (HbF), which is composed of two Î±-globin chains and two Î³-globin chains. Shortly before birth, there is a switch from HbF to adult hemoglobin, which contains two Î±-globin and two Î²-globin polypeptide chains. The switch from HbF to adult hemoglobin is mediated by a transcriptional switch from Î³-globin to Î²-globin within the Î²-globin gene cluster located on chromosome 11.

Hemoglobinopathies are disorders caused by genetic defects that affect the production or function of hemoglobin molecules. Two of the most common of the hemoglobinopathies are Î²-thalassemia and sickle cell disease (SCD).

Î²-thalassemia is one of the most common autosomal recessive disorders worldwide with high prevalence in populations in the Mediterranean (5-15%), Middle-East and West Asia (2-5%), South-East Asia (up to 10%), and South Asia (up to 18%) (Colah, R. et al. 2010 . Expert Rev Hematol 3, 103-117). Due to population migration, Î²-thalassemia is also found in Northern Europe, North and South America, Caribbean, and Australia. Currently, the worldwide living population of Î²-thalassemia major patients is estimated to be 200,000 that are registered and receiving treatment (Galanello, R., Origa, R., 2010 . Orphanet J Rare Dis 5, 11).

Î²-thalassemia is caused by a spectrum of mutations that result in reduced or absent production of adult hemoglobin (HbA). Different forms of Hb are produced during different stages of development. Fetal hemoglobin (HbF) is the predominant Hb prior to birth and extending into the newborn period. HbF is a tetrameric globin protein containing 2 Î³-globin and 2 Î±-globin chains (Î±2Î³2). After the newborn period, the main form of Hb is HbA, a heterotetramer comprised of 2 Î²-globin and 2 Î±-globin chains (Î±2Î²2). HbA normally accounts for >95% of the total Hb in the blood of adults.

Treatment of transfusion-dependent Î²-thalassemia (TDT), in particular, includes lifelong blood transfusions every 3-6 weeks. The aim of transfusion therapy is to keep Hb levelsâ¥9 g/dL in order to ameliorate the symptoms and physiologic sequela of severe anemia and to maintain normal growth and development. Though chronic blood transfusion regimens are effective at preventing the hallmark symptoms and physical manifestations of disease, they introduce a large iron overload that may lead to mortality through iron associated heart and liver toxicity. To prevent this, iron overload is managed with iron chelation regimens that are usually initiated at an early age. Poor compliance with chelation regimens remains a key challenge. Despite the improvements with current therapies, there is poor quality of life and overall survival until the age of 30 years is only 55%.

SCD is one of the most common monogenic disorders affecting millions of people. It is estimated to affect over 100,000 individuals in the US and about 42,000 individuals in Europe. The most severe and prevalent form of SCD, referred to as sickle cell anemia, is an autosomal recessive disease due to homozygous mutations in which a valine replaces a glutamic acid at position 6 in the Î²-globin protein which leads to polymerization of deoxygenated hemoglobin and red blood cell (RBC) sickling.

SCD is a chronic disease, characterized by recurrent acute VOC that lead to acute pain, chronic hemolysis, anemia, progressive tissue injury, and organ dysfunction. The disease affects multiple organs causing acute and chronic complications such as acute chest syndrome, stroke, priapism, splenic sequestration, osteonecrosis, renal failure, pulmonary hypertension, liver disease, bone damage, limited growth, increased susceptibility to infections, fatigue, and progressive cognitive decline.

About 90% of children born with SCD in the US or EU will survive into adulthood, but their lifespan is shortened by two to three decades compared to the general population with a median age of death of approximately forty to fifty years.

SUMMARY

The present disclosure provides, in some embodiments, methods and compositions for the treatment of hemoglobinopathies, such as Î²-thalassemia or sickle cell disease. A gene editing technology is used to accurately and efficiently introduce genetic changes into a non-coding erythroid lineage-specific enhancer of the BCL11A gene, thus specifically down-regulating BCL11A in erythroid precursors without affecting other hematopoietic lineages. Without being bound by theory, it is thought that this noncoding change will reactivate Î³-globin gene transcription, and elevate fetal hemoglobin (HbF) protein in red blood cells (RBCs), thereby ameliorate disease severity.

Thus, in some aspects, the present disclosure provides compositions that include CD34+ human hematopoietic stem and progenitor cells (hHSPCs) that have a genetic modification within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene. In some embodiments, the composition further comprises a serum-free cryopreservation medium, 5% dimethylsulfoxide (DMSO), dextran-40, or any combination of two or more of the foregoing reagents.

In some embodiments, the genetic modification is or comprises an (at least one) insertion, deletion, mutation, or combination thereof. In some embodiments, the genetic modification comprises an insertion and a deletion (i.e., an indel) resulting from a CRISR-Cas9-mediated modification.

In some embodiments, the genetic modification is producing by delivering to the CD34+ hHSPCs a Cas9 endonuclease (e.g., of S. pyrogenes) (or a nucleic acid encoding a Cas9 nuclease) and a guide RNA (gRNA, such as a sgRNA) (or a nucleic acid encoding a gRNA) that targets the +58 DHS within the erythroid lineage-specific enhancer of a human BCL11A gene. In some embodiments, the gRNA comprises SEQ ID NO:1 or SEQ ID NO:2 (a modified version of SEQ ID NO:1). In some embodiments, the gRNA comprises three 2â²-O-methyl-phosphorothioate residues at or near each of its 5â² and 3â² ends. In some embodiments, the endonuclease comprises a N-terminal SV40 nuclear localization signal (NLS) and/or a C-terminal SV40 nuclear localization signal (NLS). In some embodiments, the Cas9 and gRNA are delivered as a ribonucleoprotein complex, optionally wherein the weight ratio of the gRNA to said endonuclease is 1:1.

In some embodiments, a genetically modified hHSPC or population of hHSPCs exhibit(s) an increase in Î³/(Î³+Î±)-globin mRNA ratios of 0.1 to 0.5 relative to unmodified CD34+ hHSPCs, and/or wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î²)-globin mRNA ratios of 0.2 to 0.6 relative to unmodified CD34+ hHSPCs. In some embodiments, a genetically modified hHSPC or population of hHSPCs exhibit(s) a HbF mean percentage of HbF/(HbF+HbA) protein levels of 15% to 50%. In some embodiments, a genetically modified hHSPC or population of hHSPCs exhibit(s) a mean allele editing frequency of 70% to 90%.

In some embodiments, at least 75% of a population of modified CD34+ hHSPCs maintain multi-lineage potential for at least sixteen weeks after administration of the modified CD34+ hHSPCs to a subject. In some embodiments, a population of modified CD34+ hHSPCs exhibit an on-target indel rate of at least 40% or at least 80%. In some embodiments, a population of modified CD34+ hHSPCs exhibit an off-target indel rate of less than 5% or less than 1%.

In other aspects, the present disclosure provides methods that include administering (e.g., via injection/IV transfusion) to a subject (e.g., a human subject) having a hemoglobinopathy a dose of CD34+ hHSPCs that comprise a genetic modification within a +58 DHS within the erythroid lineage-specific enhancer of a human BCL11A gene, wherein the hHSPCs are administered in an effective amount to reduce the number of blood transfusions administered the subject (e.g., by 50%, by 60%, by 70%, by 80%, by 90%, and/or by 2-fold, 3-fold, 4-fold, 5-fold, or more) relative to baseline and/or to increase fetal hemoglobin (HbF) levels in the subject to at least 20%. In some embodiments, the methods are methods of treating a hemoglobinopathy in a subject, such as Î²-thalassemia or sickle cell disease. In some embodiments, the method are methods of increasing HbF in a subject.

In some embodiments, the subject is 18 years of age or older. In some embodiments, the subject is 18 to 35 years of age. In some embodiments, the subject is older than 35 years of age. In other embodiments, the subject is younger than 18 years of age. In some embodiments, the subject is 11 years of age or older. In some embodiments, the subject is 11 to 35 years of age. In some embodiments, the subject is 2 years of age or older. In some embodiments, the subject is 2 to 35 years of age.

In some embodiments, the methods include (a) mobilizing stem cells in a subject having a hemoglobinopathy; (b) collecting CD34+ hHSPCs from the subject; (c) producing modified CD34+ hHSPCs that comprise a genetic modification in within a +58 DHS within the erythroid lineage-specific enhancer of a human BCL11A gene; and (d) administering to the subject a dose of the modified CD34+ hHSPCs of step (c) in an effective amount to reduce the number of blood transfusions administered the subject (e.g., by 50%, by 60%, by 70%, by 80%, by 90%, and/or by 2-fold, 3-fold, 4-fold, 5-fold, or more) relative to baseline and/or to increase fetal hemoglobin (HbF) levels in the subject to at least 20%. In some embodiments, the methods are methods of treating a hemoglobinopathy, such as Î²-thalassemia or sickle cell disease. In some embodiments, the method are methods of increasing HbF in a subject. In some embodiments, step (a) comprises administering an inhibitor of CXCR4 chemokine receptor, optionally wherein the inhibitor of CXCR4 chemokine receptor is Plerixafor, to the subject. In some embodiments, step (a) further comprises administering granulocyte colony stimulating factor (GCSF) to the subject.

In some embodiments, methods comprise administering red blood cells to the subject, optionally before mobilizing stem cells in the subject (step (a)) and/or after collecting CD34+ hHSPCs from the subject (step (b)).

In some embodiments, at least 15Ã10 6 CD34+ hHSPCs/kg are collected from the subject, e.g., in step (b).

In some embodiments, methods comprise administering busulfan to the subject, optionally after producing modified CD34+ hHSPCs (step (c)) and before administering a dose of said modified CD34+ hHSPCs to the subject (step (d)). In some embodiments, busulfan is administered intravenously in 4 mg/kg to 5 mg/kg doses for four days or intravenously in 0.5 mg/kg to 1 mg/kg doses every six hours for four days. In some embodiments, a dose of busulfan is adjusted based on pharmacokinetic level to achieve an area under the curve (AUC) of 4500 to 5500 Î¼M/min, preferably 5000 Î¼M/min.

In some embodiments, the modification of step (c) is an indel, optionally produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease (e.g., a S. pyogenes Cas9 endonuclease) and a guide RNA (e.g., gRNA comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:2) that targets the +58 DHS of a human BCL11A gene.

In some embodiments, neutrophil engraftment occurs in the subject within 35-45 days, e.g., 42 days, after administration of the modified CD34+ hHSPCs (step (d)).

In some embodiments, a subject having a hemoglobinopathy is a subject having Î²-thalassemia. In other embodiments, a subject having a hemoglobinopathy is a subject having sickle cell disease.

In some embodiments, the subject requires fewer (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90% fewer) blood transfusions within a 2-year period of the time of administration of the modified CD34+ hHSPCs, or within a 2-year period after the time of administration of the modified CD34+ hHSPCs, relative to a 2-year period before the time of administration of the modified CD34+ hHSPCs.

In some embodiments, the subject achieves transfusion reduction or transfusion independence for at least three months, at least six months, or at least twelve months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs.

In some embodiments, the subject exhibits a decrease (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% decrease) in parameters of iron overload relative to baseline as assessed by magnetic resonance imaging (MRI) or by change in serum ferritin level over time. In some embodiments, the decrease in parameters of iron overload includes a decrease (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% decrease) in liver iron concentration (LIC) and/or cardiac iron content (CIC).

In some embodiments, the subject is no longer in need of iron chelation therapy within a 2 to 5 year period (e.g., within 2, 3, 4, or 5 years) of the time of administration of the modified CD34+ hHSPCs, or within a 2 to 5 year period after the time of administration of the modified CD34+ hHSPCs, relative to a 2 to 5 year period before the time of administration of the modified CD34+ hHSPCs.

In some embodiments, the subject exhibits HbF levels of at least 20% for at least three months, at least six months, starting at any time at or after the time of administration of the modified CD34+ hHSPCs. In some embodiments, the subject exhibits HbF levels of at least 20% for at least three months, at least six months, starting three months or six months after the time of administration of the modified CD34+ hHSPCs. In some embodiments, the subject exhibits HbF levels of at least 20% in the absence of treatment with a secondary drug, e.g., hydroxyurea (HU).

In some embodiments, the subject exhibits a relative change, e.g., a reduction, in annualized rate of severe vaso-occlusive crises (VOC) from baseline, starting six months after administration of the modified CD34+ hHSPCs. In some embodiments, the subject exhibits an absence of VOC for at least 12 months or at least 24 months, starting six months after administration of the modified CD34+ hHSPCs.

In some embodiments, the subject experiences a change in patient reported outcomes (PROs) over time, e.g., after administration of the modified CD34+ hHSPCs, using at least one of the following assays selected from: Pain scale (11 point numerical rating scale [NRS]), EuroQol Quality of Life Scale (EQ 5D 5L), functional assessment of cancer therapy-bone marrow transplant (FACT-BMT), Patient-reported Outcome Measurement Information System (PROMIS)-Fatigue, PROMIS-Cognitive function, and Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me).

In some embodiments, the subject exhibits a change in hemolytic index as measured by principal component analysi

RELATED APPLICATIONS

BACKGROUND

SUMMARY

In some embodiments, at least 15Ã10 6 CD34+ hHSPCs/kg are collected from the subject, e.g., in step (b).

In some embodiments, neutrophil engraftment occurs in the subject within 35-45 days, e.g., 42 days, after administration of the modified CD34+ hHSPCs (step (d)).

In some embodiments, a subject having a hemoglobinopathy is a subject having Î²-thalassemia. In other embodiments, a subject having a hemoglobinopathy is a subject having sickle cell disease.

In some embodiments, the subject exhibits a change in hemolytic index as measured by principal component analysis of the following four markers of hemolysis over time: reticulocyte count, serum concentrations of aspartate transaminase, lactate dehydrogenase [LDH], and total bilirubin. In some embodiments, the subject exhibits a change in tricuspid regurgitant jet velocity (TRV) over time.

In some embodiments, a method further comprises administering red blood cells to the subject, wherein the red blood cells are administered before the step of administering the modified CD34+ hHSPCs. In some embodiments, the subject has received a red blood cell (RBC) transfusion before the step of administering the modified CD34+ hHSPCs. the subject has a hemoglobin S (HbS) level of less than 30% of total Hb and/or a total Hb concentration of 11 g/dL or less.

In some embodiments, the subject exhibits an increase, optionally at least a 10% increase, in the proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) over a period of time. In some embodiments, the subject exhibits a change in inflammatory and endothelial activation markers, a change in the proportion of alleles with the genetic modification present in peripheral blood leukocytes, and/or a change in the proportion of alleles with the genetic modification present in bone marrow cells over a period of time, optionally wherein the period of time is at least three months or at least six months following administration of the modified CD34+ hHSPCs.

In some embodiments, modified CD34+ hHSPCs that are administered to a subject are modified CD34+ hHSPCs that exhibit an increase in Î³/(Î³+Î±)-globin mRNA ratios of 0.1 to 0.5 relative to unmodified CD34+ hHSPCs, and/or wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î²)-globin mRNA ratios of 0.2 to 0.6 relative to unmodified CD34+ hHSPCs. In some embodiments, modified CD34+ hHSPCs that are administered to a subject are modified CD34+ hHSPCs that exhibit a HbF mean percentage of HbF/(HbF+HbA) protein levels of 15% to 50%, exhibit a ratio of (Î³+Î²)/Î±-globin mRNA that is at or above 0.4, and/or exhibit a mean allele editing frequency of 70% to 90%. In some embodiments, at least 50% of the modified CD34+ hHSPCs that are administered to a subject maintain multi-lineage potential for at least sixteen weeks after administration to the subject. In some embodiments, modified CD34+ hHSPCs that are administered to a subject are modified CD34+ hHSPCs that exhibit an on-target indel rate of at least 40% or at least 80%.

In some embodiments, a subject does not exhibit neoplastic and/or myeloproliferative lesions resulting from administration of the modified CD34+ hHSPCs.

In some embodiments, a dose comprises at least 2Ã10 6 or at least 3Ã10 6 modified CD34+ hHSPCs/kg.

In some embodiments, a method further comprises administering plerixafor to the subject, wherein red blood cells are administered before the step of administering the modified CD34+ hHSPCs. In some embodiments, a method further comprises administering a granulocyte colony stimulating factor to the subject.

The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs showing that edited cells maintain the ability to engraft and differentiate.

FIG. 2 is a graph showing the globin mRNA ratio after gene editing of Î²-thalassemia patient samples.

FIG. 3 is a graph showing the globin mRNA ratio after gene editing of Î²-thalassemia patient samples with the data separated by genotype.

FIG. 4 is a series of graphs showing that HbF protein increases after editing of SCD patient samples.

DETAILED DESCRIPTION

Currently, the only curative treatment option for transfusion-dependent Î²-thalassemia (TDT) is allogeneic hematopoietic stem cell transplant (allo-HSCT). There are significant risks associated with allo-HSCT such as serious infections, graft failure and graft-versus-host disease (GvHD), some of which can be fatal. As such, transplants are infrequently performed, and are offered primarily to subjects who have available human leukocyte antigen (HLA)-matched sibling donors, who are young (<16 years of age), and who do not have significant iron overload. Because of the need of a HLA-matched sibling donor, allo-HSCT is available to only <25% of eligible patients with remainder of the patients requiring lifelong transfusions and chelation. Transplants using alternative donor sources such as unrelated cord blood and haploidentical donors remain experimental due to high risk of engraftment failure and GvHD. The absence of suitable donors, the significant risks associated with transplantation, and the requirement for post-transplant immunosuppression therapy to prevent GvHD indicate an unmet medical need for novel therapies with transformative potential for subjects with TDT.

Approved therapies to prevent complications of sickle cell disease (SCD) include hydroxyurea (HU) in the US and EU and L-glutamine oral powder in the US. These therapies reduce complications of SCD; however, patients can still have breakthrough vaso-occlusive crisis (VOC). Furthermore, HU is not effective in all patients, is not well-tolerated and has carcinogenic and teratogenic risks. Allogeneic hematopoietic stem cell transplant (HSCT) is the only known cure for SCD, but HSCT is only available to about 20% of patients who have a matched donor, and graft versus-host disease (GvHD) is a known risk. Therefore, there is also significant unmet medical need for the treatment of SCD and other hemoglobinopathies.

Gene editing with the methods and compositions provided herein induce changes in the DNA sequence in autologous CD34 + hHSPCs such that upon erythroid differentiation in a patient, the expression of Î³-globin is increased, leading to an increase in HbF expression levels in adult erythroid cells. The increase in HbF ameliorates the clinical manifestations of Î²-thalassemia and SCD.

The CRISPR-Cas9 editing therapeutic approach of the present disclosure is to restore HbF production through editing of a non-coding region in the BCL11A gene. BCL11A is a transcriptional silencer of Î³-globin gene expression and hence a negative modulator of HbF.

Hemoglobinopathies

Î² Thalassemia

Aspects of the present disclosure provide methods for treating (e.g., ameliorating the symptoms and/or clinical manifestations) of beta thalassemia (Î² thalassemia). Beta thalassemia is a blood disorder that reduces the production of hemoglobin. Hemoglobin is the iron-containing protein in red blood cells that carries oxygen to cells throughout the body. A lack of beta-globin leads to a reduced amount of functional hemoglobin.

In a subject with beta thalassemia, low levels of hemoglobin lead to a lack of oxygen in many parts of the body. Without sufficient hemoglobin, red blood cells do not develop normally, causing a shortage of mature red blood cells. The low number of mature red blood cells leads to anemia, which can cause pale skin, weakness, fatigue, and more serious complications. People with beta thalassemia are at an increased risk of developing abnormal blood clots.

Beta thalassemia is classified into two types depending on the severity of symptoms: thalassemia major (also known as Cooley's anemia) and thalassemia intermedia . Of the two types, thalassemia major is more severe.

The signs and symptoms of thalassemia major appear within the first two years of life. Children develop life-threatening anemia. They do not gain weight and grow at the expected rate (failure to thrive) and may develop yellowing of the skin and whites of the eyes (jaundice). Affected individuals may have an enlarged spleen, liver, and heart, and their bones may be misshapen. Some adolescents with thalassemia major experience delayed puberty. Many people with thalassemia major have such severe symptoms that they need frequent blood transfusions to replenish their red blood cell supply. Over time, an influx of iron-containing hemoglobin from chronic blood transfusions can lead to a buildup of iron in the body, resulting in liver, heart, and hormone problems.

Thalassemia intermedia is milder than thalassemia major. The signs and symptoms of thalassemia intermedia appear in early childhood or later in life. Affected individuals have mild to moderate anemia and may also have slow growth and bone abnormalities.

Mutations in the hemoglobin gene cause beta thalassemia. Some mutations in the hemoglobin gene prevent the production of any beta-globin. The absence of beta-globin is referred to as beta-zero (Î²0) thalassemia. Other hemoglobin gene mutations allow some beta-globin to be produced but in reduced amounts. A reduced amount of beta-globin is called beta-plus (Î²+) thalassemia. The degree of impaired HbA production, resulting from the extent of incomplete (Î²+) or absent (Î²0) Î²-globin expression, determines the severity of Î²-thalassemia. Reduction in Î²-globin production results in an accumulation of excess, uncomplexed Î±-globin in erythroblasts. The clinical implications of this Î±-globin/Î²-globin imbalance are:

1) Hemolysis leading to a lack of sufficient erythrocytes and Hb to effectively transport oxygen throughout the body; 2) Oxidative damage of the cell membrane, thereby resulting in apoptosis of erythrocyte precursors and therefore ineffective erythropoiesis; and 3) Ineffective erythropoiesis which leads to morbidities such as splenomegaly, bone marrow expansion, concomitant bone deformities, and iron overload.

Having either B0 or B+ thalassemia does not necessarily predict disease severity, however; people with both types have been diagnosed with thalassemia major and thalassemia intermedia.

Patients with the HPFH phenotype have sustained HbF levels of 10% to 30% of total Hb throughout their lives, with often a pan-cellular distribution of HbF. A number of people with HPFH also carry the genetic defects for Î²-thalassemia or sickle cell disease. These patients who co-inherit both the HPFH and a Î² globin mutation have no clinical symptoms of their underlying Î²-thalassemia or sickle cell disease or suffer a mild form of the disease.

An increased HbF level is an ameliorating and protecting factor in Î² thalassemia in patients with non-transfusion-dependent thalassemia (NTDT) where HbF levels can be measured (Musallam, K. M. et al. 2012. Blood 119, 364-367).

Increased Î³-globin production mitigates the pathology resulting from excess unpaired Î±-globin and the Î±/Î²-protein imbalance that is a hallmark of Î²-thalassemia. As a result, there are improvements in the ineffective erythropoiesis seen in the disease, decreased hemolysis, and increased total hemoglobin levels from the improved survival of erythrocytes containing higher levels of HbF. There appears to be no minimum threshold of HbF that is associated with lower morbidity in patients with Î² thalassemia, as any amount of HbF appeared to be beneficial in non-transfusion-dependent patients with Î²-thalassemia intermedia (Musallam, K. M. et al. 2013 . Blood 121, 2199-2212). Resultant decrease in ineffective erythropoiesis due to increased HbF levels may also have a positive effect on iron overload and end-organ damage (Tanno, T. and Miller, J. L., 2010 . Adv Hematol 358283).

Treatment of transfusion-dependent Î²-thalassemia (TDT), in particular, includes lifelong blood transfusions every 3-6 weeks. The aim of transfusion therapy is to keep Hb levels â¥9 g/dL in order to ameliorate the symptoms and physiologic sequela of severe anemia and to maintain normal growth and development. Though chronic blood transfusion regimens are effective at preventing the hallmark symptoms and physical manifestations of disease, they introduce a large iron overload that may lead to mortality through iron associated heart and liver toxicity. To prevent this, iron overload is managed with iron chelation regimens that are usually initiated at an early age. Poor compliance with chelation regimens remains a key challenge. Despite the improvements with current therapies, there is poor quality of life and overall survival until the age of 30 years is only 55%.

Sickle Cell Disease

Aspects of the present disclosure provide methods for treating (e.g., ameliorating the symptoms and/or clinical manifestations) of sickle cell disease (SCD). Sickle cell disease is a group of disorders that affects hemoglobin, the molecule in red blood cells that delivers oxygen to cells throughout the body. Subjects with this disorder have atypical hemoglobin molecules called hemoglobin S, which can distort red blood cells into a sickle, or crescent, shape.

Signs and symptoms of SCD usually begin in early childhood. Characteristic features of this disorder include a low number of red blood cells (anemia), repeated infections, and periodic episodes of pain. The severity of symptoms varies from person to person. Some subjects have mild symptoms, while others are frequently hospitalized for more serious complications.

The signs and symptoms of SCD are caused by the sickling of red blood cells. When red blood cells sickle, they break down prematurely, which can lead to anemia. Anemia can cause shortness of breath, fatigue, and delayed growth and development in children. The rapid breakdown of red blood cells may also cause yellowing of the eyes and skin, which are signs of jaundice. Painful episodes can occur when sickled red blood cells, which are stiff and inflexible, get stuck in small blood vessels. These episodes deprive tissues and organs of oxygen-rich blood and can lead to organ damage, especially in the lungs, kidneys, spleen, and brain. A particularly serious complication of SCD is high blood pressure in the blood vessels that supply the lungs (pulmonary hypertension). Pulmonary hypertension occurs in about one-third of adults with SCD and can lead to heart failure.

Mutations in the hemoglobin gene cause SCD. Hemoglobin consists of four protein subunits, typically, two subunits called alpha-globin and two subunits called beta-globin. The hemoglobin gene provides instructions for making beta-globin. Beta-globin is a component (subunit) of hemoglobin. Hemoglobin consists of four protein subunits, typically two subunits of beta-globin and two subunits of another protein called alpha-globin. Various versions of beta-globin result from different mutations in the hemoglobin gene. One particular hemoglobin gene mutation produces an abnormal version of beta-globin known as hemoglobin S (HbS). Other mutations in the hemoglobin gene lead to additional abnormal versions of beta-globin such as hemoglobin C (HbC) and hemoglobin E (HbE).

Fetal Hemoglobin

Some aspects of the present disclosure provide methods that elevate fetal hemoglobin levels in a subject, e.g., a subject having Î²-thalassemia, sickle cell disease, or other hemoglobinopathy. Red blood cells function mainly to transport gases into and out of cells. This is facilitated by a structural component of hemoglobin, which has the ability to bind with gases. Three types of hemoglobin are synthesized in humans depending on the stage of development. Embryonic hemoglobin is produced before birth, fetal hemoglobin (HbF) during fetal life, and adult hemoglobin after birth. Fetal hemoglobin (HbF, Î± 2 Î³ 2 ) is the main oxygen transport protein in a human fetus and includes alpha (Î±) and gamma (Î³) subunits. HbF expression ceases about 6 months after birth. Adult hemoglobin (HbA, Î± 2 Î² 2 ) is the main oxygen transport protein in a human after Ë34 weeks from birth, and includes alpha (Î±) and beta (Î²) subunits. After 34 weeks, a developmental switch results in decreased transcription of the Î³-globin genes and increased transcription of Î²-globin genes. A replacement of glutamic acid of the beta chain by valine at the 6th position gives rise to a sickle cell disorder. This change, called hemoglobin S (HbS), is an abnormal hemoglobin. On exposure to low oxygen concentration, the HbS precipitates into elongated crystals appearing as sickled, instead of a biconcave disc. Sickle cell disease is characterized by occlusion events in the vascular that results in pain, organ failure and, occasionally, death. Since many of the forms of hemoglobinopathies are a result of the failure to produce normal Î²-globin protein in sufficient amounts or failure to produce normal Î²-globin protein entirely, increased expression of Î³-globin (HbF) will ameliorate Î²-globin disease severity.

BCL11A Erythroid-Lineage Specific Enhancer

In some embodiments, cells of the present disclosure (e.g., CD34+ hHSPCs) comprise a genetic modification within the +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene. BCL11A is a transcriptional silencer of Î³ globin gene expression and hence a negative modulator of HbF (see Menzel S et al. Nature Genetics. 2007; 39 (10): 1197-9; Lettre G et al. PNAS. 2008; 105 (33): 11869-74; and Uda M et al. PNAS. 2008; 105 (5): 1620-5, each of which is incorporated herein in its entirety). BCL11A is located on Chromosome 2 and ranges from 60,451,167-60,553,567 base pairs (bp) (GRCh38). This gene encodes a zinc finger transcription factor that represses fetal hemoglobin (HbF) and downregulates HbF expression starting at about 6 weeks after birth. The BCL11A gene contains four exons, spanning 102.4 kb of genomic DNA and includes a binding domain in intron 2 for the transcription factor GATA-1. GATA-1 binding enhances BCL11A expression which, in turn, represses HbF expression. Intron 2 contains multiple DNase I hypersensitive sites (DHS), including sites referred to as +55, +58, and +62 based on the distance in kilobases from the transcriptional start site. Naturally occurring SNPs within this region are associated with decreased BCL11A expression and increased fetal Hb levels. These SNPs are organized around three DNA Hypersensitivity sites, +55DHS, +58DHS and +62DHS. Of the three regions, the +58 DHS region, appears to be the key region associated with increased fetal Hb levels and also harbors a GATA1 transcriptional control region.

In some embodiments, the gene editing strategies, e.g., CRISPR-Cas9 gene editing strategies, of the present disclosure (for instance, through the NHEJ repair process discussed below) generate indels within the non-coding BCL11A erythroid lineage-specific enhancer on chromosome 2, thus down-regulating BCL11A in erythroid precursors with no effect expected in other hematopoietic lineages. Thus, in some embodiments, the genetic modification within the +58 DHS of a human BCL11A gene comprises at least one (on or more) indel. Without being bound by theory, it is thought that this noncoding change will reactivate Î³-globin gene transcription, and elevate HbF protein in RBCs.

The transcriptional control sequence of the BCL11A gene can also be modulated or inactivated by inserting a wild-type BCL11A gene or cDNA comprising a modified transcriptional control sequence. For example, the donor for modulating or inactivating by homology directed repair (HDR) contains the modified transcriptional control sequence of the BCL11A gene with small or large flanking homology arms to allow for annealing. HDR is essentially an error-free mechanism that uses a supplied homologous DNA sequence as a template during DSB repair. The rate of homology directed repair (HDR) is a function of the distance between the transcriptional control sequence and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing.

In addition to deleting, modulating, or inactivating the transcriptional control sequence of the BCL11A gene by NHEJ or HDR, a range of other options are possible. If there are small or large deletions, a cDNA can be knocked in that contains a modified transcriptional control sequence of the BCL11A gene. A full length cDNA can be knocked into any âsafe harborââi.e., non-deleterious insertion point that is not the BCL11A gene itselfâ, with or without suitable regulatory sequences. If this construct is knocked-in near the BCL11A regulatory elements, it should have physiological control, similar to the normal gene. Two or more (e.g., a pair) nucleases can be used to delete transcriptional control sequence regions, though a donor would usually have to be provided to modulate or inactivate the function. In this case two gRNA and one donor sequence would be supplied.

Provided herein are cellular, ex vivo and in vivo methods for using genome engineering tools to create permanent changes to the genome by: 1) modulating or inactivating the transcriptional control sequence of the BCL11A gene, by deletions that arise due to the NHEJ pathway; 2) modulating or inactivating the transcriptional control sequence of the BCL11A gene, by HDR; 3) modulating or inactivating the transcriptional control sequence of the BCL11A gene, by deletions of at least a portion of the transcriptional control sequence and/or knocking-in a wild-type BCL11A gene or cDNA comprising a modified transcriptional control sequence into the gene locus or a safe harbour locus. Such methods use endonucleases, such as CRISPR-associated (Cas9, Cpf1 and the like) nucleases, to permanently delete, insert, or edit the transcriptional control sequence within or near the genomic locus of the BCL11A gene or other DNA sequence that encodes a regulatory element of the BCL11A gene. In this way, examples set forth in the present disclosure can help to delete, modulate, or inactivate the transcriptional control sequence of the BCL11A gene with a single treatment or a limited number of treatments (rather than deliver potential therapies for the lifetime of the patient).

Genome Editing

Genome editing generally refers to the process of modifying the nucleotide sequence of a genome, preferably in a precise or pre-determined manner. Examples of methods of genome editing described herein include methods of using site-directed nucleases to cut deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and NHEJ, as recently reviewed in Cox et al., Nature Medicine 21 (2), 121-31 (2015). These two main DNA repair processes consist of a family of alternative pathways. NHEJ directly joins the DNA ends resulting from a double-strand break, sometimes with the loss or addition of nucleotide sequence, which may disrupt or enhance gene expression. HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a double-stranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as âAlternative NHEJâ, in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few basepairs flanking the DNA break site to drive a more favored DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online doi: 10.1038/nsmb.2961 (2015); Mateos-Gomez et al., Nature 518, 254-57 (2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances it may be possible to predict likely repair outcomes based on analysis of potential microhomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desired genomic alterations. A step in the genome editing process can be to create one or two DNA breaks, the latter as double-strand breaks or as two single-stranded breaks, in the target locus as near the site of intended mutation. This can be achieved via the use of site-directed polypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introduce double-strand breaks or single-strand breaks in nucleic acids, e.g., genomic DNA. The double-strand break can stimulate a cell's endogenous DNA-repair pathways (e.g., homology-dependent repair or non-homologous end joining or alternative non-homologous end joining (A-NHEJ) or microhomology-mediated end joining). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the site of cleavage, and can lead to disruption or alteration of gene expression. HDR can occur when a homologous repair template, or donor, is available. The homologous donor template can comprise sequences that can be homologous to sequences flanking the target nucleic acid cleavage site. The sister chromatid can be used by the cell as the repair template. However, for the purposes of genome editing, the repair template can be supplied as an exogenous nucleic acid, such as a plasmid, duplex oligonucleotide, single-strand oligonucleotide, double-stranded oligonucleotide, or viral nucleic acid. With exogenous donor templates, an additional nucleic acid sequence (such as a transgene) or modification (such as a single or multiple base change or a deletion) can be introduced between the flanking regions of homology so that the additional or altered nucleic acid sequence also becomes incorporated into the target locus. MMEJ can result in a genetic outcome that is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few basepairs flanking the cleavage site to drive a favored end-joining DNA repair outcome. In some instances it may be possible to predict likely repair outcomes based on analysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. An exogenous polynucleotide sequence is termed a donor polynucleotide (or donor or donor sequence or polynucleotide donor template) herein. The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not naturally occur at the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocations and/or gene mutation. The processes of deleting genomic DNA and integrating non-native nucleic acid into genomic DNA are examples of genome editing.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic locus can be found in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, the CRISPR locus encodes products that function as a type of immune system to help defend the prokaryotes against foreign invaders, such as virus and phage. There are three stages of CRISPR locus function: integration of new sequences into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invader nucleic acid. Five types of CRISPR systems (e.g., Type I, Type II, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referred to as ârepeats.â When expressed, the repeats can form secondary structures (e.g., hairpins) and/or comprise unstructured single-stranded sequences. The repeats usually occur in clusters and frequently diverge between species. The repeats are regularly interspaced with unique intervening sequences referred to as âspacers,â resulting in a repeat-spacer-repeat locus architecture. The spacers are identical to or have high homology with known foreign invader sequences. A spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit. A crRNA comprises a âseedâ or spacer sequence that is involved in targeting a target nucleic acid (in the naturally occurring form in prokaryotes, the spacer sequence targets the foreign invader nucleic acid). A spacer sequence is located at the 5â² or 3â² end of the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPR Associated (Cas) genes. Cas genes encode endonucleases involved in the biogenesis and the interference stages of crRNA function in prokaryotes. Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNaseIII, and then hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous RNaseIII can be recruited to cleave the pre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimming to produce the mature crRNA form (e.g., 5â² trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid in a Type II CRISPR system is referred to as a protospacer adjacent motif (PAM). In nature, the PAM is essential to facilitate binding of a site-directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B (CASS4a). Jinek et al., Science, 337 (6096): 816-821 (2012) showed that the CRISPR/Cas9 system is useful for RNA-programmable genome editing, and international patent application publication number WO2013/176772 provides numerous examples and applications of the CRISPR/Cas endonuclease system for site-specific gene editing.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG. 1 of Fonfara et al., Nucleic Acids Research, 42:2577-2590 (2014). The CRISPR/Cas gene naming system has undergone extensive rewriting since the Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAM sequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing to cleave DNA. The site-directed nuclease or polypeptide can be administered to a cell or a patient as either: one or more polypeptides, or one or more mRNAs encoding the polypeptide.

In the context of a CRISPR/Cas system, the site-directed polypeptide can bind to a guide RNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed. In the CRISPR/Cas system disclosed herein, the site-directed polypeptide can be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprises a plurality of nucleic acid-cleaving (i.e., nuclease) domains. Two or more nucleic acid-cleaving domains can be linked together via a linker. For example, the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type

CLAIMS

Claims ( 85 )

What is claimed is:

1 . A composition comprising CD34+ human hematopoietic stem and progenitor cells (hHSPCs) that comprise a genetic modification within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene.

2 . The composition of claim 1 , further comprising a cryopreservation medium substantially free of serum, 5% dimethylsulfoxide (DMSO), and/or dextran-40.

3 . The composition of claim 1 or 2 , wherein the genetic modification comprises an indel.

4 . The composition of any one of claims 1-3 , wherein the genetic modification is producing by delivering to CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) that targets the +58 DHS of a human BCL11A gene.

5 . The composition of claim 4 , wherein the gRNA comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

6 . The composition of claim 5 , wherein the gRNA comprises three 2â²-O-methyl-phosphorothioate residues at or near each of its 5â² and 3â² ends.

7 . The composition of any one of claims 4-6 , wherein the Cas9 endonuclease is an S. pyogenes Cas9 endonuclease or variant thereof.

8 . The composition of any one of claims 1-7 , wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î±)-globin mRNA ratios of 0.1 to 0.5 and/or wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î²)-globin mRNA ratios of 0.2 to 0.6.

9 . The composition of any one of claims 1-8 , wherein the modified CD34+ hHSPCs exhibit a HbF mean percentage of HbF/(HbF+HbA) protein levels of 15% to 50%.

10 . The composition of any one of claims 1-9 , wherein the modified CD34+ hHSPCs exhibit a mean allele editing frequency of 70% to 90%.

11 . The composition of any one of claims 1-10 , wherein at least 75% of the modified CD34+ hHSPCs maintain multi-lineage potential for at least sixteen weeks after administration of the modified CD34+ hHSPCs to a subject.

12 . The composition of any one of claims 1-11 , wherein the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 40%.

13 . The composition of claim 12 , wherein the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 80%.

14 . The composition of any one of claims 1-13 , wherein the modified CD34+ hHSPCs exhibit an off-target indel rate of less than 5%.

15 . The composition of any one of claims 1-14 , wherein the modified CD34+ hHSPCs exhibit an off-target indel rate of less than 1%.

16 . The composition of any one of claims 1-15 comprising a single dose of the modified CD34+ hHSPCs.

17 . The composition of claim 16 , wherein the single dose comprises at least at least 2Ã10 6 modified CD34+ hHSPCs/kg.

18 . The composition of any one of claims 1-17 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a S. pyogenes Cas9 endonuclease or a variant thereof comprising a N-terminal SV40 nuclear localization signal (NLS).

19 . The composition of claim 18 , wherein the S. pyogenes Cas9 endonuclease or a variant thereof further comprises a C-terminal NLS, optionally a C-terminal SV40 NLS.

20 . The composition of claim 18 or 19 further comprising a gRNA (gRNA) comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

21 . The composition of claim 20 , wherein the weight ratio of the gRNA to said endonuclease is 1:1.

22 . A method comprising administering to a subject having a hemoglobinopathy a dose of CD34+ human hematopoietic stem and progenitor cells (hHSPCs) that comprise a genetic modification within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human B-cell lymphoma 11A (BCL11A) gene, wherein the hHSPCs are administered in an effective amount to reduce the number of blood transfusions administered to the subject relative to baseline and/or to increase fetal hemoglobin (HbF) levels in the subject to at least 20%.

23 . A method comprising:

(a) mobilizing stem cells in a subject having a hemoglobinopathy; (b) collecting CD34+ hHSPCs from the subject after step (a); (c) producing modified CD34+ hHSPCs that comprise a genetic modification in within a +58 DNase I hypersensitive site (DHS) within the erythroid lineage-specific enhancer of a human BCL11A gene; and (d) administering to the subject a dose of the modified CD34+ hHSPCs of step (c) in an effective amount to reduce the number of blood transfusions administered to the subject relative to baseline and/or to increase fetal hemoglobin (HbF) levels in the subject to at least 20%.

24 . The method of claim 23 , wherein step (a) comprises administering an inhibitor of CXCR4 chemokine receptor, optionally wherein the inhibitor of CXCR4 chemokine receptor is Plerixafor, to the subject.

25 . The method of claim 23 or 24 , wherein step (a) further comprises administering granulocyte colony stimulating factor to the subject.

26 . The method of any one of claims 22-24 further comprising administering red blood cells to the subject.

27 . The method of claim 26 , wherein the red blood cells are administered before step (a) and/or after step (b).

28 . The method of any one of claims 23-27 , wherein at least 15Ã10 6 CD34+ hHSPCs/kg are collected in step (b).

29 . The method of any one of claims 22-28 further comprising administering busulfan to the subject.

30 . The method of claim 29 , wherein the busulfan is administered after step (c) and before step (d).

31 . The method of claim 30 , wherein an intravenous 4 mg/kg to 5 mg/kg dose of busulfan is administered daily for four days or an intravenous 0.5 mg/kg to 1 mg/kg dose is administered every six hours for four days.

32 . The method of claim 30 , wherein the dose is adjusted based on pharmacokinetic level to achieve an area under the curve (AUC) of 4500 to 5500 Î¼M/min, preferably 5000 Î¼M/min.

33 . The method of any one of claims 22-32 , wherein the genetic modification is an indel.

34 . The method of any one of claims 22-31 , wherein the genetic modification is produced by delivering to the CD34+ hHSPCs a Cas9 endonuclease and a guide RNA (gRNA) that targets the +58 DHS of a human BCL11A gene.

35 . The method of claim 34 , wherein the gRNA comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

36 . The method of claim 35 , wherein the gRNA comprises three 2â²-O-methyl-phosphorothioate residues at or near each of its 5â² and 3â² ends.

37 . The method of any one of claims 34-36 , wherein the Cas9 endonuclease is an S. pyogenes Cas9 endonuclease.

38 . The method of any one of claims 22-37 , wherein neutrophil engraftment occurs in the subject within 35-45 days after administration of the modified CD34+ hHSPCs.

39 . The method of claim 38 , wherein neutrophil engraftment occurs in the subject within 42 days after administration of the modified CD34+ hHSPCs.

40 . The method of any one of claims 22-39 , wherein the hemoglobinopathy is Î²-thalassemia.

41 . The method of any one of claims 22-40 , wherein the subject requires fewer blood transfusions within a two-year period of the time of administration of the modified CD34+ hHSPCs, or within a two-year period after the time of administration of the modified CD34+ hHSPCs, relative to a two-year period before the time of administration of the modified CD34+ hHSPCs.

42 . The method of any one of claims 22-41 , wherein the subject achieves transfusion reduction or transfusion independence for at least three months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs.

43 . The method of any one of claims 22-42 , wherein the subject achieves transfusion reduction or transfusion independence for at least six months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs.

44 . The method of any one of claims 22-43 , wherein the subject achieves transfusion reduction or transfusion independence for at least twelve months following administration of the modified CD34+ hHSPCs starting three months after administration of the modified CD34+ hHSPCs.

45 . The method of any one of claims 22-44 , wherein in the subject there is a change in patient reported outcomes (PROs) over time using at least one of the following assays selected from: Pain scale (11 point numerical rating scale [NRS]), EuroQol Quality of Life Scale (EQ 5D 5L), functional assessment of cancer therapy-bone marrow transplant (FACT-BMT), Patient-reported Outcome Measurement Information System (PROMIS)-Fatigue, PROMIS-Cognitive function, and Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me).

46 . The method of any one of claims 22-45 , wherein in the subject there is a decrease in parameters of iron overload relative to baseline as assessed by magnetic resonance imaging (MRI).

47 . The method of any one of claims 22-46 , wherein in the subject there is a decrease in parameters of iron overload relative to baseline as assessed by change in serum ferritin level over time.

48 . The method of claim 46 or 47 , wherein the decrease in parameters of iron overload includes a decrease in liver iron concentration (LIC) and/or cardiac iron content (CIC).

49 . The method of any one of claims 22-48 , wherein the subject is no longer in need of iron chelation therapy within a 2 to 5 year period of the time of administration of the modified CD34+ hHSPCs, or within a 2 to 5 year period after the time of administration of the modified CD34+ hHSPCs, relative to a 2 to 5 year period before the time of administration of the modified CD34+ hHSPCs.

50 . The method of any one of claims 22-39 , wherein the hemoglobinopathy is sickle cell disease.

51 . The method of any one of claim 22-39 or 50 , wherein the HbF level in the subject is at least 20% for at least three months starting at any time at or after the time of administration of the modified CD34+ hHSPCs.

52 . The method of any one of claim 22-39 or 50-51 , wherein the HbF level in the subject is at least 20% for at least six months starting at any time from at the time of administration of the modified CD34+ hHSPCs.

53 . The method any one of claim 22-39 or 50-52 , wherein the HbF level in the subject is at least 20% for at least three months starting three months after administration of the modified CD34+ hHSPCs.

54 . The method any one of claim 22-39 or 50-53 , wherein the HbF level in the subject is at least 20% for at least three months starting six months after administration of the modified CD34+ hHSPCs.

55 . The method of any one of claim 22-39 or 50-54 , wherein the HbF level in the subject is at least 20% in the absence of treatment with a secondary drug.

56 . The method of claim 55 , wherein said secondary drug is hydroxyurea (HU).

57 . The method of any one of claims any one of claim 22-39 or 50-56 , wherein there is a relative change in annualized rate of severe vaso-occlusive crises (VOC) from baseline, starting six months after administration of the modified CD34+ hHSPCs.

58 . The method of claim 57 , wherein there is a reduction in annualized rate of VOC from baseline by at least 50%, starting six months after administration of the modified CD34+ hHSPCs.

59 . The method of claim 57 or 58 , wherein there is an absence of VOC for at least 12 months, starting six months after administration of the modified CD34+ hHSPCs.

60 . The method of claim 59 , wherein there is an absence of VOC for at least 24 months, starting six months after administration of the modified CD34+ hHSPCs.

61 . The method of any one of claim 22-39 or 50-60 , wherein in the subject there is a change in patient reported outcomes (PROs) over time using at least one of the following assays selected from: Pain scale (11 point numerical rating scale [NRS]), EuroQol Quality of Life Scale (EQ 5D 5L), functional assessment of cancer therapy-bone marrow transplant (FACT-BMT), Patient-reported Outcome Measurement Information System (PROMIS)-Fatigue, PROMIS-Cognitive function, and Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me).

62 . The method of any one of claim 22-39 or 50-61 , wherein in the subject there is a change in hemolytic index as measured by principal component analysis of the following four markers of hemolysis over time: reticulocyte count, serum concentrations of aspartate transaminase, lactate dehydrogenase [LDH], and total bilirubin.

63 . The method of any one of claim 22-39 or 50-62 , wherein in the subject there is a change in tricuspid regurgitant jet velocity (TRV) over time.

64 . The method of any one of claim 22-39 or 50-63 further comprising administering red blood cells to the subject, wherein the red blood cells are administered before the step of administering the modified CD34+ hHSPCs.

65 . The method of any one of claim 22-39 or 50-64 , wherein the subject has received a red blood cell (RBC) transfusion before the step of administering the modified CD34+ hHSPCs.

66 . The method of any one of claim 22-39 or 50-65 , wherein the subject has a hemoglobin S (HbS) level of less than 30% of total Hb.

67 . The method of any one of claim 22-39 or 50-66 , wherein the subject has a total Hb concentration of 11 g/dL or less.

68 . The method of any one of claims 22-67 , wherein in the subject there is an increase, optionally at least a 10% increase, in the proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) over a period of time.

69 . The method of any one of claims 22-68 , wherein in the subject there is a change in inflammatory and endothelial activation markers over a period of time.

70 . The method of claim 69 , wherein in the subject there is a change in the proportion of alleles with the genetic modification present in peripheral blood leukocytes over a period of time.

71 . The method of any one of claims 22-70 , wherein in the subject there is a change in the proportion of alleles with the genetic modification present in bone marrow cells over a period of time.

72 . The method of any one of claims 68-71 , wherein the period of time is at least three months following administration of the modified CD34+ hHSPCs.

73 . The method of claim 72 , wherein the period of time is at least six months following administration of the modified CD34+ hHSPCs.

74 . The method of any one of claims 22-73 , wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î±)-globin mRNA ratios of 0.1 to 0.5 and/or wherein the modified CD34+ hHSPCs exhibit an increase in Î³/(Î³+Î²)-globin mRNA ratios of 0.2 to 0.6.

75 . The method of any one of claims 22-74 , wherein the modified CD34+ hHSPCs exhibit a HbF mean percentage of HbF/(HbF+HbA) protein levels of 15% to 50%.

76 . The method of any one of claims 22-75 , wherein the modified CD34+ hHSPCs exhibit a ratio of (Î³+Î²)/Î±-globin mRNA that is at or above 0.4.

77 . The method of any one of claims 22-76 , wherein the modified CD34+ hHSPCs exhibit a mean allele editing frequency of 70% to 90%.

78 . The method of any one of claims 22-77 , wherein at least 50% of the modified CD34+ hHSPCs maintain multi-lineage potential for at least sixteen weeks after administration of the modified CD34+ hHSPCs.

79 . The method of any one of claims 22-78 , wherein the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 40%.

80 . The method of claim 79 , wherein the modified CD34+ hHSPCs exhibit an on-target indel rate of at least 80%.

81 . The method of any one of claims 22-80 , wherein the subject does not exhibit neoplastic and/or myeloproliferative lesions resulting from administration of the modified CD34+ hHSPCs.

82 . The method of any one of claims 22-81 , wherein the dose is at least 2Ã10 6 modified CD34+ hHSPCs/kg.

83 . The method of claim 82 , wherein the dose is at least 3Ã10 6 to modified CD34+ hHSPCs/kg.

84 . The method of any one of claims 22-83 further comprising administering plerixafor to the subject, wherein red blood cells are administered before the step of administering the modified CD34+ hHSPCs.

85 . The method of any one of claims 22-84 further comprising administering a granulocyte colony stimulating factor to the subject.

US18/929,289

2017-12-05

2024-10-28

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and Uses Thereof

Pending

US20250041349A1

( en )

Priority Applications (1)

Application Number

Priority Date

Filing Date

Title

US18/929,289

US20250041349A1

( en )

2017-12-05

2024-10-28

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and Uses Thereof

Applications Claiming Priority (8)

Application Number

Priority Date

Filing Date

Title

US201762594689P

2017-12-05

US201862664023P

2018-04-27

US201862671770P

2018-05-15

US201862734543P

2018-09-21

US201862734431P

2018-09-21

PCT/US2018/063973

WO2019113149A1

( en )

2017-12-05

2018-12-05

Crispr-cas9 modified cd34+ human hematopoietic stem and progenitor cells and uses thereof

US202016769926A

2020-06-04

US18/929,289

US20250041349A1

( en )

2017-12-05

2024-10-28

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and Uses Thereof

Related Parent Applications (2)

Application Number

Title

Priority Date

Filing Date

PCT/US2018/063973

Continuation

WO2019113149A1

( en )

2017-12-05

2018-12-05

Crispr-cas9 modified cd34+ human hematopoietic stem and progenitor cells and uses thereof

US16/769,926

Continuation

US12161674B2

( en )

2017-12-05

2018-12-05

CRISPR-CAS9 modified CD34+ human hematopoietic stem and progenitor cells and uses thereof

Publications (1)

Publication Number

Publication Date

US20250041349A1

true

US20250041349A1 ( en )

2025-02-06

Family

ID=64902426

Family Applications (2)

Application Number

Title

Priority Date

Filing Date

US16/769,926

Active

2040-11-04

US12161674B2

( en )

2017-12-05

2018-12-05

CRISPR-CAS9 modified CD34+ human hematopoietic stem and progenitor cells and uses thereof

US18/929,289

Pending

US20250041349A1

( en )

2017-12-05

2024-10-28

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and Uses Thereof

Family Applications Before (1)

Application Number

Title

Priority Date

Filing Date

US16/769,926

Active

2040-11-04

US12161674B2

( en )

2017-12-05

2018-12-05

CRISPR-CAS9 modified CD34+ human hematopoietic stem and progenitor cells and uses thereof

Country Status (13)

Country

Link

( 2 )

US12161674B2

( en )

( 1 )

EP3720473A1

( en )

( 1 )

JP2021505587A

( en )

( 2 )

KR20200106159A

( en )

( 1 )

CN111629747A

( en )

( 1 )

AU2018378479B2

( en )

( 1 )

BR112020011255A2

( en )

( 1 )

CA3084955A1

( en )

( 1 )

IL275037A

( en )

( 1 )

MA51107A

( en )

( 1 )

MX2020005849A

( en )

( 1 )

SG11202005147WA

( en )

( 1 )

WO2019113149A1

( en )

Families Citing this family (15)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

JP6974349B2

( en )

2016-04-18

2021-12-01

ã¯ãªã¹ãã¼ ã»ã©ãã¥ã¼ãã£ã¯ã¹ ã¢ã¯ãã§ã³ã²ã¼ã«ã·ã£ãã

Materials and methods for the treatment of hemoglobin abnormalities

WO2019014564A1

( en )

2017-07-14

2019-01-17

Editas Medicine, Inc.

Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites

US12522811B2

( en )

2018-05-01

2026-01-13

The Children's Medical Center Corporation

Enhanced BCL11A RNP / CRISPR delivery and editing using a 3XNLS-CAS9

IL282290B1

( en )

2018-10-16

2026-04-01

Blueallele Llc

Methods for targeted insertion of dna in genes

WO2021067233A1

( en )

2019-09-30

2021-04-08

The Children's Medical Center Corporation

Bcl11a guide and base editor delivery

GB202017555D0

( en )

2020-11-06

2020-12-23

Harbour Antibodies Bv

Antibody-conjugated nanoparticles

AU2021397627A1

( en )

2020-12-08

2023-07-20

Todd Frank OVOKAITYS

Methods and systems for increased production of stem cells

AU2021413252A1

( en )

2020-12-31

2023-06-08

Vor Biopharma Inc.

Compositions and methods for cd34 gene modification

WO2022240787A1

( en )

2021-05-13

2022-11-17

The Children's Medical Center Corporation

Methods for stratifying subjects for fetal hemoglobin reinduction

US20250381304A1

( en )

2022-06-21

2025-12-18

Crispr Therapeutics Ag

Methods and compositions for in vivo editing of stem cells

US20250368954A1

( en )

2022-10-05

2025-12-04

Stratus Therapeutics, Inc.

Myeloid lineages derived from pluripotent cells

WO2024211616A1

( en )

2023-04-04

2024-10-10

Ovokaitys Todd Frank

Methods and systems for improved therapies of genetic diseases using photo-activated allogenic stem cells

CN117338902B

( en )

2023-10-25

2024-07-09

åäº¬å»ç§å¤§å¦

A compound preparation for treating egg maturation disorder

WO2025094917A1

( en )

2023-10-30

2025-05-08

ï¼£ï¼ï¼µæ ªå¼ä¼ç¤¾

Method for producing hematopoietic stem cells in which bcl11a gene enhancer is partially or entirely destroyed, and kit for use in the method

TW202606758A

( en )

2024-05-31

2026-02-16

ç¾åç¶æ³°åæ¯è£½è¥å¬å¸

Anti-cd74 antibody compositions and methods

Family Cites Families (148)

* Cited by examiner, â Cited by third party

Publication number

Priority date

Publication date

Assignee

Title

US3687808A

( en )

1969-08-14

1972-08-29

Univ Leland Stanford Junior

Synthetic polynucleotides

US4469863A

( en )

1980-11-12

1984-09-04

Ts O Paul O P

Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof

US5023243A

( en )

1981-10-23

1991-06-11

Molecular Biosystems, Inc.

Oligonucleotide therapeutic agent and method of making same

US4476301A

( en )

1982-04-29

1984-10-09

Centre National De La Recherche Scientifique

Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon

JPS5927900A

( en )

1982-08-09

1984-02-14

Wakunaga Seiyaku Kk

Oligonucleotide derivative and its preparation

FR2540122B1

( en )

1983-01-27

1985-11-29

Centre Nat Rech Scient

NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION

US4605735A

( en )

1983-02-14

1986-08-12

Wakunaga Seiyaku Kabushiki Kaisha

Oligonucleotide derivatives

US4948882A

( en )

1983-02-22

1990-08-14

Syngene, Inc.

Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis

US4824941A

( en )

1983-03-10

1989-04-25

Julian Gordon

Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems

US4587044A

( en )

1983-09-01

1986-05-06

The Johns Hopkins University

Linkage of proteins to nucleic acids

US5118802A

( en )

1983-12-20

1992-06-02

California Institute Of Technology

DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside

US5550111A

( en )

1984-07-11

1996-08-27

Temple University-Of The Commonwealth System Of Higher Education

Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof

US5367066A

( en )

1984-10-16

1994-11-22

Chiron Corporation

Oligonucleotides with selectably cleavable and/or abasic sites

US5430136A

( en )

1984-10-16

1995-07-04

Chiron Corporation

Oligonucleotides having selectably cleavable and/or abasic sites

US5258506A

( en )

1984-10-16

1993-11-02

Chiron Corporation

Photolabile reagents for incorporation into oligonucleotide chains

US4828979A

( en )

1984-11-08

1989-05-09

Life Technologies, Inc.

Nucleotide analogs for nucleic acid labeling and detection

FR2575751B1

( en )

1985-01-08

1987-04-03

Pasteur Institut

NOVEL ADENOSINE DERIVATIVE NUCLEOSIDES, THEIR PREPARATION AND THEIR BIOLOGICAL APPLICATIONS

US5034506A

( en )

1985-03-15

1991-07-23

Anti-Gene Development Group

Uncharged morpholino-based polymers having achiral intersubunit linkages

US5185444A

( en )

1985-03-15

1993-02-09

Anti-Gene Deveopment Group

Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages

US5235033A

( en )

1985-03-15

1993-08-10

Anti-Gene Development Group

Alpha-morpholino ribonucleoside derivatives and polymers thereof

US5405938A

( en )

1989-12-20

1995-04-11

Anti-Gene Development Group

Sequence-specific binding polymers for duplex nucleic acids

US5166315A

( en )

1989-12-20

1992-11-24

Anti-Gene Development Group

Sequence-specific binding polymers for duplex nucleic acids

US4762779A

( en )

1985-06-13

1988-08-09

Amgen Inc.

Compositions and methods for functionalizing nucleic acids

US5317098A

( en )

1986-03-17

1994-05-31

Hiroaki Shizuya

Non-radioisotope tagging of fragments

JPS638396A

( en )

1986-06-30

1988-01-14

Wakunaga Pharmaceut Co Ltd

Poly-labeled oligonucleotide derivative

US5264423A

( en )

1987-03-25

1993-11-23

The United States Of America As Represented By The Department Of Health And Human Services

Inhibitors for replication of retroviruses and for the expression of oncogene products

US5276019A

( en )

1987-03-25

1994-01-04

The United States Of America As Represented By The Department Of Health And Human Services

Inhibitors for replication of retroviruses and for the expression of oncogene products

US4904582A

( en )

1987-06-11

1990-02-27

Synthetic Genetics

Novel amphiphilic nucleic acid conjugates

DE3851889T2

( en )

1987-06-24

1995-04-13

Florey Howard Inst

NUCLEOSIDE DERIVATIVES.

US5585481A

( en )

1987-09-21

1996-12-17

Gen-Probe Incorporated

Linking reagents for nucleotide probes

US4924624A

( en )

1987-10-22

1990-05-15

Temple University-Of The Commonwealth System Of Higher Education

2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof

US5188897A

( en )

1987-10-22

1993-02-23

Temple University Of The Commonwealth System Of Higher Education

Encapsulated 2',5'-phosphorothioate oligoadenylates

US5525465A

( en )

1987-10-28

1996-06-11

Howard Florey Institute Of Experimental Physiology And Medicine

Oligonucleotide-polyamide conjugates and methods of production and applications of the same

DE3738460A1

( en )

1987-11-12

1989-05-24

Max Planck Gesellschaft

MODIFIED OLIGONUCLEOTIDS

US5082830A

( en )

1988-02-26

1992-01-21

Enzo Biochem, Inc.

End labeled nucleotide probe

WO1989009221A1

( en )

1988-03-25

1989-10-05

University Of Virginia Alumni Patents Foundation

Oligonucleotide n-alkylphosphoramidates

US5278302A

( en )

1988-05-26

1994-01-11

University Patents, Inc.

Polynucleotide phosphorodithioates

US5109124A

( en )

1988-06-01

1992-04-28

Biogen, Inc.

Nucleic acid probe linked to a label having a terminal cysteine

US5216141A

( en )

1988-06-06

1993-06-01

Benner Steven A

Oligonucleotide analogs containing sulfur linkages

US5175273A

( en )

1988-07-01

1992-12-29

Genentech, Inc.

Nucleic acid intercalating agents

US5262536A

( en )

1988-09-15

1993-11-16

E. I. Du Pont De Nemours And Company

Reagents for the preparation of 5'-tagged oligonucleotides

US5512439A

( en )

1988-11-21

1996-04-30

Dynal As

Oligonucleotide-linked magnetic particles and uses thereof

US5599923A

( en )

CRISPR-CAS9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells and … — Vertex Pharmaceuticals Incorporated (US20250041349A1)

Related documents