JP2023521524A - Modified cells and methods for the treatment of hemoglobinopathies - Google Patents

Abstract

Genome editing system, guide RNA and CRISPR-mediated modification for intracellular modification of a portion of the HBG1 and HBG2 loci, a portion of the erythrocyte-specific enhancer of the BCL11A gene, or a combination thereof, and increased expression of fetal hemoglobin. A method is provided.

Description

PRIORITY CLAIM This application is based on U.S. Provisional Patent Application No. 62/945,190, filed Dec. 8, 2019, both of which are hereby incorporated by reference in their entireties, including drawings. and claims priority from U.S. Provisional Patent Application No. 63/115,518, filed November 18, 2020.

SEQUENCE LISTING This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web, the entire contents of which are incorporated by reference. The ASCII copy created on Dec. 8, 2020 is SequenceListing. txt and has a size of 699 KB.

The present disclosure provides genome editing systems and methods for modifying a target nucleic acid sequence or modulating expression of a target nucleic acid sequence and those related to modification of genes encoding hemoglobin subunits and/or treatment of hemoglobinopathies. Regarding the use of

Hemoglobin (Hb) carries oxygen in red blood cells (RBCs) from the lungs to the tissues. During prenatal development and shortly after birth, hemoglobin is transformed into fetal hemoglobin (HbF), a tetrameric protein composed of two alpha (α)-globin chains and two gamma (γ)-globin chains. Exists in form. Through a process known as globin switching, HbF is largely replaced by adult hemoglobin (HbA), a tetrameric protein in which the γ-globin chains of HbF are replaced by beta (β)-globin chains. . The average adult makes less than 1% HbF from total hemoglobin (Thein 2009). The α-hemoglobin gene resides on chromosome 16, while the β-hemoglobin gene (HBB), the Aγ-globin chain (HBG1, also known as γ-globin A) and the Gγ-globin chain (HBG2, also known as γ-globin G) ) is located on chromosome 11 within the globin gene cluster (also called the globin locus).

Mutations in HBB can lead to hemoglobinopathies (ie, hemoglobinopathies), including sickle cell disease (SCD) and β-thalassemia (β-Thal). Approximately 93,000 people in the United States have been diagnosed with hemoglobinopathies. Worldwide, 300,000 children are born with hemoglobinopathies each year (Angastiniotis 1998). Since these pathologies are associated with HBB mutations, these symptoms typically do not become apparent until globin switches from HbF to HbA.

SCD is the most common inherited blood disorder in the United States, affecting approximately 80,000 people (Brousseau 2010). SCD is most common in people of African ancestry, with a prevalence of SCD of 1 in 500 people. In Africa, the prevalence of SCD is 15 million (Aliyu 2008). SCD is also more common in people of Indian, Saudi and Mediterranean descent. Among Hispanic Americans, the prevalence of sickle cell disease is 1 in 1,000 (Lewis 2014).

SCD is the c. Caused by one homozygous mutation of 17A>T (HbS mutation). A sickle mutation is a point mutation (GAG>GTG) on HBB that results in a substitution of valine for glutamic acid at amino acid position 6 of exon 1. The valine at position 6 of the β-hemoglobin chain is hydrophobic and causes a conformational change in the β-globin protein when not oxygen bound. This conformational change causes polymerization of the HbS protein in the absence of oxygen, resulting in RBC deformation (ie, sickling). SCD is inherited autosomal recessively, so only patients with two HbS alleles have the disease. Heterozygous subjects have sickle cell trait and may suffer from anemia and/or painful episodes if left severely dehydrated or oxygen deprived.

Sickle RBCs cause multiple symptoms including anemia, sickle cell crisis, episodes of vascular occlusion, aplastic episodes and acute chest syndrome. Sickle-shaped RBCs are less elastic than wild-type erythrocytes and cannot easily pass through capillary beds, causing obstruction and ischemia (ie, vascular occlusion). Vascular occlusion occurs when sickle cells block blood flow in the capillary beds of an organ, resulting in pain, ischemia and necrosis. These episodes typically last 5-7 days. The spleen plays a role in removing dysfunctional RBCs and is therefore typically enlarged in early childhood and frequently suffers episodes of vascular occlusion. By the end of childhood, the spleen of SCD patients is often infarcted, leading to splenic crush. Hemolysis is a constant feature of SCD and leads to anemia. Sickle cells survive 10-20 days in circulation, while healthy red blood cells survive 90-120 days. SCD subjects are transfused as needed to maintain adequate hemoglobin levels. Frequent blood transfusions put subjects at risk for HIV, hepatitis B and hepatitis C infection. Subjects may also suffer from acute chest attacks and infarcts of the extremities, end organs and central nervous system.

Subjects with SCD have a reduced life expectancy. The prognosis of patients with SCD is steadily improving with careful lifelong management of onset and anemia. As of 2001, life expectancy for subjects with sickle cell disease is in the mid to late fifties. Current treatment of SCD involves hydration and pain control during episodes and blood transfusions to correct anemia as needed.

Thalassemias (eg β-Thal, δ-Thal and β/δ-Thal) cause chronic anemia. β-Thal is estimated to affect approximately 1 in 100,000 people worldwide. Its prevalence is high in certain populations, including Europeans, with a prevalence of approximately 1 in 10,000. β-Thal major is the more severe form and is life threatening unless treated with lifelong blood transfusions and chelation therapy. In the United States, there are about 3,000 subjects with β-Thal major. β-Thal intermediate does not require transfusions, but it can cause growth retardation and profound systemic abnormalities, often requiring lifelong chelation therapy. HbA accounts for the majority of hemoglobin in adult RBCs, but about 3% of adult hemoglobin is a HbA variant in which two γ-globin chains are replaced by two δ(Δ)-globin chains. It is the form of HbA2. Delta-Thal is associated with mutations in the delta hemoglobin gene (HBD) that cause loss of HBD expression. Co-inheritance of HBD mutations may mask the diagnosis of β-Thal (ie β/δ-Thal) by reducing the level of HbA2 to the normal range (Bouva 2006). β/δ-Thal is usually caused by deletion of HBB and HBD sequences in both alleles. In homozygous (δo/δo βo/βo) patients, HBG is expressed leading to the production of HbF only.

Like SCD, β-Thal is caused by mutations in the HBB gene. The most common HBB mutations resulting in β-Thal are c. -136C>G, c. 92+1G>A, c. 92+6T>C, c. 93-21G>A, c. 118C>T, c. 316-106C>G, c. 25_26delAA, c. 27_28insG, c. 92+5G>C, c. 118C>T, c. 135 del C, c. 315+1G>A, c. -78A>G, c. 52A>T, c. 59A>G, c. 92+5G>C, c. 124_127delTTCT, c. 316-197C>T, c. -78A>G, c. 52A>T, c. 124_127delTTCT, c. 316-197C>T, c. −138C>T, c. -79A>G, c. 92+5G>C, c. 75T>A, c. 316-2A>G and c. 316-2A>C. These and other mutations associated with β-Thal cause mutations or deletions in the β-globin chain, which cause perturbations in the ratio of normal Hb α-hemoglobin to β-hemoglobin. Excess α-globin chains are deposited in erythroid progenitors in the bone marrow.

In β-Thal major, both alleles of HBB contain a nonsense, frameshift or splicing mutation that results in a complete lack of β-globin production (denoted as β ⁰ /β ⁰ ). β-Thal major results in severe depletion of β-globin chains, leading to marked precipitation of α-globin chains in RBCs and high-severity anemia.

β-Thal intermediates arise from mutations in the 5' or 3' untranslated region of HBB, mutations in the promoter region or polyadenylation signal of HBB, or splicing mutations within the HBB gene. A patient's genotype is designated as βo/β+ or β+/β+. βo represents the absence of expression of β-globin chains; β+ represents dysfunctional but present β-globin chains. Phenotypic expression varies between patients. β-Thal intermediate has some production of β-globin, resulting in less α-globin chain precipitation in erythroid precursors and less severe anemia than β-Thal major. However, proliferation of the erythroid lineage secondary to chronic anemia has more serious consequences.

Subjects with β-Thal major develop between 6 months and 2 years of age and suffer from failure to thrive, fever, hepatosplenomegaly and diarrhea. Appropriate treatment includes regular blood transfusions. Treatment for β-Thal major also includes splenectomy and treatment with hydroxyurea. If patients receive regular blood transfusions, they develop normally into their early twenties. At that point, patients require chelation therapy (in addition to ongoing blood transfusions) to prevent complications of iron overload. Iron overload can be manifested as retarded growth or delayed sexual maturity. In adulthood, inadequate chelation therapy can lead to cardiomyopathy, cardiac arrhythmias, liver fibrosis and/or cirrhosis, diabetes, thyroid and parathyroid abnormalities, thrombosis and osteoporosis. Frequent blood transfusions also put subjects at risk for HIV, hepatitis B and hepatitis C infection.

Subjects with β-Thal intermediate form are generally present between the ages of 2-6 years. Subjects generally do not require blood transfusions. However, bone abnormalities occur due to chronic hypertrophy of the erythroid lineage to compensate for chronic anemia. The subject may also have a long bone fracture due to osteoporosis. Extramedullary erythropoiesis is common, resulting in swelling of the spleen, liver and lymph nodes. This can lead to spinal cord compression and neurological problems. Subjects also suffer from leg ulcers and are at increased risk of thrombotic events including stroke, pulmonary embolism and deep vein thrombosis. Treatments for β-Thal intermediate include splenectomy, folic acid supplementation, hydroxyurea therapy, and radiation therapy for extramedullary masses. Chelation therapy is used in subjects who develop iron overload.

β-Thal patients often have a reduced life expectancy. Subjects with β-Thal major who do not receive transfusion therapy generally die in their twenties or thirties. Subjects with β-Thal major who receive regular blood transfusions and appropriate chelation therapy can live well into their 50s. Heart failure secondary to iron toxicity is the leading cause of death due to iron toxicity in β-Thal major subjects.

Currently, various new treatments for SCD and β-Thal are being developed. Delivery of the anti-sickling HBB gene via gene therapy is currently being investigated in clinical trials. However, the long-term efficacy and safety of this approach are unknown. Transplantation of hematopoietic stem cells (HSC) from HLA-matched allogeneic stem cell donors has been demonstrated to cure SCD and β-Thal, but this procedure is necessary to prepare the subject for transplantation. It carries risks, including those associated with ablation therapy, and increases the risk of life-threatening opportunistic infections and post-transplant graft-versus-host disease. Moreover, often a matching allogeneic donor cannot be identified. Accordingly, there is a need for improved methods for managing these and other hemoglobinopathies.

Provided herein in certain embodiments is a first population of modified cells comprising a plurality of modified CD34+ or hematopoietic stem cells having one or more indels in the HBG gene promoter. In some of these embodiments, the plurality of modified cells contains an indel in the CCAAT box target region.

In certain embodiments of the first population of engineered cells provided herein, one or more of the cells in the plurality of engineered cells comprise HBG1/2c. Contains deletions from -104 to -121. In some of these embodiments, HBG1/2c. -104 to -121 deletion is ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3%, ≥3.5% of indels in multiple modified cells as a whole , 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, 7.5% or more, 8% or more, 8.5% or more , 9% or more, 9.5% or more, 10% or more, 10.5% or more, 11% or more, 11.5% or more, 12% or more, 12.5% or more, 13% or more, 13.5% or more , 14% or more, 14.5% or more, or 15% or more, or 15.5% or more. In some of these embodiments, HBG1/2c. Deletions from -104 to -121 collectively constitute less than 25% of the indels in multiple modified cells.

In certain embodiments of the first population of modified cells provided herein, 60% or more, 65% or more, 70% or more, 75% or more, 80% of the indels in the plurality of modified cells as a whole Greater than or equal to 85%, 90% or more, or 95% or more are deletions of at least 4 base pairs.

In certain embodiments of the first population of modified cells provided herein, 25% or more, 30% or more, 35% or more, 40% or more, 45% of the indels in the plurality of modified cells as a whole Greater than or equal to 50% or more, 55% or more, 60% or more, or 65% or more are deletions of at least 4 base pairs introduced by a repair mechanism other than microhomology-mediated end joining (MMEJ) repair.

In certain embodiments of the first population of modified cells provided herein, 25% or more, 30% or more, 35% or more, 40% or more, 45% of the indels in the plurality of modified cells as a whole Greater than or equal to 50%, 55% or more, 60% or more, or 65% or more are deletions of at least 4 base pairs introduced by non-homologous end joining (NHEJ) repair, eg, standard NHEJ repair.

In certain embodiments of the first population of modified cells provided herein, 50% or more, 55% or more, 60% or more, 65% or more, 70% of the indels in the plurality of modified cells as a whole Greater than or equal to 75%, 80% or more, 85% or more, 90% or more, or 92% or more are deletions of 1-25 base pairs.

In certain embodiments of the first population of modified cells provided herein, 50% or more, 55% or more, 60% or more, 65% or more, 70% of the indels in the plurality of modified cells as a whole Greater than 75%, 80% or more, or 85% or more are deletions of 3-25 base pairs.

In certain embodiments of the first population of modified cells provided herein, 50% or more, 55% or more, 60% or more, 65% or more, 70% of the indels in the plurality of modified cells as a whole Greater than, 75% or more, or 80% or more are deletions of 4-25 base pairs.

In certain embodiments of the first population of modified cells provided herein, 50% or more, 55% or more, 60% or more, 65% or more, 70% of the indels in the plurality of modified cells as a whole more, or more than 72%, are deletions of 5-25 base pairs.

In certain embodiments of the first population of engineered cells provided herein, the engineered cells comprise a first gRNA comprising a first gRNA targeting domain and a Cpf1 RNA-directed nuclease or a modified Cpf1 RNA-directed nuclease. to a first population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells to generate indels. In some of these embodiments, the first RNP complex is delivered to the first population of unmodified cells by electroporation. In certain embodiments, the first population of unmodified cells is from a subject with sickle cell disease. In certain embodiments, the first gRNA comprises a 5' end and a 3' end and has a DNA extension at the 5' end and a 2'-O-methyl-3'-phosphorothioate modification at the 3' end. . In some of these embodiments, the 5' terminal DNA extension comprises a sequence set forth in any of SEQ ID NOS: 1235-1250. In certain embodiments, the first gRNA targeting domain comprises the sequence set forth in SEQ ID NO:1254. In certain embodiments, the first gRNA comprises the sequence set forth in SEQ ID NO:1051. In certain embodiments, the modified Cpf1 RNA-inducing nuclease comprises the sequence set forth in SEQ ID NO:1097. In certain embodiments, the first population of modified cells has a higher fetal hemoglobin (HbF) level than the first population of unmodified cells.

In certain embodiments of the first population of engineered cells provided herein, one or more of the cells in the plurality of engineered cells has (a) HBG1/HBG1 at the HBG1 promoter, the HBG2 promoter, or both. 2c. -104 to -121 deletion; (b) HBG1/2 c. -110 to -115 deletion; (c) HBG1/2 c. -112 to -115; (d) HBG1/2 c. -113 to -115 deletion; (e) HBG1/2 c. -111 to -115 deletion; (f) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. -111 to -117 deletion; (g) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. -102 to -114 deletion; (h) HBG1/2c in HBG1 promoter, HBG2 promoter, or both. -114 to -118 deletion; (i) HBG1/2c in HBG1 promoter, HBG2 promoter, or both. -112 to -116; or (j) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. Contains deletions from -113 to -117.

In certain embodiments of the first population of engineered cells provided herein, the plurality of engineered cells as a whole comprises (a) HBG1/2c. -104 to -121; and (b) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. Contains deletions from -110 to -115. In certain embodiments, HBG1/2c. -104 to -121 deletion is ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3%, ≥3.5% of indels in multiple modified cells as a whole , 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, 7.5% or more, 8% or more, 8.5% or more , 9% or more, 9.5% or more, 10% or more, 10.5% or more, 11% or more, 11.5% or more, 12% or more, 12.5% or more, 13% or more, 13.5% or more , 14% or more, 14.5% or more, or 15% or more. In certain embodiments, HBG1/2c. -104 to -121 deletions collectively constitute 1% to 15.5% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -110 to -115 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. The −110 to −115 deletions collectively constitute 0.5% to 6% of the indels in multiple modified cells.

In certain embodiments of the first population of engineered cells provided herein, the plurality of engineered cells as a whole comprises (a) HBG1/2c. -104 to -121 deletion; (b) HBG1/2 c. -110 to -115 deletion; (c) HBG1/2 c. -112 to -115; (d) HBG1/2 c. -113 to -115 deletion; (e) HBG1/2 c. -111 to -115 deletion; (f) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. -111 to -117 deletion; (g) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. -102 to -114 deletion; (h) HBG1/2c in HBG1 promoter, HBG2 promoter, or both. -114 to -118 deletion; (i) HBG1/2c in HBG1 promoter, HBG2 promoter, or both. -112 to -116; and (j) HBG1/2c in the HBG1 promoter, HBG2 promoter, or both. Contains deletions from -113 to -117. In certain embodiments, HBG1/2c. -104 to -121 deletion is ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3%, ≥3.5% of indels in multiple modified cells as a whole , 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more, 7.5% or more, 8% or more, 8.5% or more , 9% or more, 9.5% or more, 10% or more, 10.5% or more, 11% or more, 11.5% or more, 12% or more, 12.5% or more, 13% or more, 13.5% or more , 14% or more, 14.5% or more, or 15% or more. In certain embodiments, HBG1/2c. -104 to -121 deletions collectively constitute 1% to 15.5% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -110 to -115 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. The −110 to −115 deletions collectively constitute 0.5% to 6% of the indels in multiple modified cells. In certain embodiments, HBG1/2c. -112 to -115 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −112 to −115 collectively constitute 0.5% to 6% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -113 to -115 deletion is 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. The −113 to −115 deletion collectively constitutes 0.5% to 6% of the indels in multiple modified cells. In certain embodiments, HBG1/2c. -111 to -115 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −111 to −115 collectively constitute 0.5% to 6% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -111 to -117 deletion is 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −111 to −117 collectively constitute 0.5% to 6% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -102 to -114 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −102 to −114 collectively constitute 0.5% to 6% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -114 to -118 deletion is 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. The −114 to −118 deletion collectively constitutes 0.5% to 6% of the indels in multiple modified cells. In certain embodiments, HBG1/2c. -112 to -116 deletion is ≥0.5%, ≥1%, ≥1.5%, ≥2%, ≥2.5%, ≥3% of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −112 to −116 collectively constitute 0.5% to 6% of indels in multiple modified cells. In certain embodiments, HBG1/2c. -113 to -117 deletion is 0.5% or more, 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more of indels in multiple modified cells as a whole , 3.5% or more, 4% or more, 4.5% or more, 5% or more, or 5.5% or more. In certain embodiments, HBG1/2c. Deletions of −113 to −117 collectively constitute 0.5% to 6% of indels in multiple modified cells.

In certain embodiments of the first population of modified cells provided herein, the plurality of modified cells as a whole comprises 108 deletions present in all 14 samples of Table 25.

In certain embodiments of the first population of modified cells provided herein, the plurality of modified cells as a whole is identified in Table 25 as having an "average % in indels" of 0.1% or greater. including indels that are In some of these embodiments, the plurality of modified cells as a whole comprises all of the indels of Table 25.

In certain embodiments of the first population of engineered cells provided herein, the plurality of engineered cells collectively comprises a plurality of engineered CD34+ or hematopoietic stem cells, and a plurality of engineered CD34+ or hematopoietic stem cells and at least 4 base pair deletions at least 10% more than the second population of modified cells with one or more indels in the HBG gene promoter, wherein The indels induced a second RNP complex comprising a second gRNA with a gRNA targeting domain comprising SEQ ID NO: 339 and a Cas9 RNA-guided nuclease into multiple unmodified CD34+ or unmodified cells, including hematopoietic stem cells. Produced by delivering to a population. In some of these embodiments, the second RNP complex is delivered to the second population of unmodified cells by electroporation. In certain embodiments, the second population of unmodified cells is from a subject with sickle cell disease. In certain embodiments, the first population of modified cells has a higher HbF level than the second population of modified cells. In some of these embodiments, the plurality of modified cells in the second population contain indels within the CCAAT box target region.

Provided herein in certain embodiments is the induction of HbF expression in a first population of modified cells comprising a plurality of modified CD34+ or hematopoietic stem cells having one or more indels in the HBG gene promoter. wherein a first RNP complex comprising a first gRNA comprising a first gRNA targeting domain and a Cpf1 RNA-guided nuclease or a modified Cpf1 RNA-guided nuclease is administered to a plurality of unmodified CD34+ or hematopoietic delivering to a first population of unmodified cells comprising stem cells to generate indels. In certain embodiments, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% of the resulting indels in the plurality of modified cells as a whole % or more are deletions of at least 4 base pairs. In certain embodiments, the first population of modified cells exhibits increased HbF levels compared to the first population of unmodified cells. In certain embodiments, the first RNP complex is delivered to the first population of unmodified cells by electroporation.

In certain embodiments, provided herein are red blood cells (RBCs) cultured from a first population of modified cells comprising a plurality of modified CD34+ cells having one or more indels in the HBG gene promoter comprising a first gRNA comprising a first gRNA targeting domain and a Cpf1 RNA-guided nuclease or a modified Cpf1 RNA-guided nuclease. Delivering RNP complexes to a first population of unmodified cells comprising a plurality of unmodified CD34+ cells to generate indels, then culturing a first population of RBCs from the first population of modified cells Including. In certain embodiments, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% of the resulting indels in the plurality of modified cells as a whole % or more are deletions of at least 4 base pairs. In certain embodiments, the first population of RBCs exhibits significantly reduced sickling upon deoxygenation compared to a second population of RBCs cultured from the first population of unmodified cells. In certain embodiments, the first population of RBCs sickles at a significantly lower partial pressure of oxygen than the second population of RBCs, eg, as measured by relative oxygen tension. In certain embodiments, the first population of RBCs has a significantly higher minimal elongation index upon deoxygenation than the second population of RBCs. In certain embodiments, the first population of RBCs has a significantly higher velocity upon deoxygenation than the second population of RBCs. In certain embodiments, the first population of RBCs has a higher HbF level than the second population of RBCs.

Provided herein in certain embodiments are a plurality of first RNP complexes comprising a first gRNA comprising a first targeting domain and a Cpf1 RNA-guided nuclease or modified Cpf1 RNA-guided nuclease. to a first population of unmodified cells comprising unmodified CD34+ or hematopoietic stem cells, a second population of modified cells comprising a plurality of modified CD34+ or hematopoietic stem cells, and containing one or more indels in the HBG gene promoter; generating one population and then administering the resulting first population of modified cells to a subject to alleviate one or more symptoms of sickle cell disease A method of alleviating one or more symptoms of sickle cell disease in a subject. In certain embodiments, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the obtained indels in the plurality of modified CD34+ cells or hematopoietic stem cells obtained. % or more, or 95% or more are deletions of at least 4 base pairs. In certain embodiments, the method is about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks, 1 week after administration. 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, more than 5 years and detecting a population of modified red blood cell progeny cells comprising a plurality of modified red blood cell progeny cells cultured from the first population of modified cells. In certain embodiments, the cultured cells are bone marrow (BM)-engrafted CD34+ hematopoietic stem cells or blood cells derived therefrom, e.g., myeloid progenitor cells or differentiated myeloid cells, e.g., erythrocytes, mast cells, myoblasts; It may also include progenitor cells or differentiated lymphocytic cells such as T lymphocytes or B lymphocytes or NK cells. In certain embodiments, the method comprises, for example, at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, 16 weeks, 20 weeks after administration, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 5 years , resulting in long-term engraftment of multiple HSC clones in bone marrow. In certain embodiments, the method comprises long-term reduction of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of total hemoglobin compared to healthy subjects. bring about expression. In certain embodiments, the method results in reconstitution of all hematopoietic cell lineages, eg, without a differentiation bias, eg, without a differentiation bias of the erythroid lineage.

Provided herein, in certain embodiments, is a cell population comprising a plurality of red blood cells (RBCs) cultured from a plurality of modified CD34+ cells from a subject with sickle cell disease, each modified cell comprising , contains an indel in the HBG gene promoter. In certain embodiments, the plurality of modified cells as a whole comprises 108 deletions present in all 14 samples of Table 25. In certain embodiments, the cell population comprises a RNP complex comprising a gRNA comprising a first targeting domain and a Cpf1 RNA-inducing nuclease or modified Cpf1 RNA-inducing nuclease from a plurality of untreated subjects with sickle cell disease. delivered to modified CD34+ cells to generate indels; manufactured by culturing RBCs from multiple modified CD34+ cells.

Provided herein, in certain embodiments, is a cell population comprising a plurality of red blood cells (RBCs) cultured from a plurality of modified CD34+ cells from a subject with sickle cell disease, each modified cell comprising , contains an indel in the HBG gene promoter. In certain embodiments, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the indels in the HBG gene promoter have at least 4 bases paired deletions. In certain embodiments, the cell population comprises a RNP complex comprising a gRNA comprising a first targeting domain and a Cpf1 RNA-inducing nuclease or modified Cpf1 RNA-inducing nuclease from a plurality of untreated subjects with sickle cell disease. delivered to modified CD34+ cells to generate indels; manufactured by culturing RBCs from multiple modified CD34+ cells.

In certain embodiments, the cell population comprises a plurality of RBCs provided herein, wherein the plurality of RBCs is more than a population of RBCs cultured from unmodified CD34+ cells of a subject with sickle cell disease. For example, a minimal elongation index upon deoxygenation than a population of RBCs cultured from unmodified CD34+ cells of a subject with sickle cell disease, sickling at significantly lower partial pressures of oxygen as measured by relative oxygen tension and/or have significantly higher velocities upon deoxygenation than populations of RBCs cultured from unmodified CD34+ cells of subjects with sickle cell disease.

Genome editing systems for modifying the promoter region of one or more γ-globin genes (e.g., HBG1, HBG2 or HBG1 and HBG2) to increase the expression of fetal hemoglobin (HbF), Cpf1 proteins, including ribonucleoprotein (RNP) complexes, guide RNAs, modified Cpf1 proteins (Cpf1 variants) and CRISPR-mediated methods are provided. In certain embodiments, the RNP complex may comprise a guide RNA (gRNA) complexed with wild-type Cpf1 or a modified Cpf1 RNA-inducing nuclease (modified Cpf1 protein). In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, a gRNA can include a gRNA targeting domain. In certain embodiments, the gRNA targeting domain may comprise a sequence selected from the group consisting of SEQ ID NOs: 1002, 1254, 1258, 1260, 1262 and 1264. In certain embodiments, the gRNA may comprise a gRNA sequence listed in Table 13. In certain embodiments, the gRNA may comprise a sequence selected from the group consisting of SEQ ID NOs: 1022, 1023, 1041-1105. In certain embodiments, the RNP complexes can include RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15).

The inventors have discovered herein that delivery of RNP complexes comprising gRNA complexed to modified Cpf1 proteins can result in increased editing of target nucleic acids. In certain embodiments, a modified Cpf1 protein may contain one or more modifications. In certain embodiments, the one or more modifications include, without limitation, one or more mutations of the wild-type Cpf1 amino acid sequence, one or more mutations of the wild-type Cpf1 nucleic acid sequence, nuclear localization One or more mutations in the signal (NLS), one or more mutations in the purification tag (eg His tag) or combinations thereof may be included. In certain embodiments, the modified Cpf1 comprises SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110-17 ( Cpf1 polynucleotide sequences). In certain embodiments, the gRNA can be modified or unmodified gRNA. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, RNP complexes comprising modified Cpf1 proteins can increase editing of target nucleic acids. In certain embodiments, RNP complexes containing modified Cpf1 proteins can increase editing resulting in increased productive indels. In various embodiments, increased editing of the target nucleic acid includes, but is not limited to, PCR amplification of the target nucleic acid followed by sequence analysis (e.g., Sanger sequencing, next generation sequencing). can be evaluated by any means known to those skilled in the art.

The inventors have also discovered herein that delivery of RNP complexes comprising modified gRNA complexed to unmodified or modified Cpf1 protein can result in increased editing of target nucleic acids. In certain embodiments, the modified gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2′-O-methyl modifications, one or more, or one A series of deoxyribonucleic acid (DNA) bases (also referred to herein as "DNA extensions"), one or more, or a stretch of ribonucleic acid (RNA) bases (also referred to herein as "RNA extensions") ) or combinations thereof. In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. For example, in certain embodiments, the DNA extension can include sequences set forth in SEQ ID NOS:1235-1250. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. For example, in certain embodiments, the RNA extension can comprise sequences set forth in SEQ ID NOs: 1231-1234, 1251-1253. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes can include RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, RNP complexes comprising modified gRNAs can increase editing of target nucleic acids. In certain embodiments, RNP complexes containing modified gRNAs can increase editing resulting in increased productive indels.

In certain embodiments, RNP complexes comprising modified gRNAs and modified Cpf1 proteins can increase editing of target nucleic acids. In certain embodiments, RNP complexes comprising modified gRNAs and modified Cpf1 proteins can increase editing resulting in increased productive indels.

We found that co-delivery of an RNP complex comprising a gRNA complexed to a Cpf1 molecule (e.g., "gRNA-Cpf1-RNP") and a "booster element" could result in increased editing of the target nucleic acid. also found. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). As used herein, the term "booster element" refers to a target without a booster element when co-delivered with an RNP complex comprising gRNA complexed to an RNA-guided nuclease ("gRNA-nuclease-RNP"). Refers to an element that increases target nucleic acid editing relative to nucleic acid editing. In certain embodiments, one or more booster elements may be co-delivered with the gRNA-nuclease-RNP complex to increase editing of the target nucleic acid. In certain embodiments, co-delivery of booster elements may increase editing resulting in increased productive indels. In various embodiments, increased editing of the target nucleic acid includes, but is not limited to, PCR amplification of the target nucleic acid followed by sequence analysis (e.g., Sanger sequencing, next generation sequencing). can be evaluated by any means known to those skilled in the art.

In certain embodiments, the gRNA-nuclease-RNP can comprise gRNA-Cpf1-RNP. In certain embodiments, the Cpf1 molecule of the gRNA-Cpf1-RNP complex can be wild-type Cpf1 or modified Cpf1. In certain embodiments, the Cpf1 molecule of gRNA-Cpf1-RNP has SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019- 1021, 1110-17 (Cpf1 polynucleotide sequences). In certain embodiments, the gRNA-Cpf1-RNP complex can comprise a gRNA comprising a targeting domain listed in Table 6 or Table 12. In certain embodiments, a gRNA-Cpf1-RNP complex can comprise a gRNA comprising a sequence listed in Table 13. In certain embodiments, the gRNA can be modified or unmodified gRNA.

In certain embodiments, the booster component may comprise a dead RNP comprising a dead gRNA molecule complexed to an RNA-guided nuclease molecule (“dead gRNA-nuclease-RNP”). In certain embodiments, the dead gRNA-nuclease-RNP is a dead gRNA complexed to a wild-type (WT) Cas9 molecule (“dead gRNA-Cas9-RNP”), a dead gRNA complexed to a Cas9 nickase molecule ( “dead gRNA-nickase-RNP”) or dead gRNA complexed to an enzymatically inactive (ei) Cas9 molecule (“dead gRNA-eiCas9-RNP”). In certain embodiments, a dead gRNA-nuclease-RNP complex may have reduced or no nuclease activity. In certain embodiments, the dead gRNA of the dead gRNA-nuclease-RNP complex can comprise any of the dead gRNAs described herein. For example, a dead gRNA that can include a targeting domain can be identical to a dead gRNA targeting domain listed in Table 8 or Table 9, or can differ by no more than 3 nucleotides. In certain embodiments, a dead gRNA may comprise a targeting domain comprising a truncation of the gRNA targeting domain. In certain embodiments, the gRNA targeting domain that is truncated can be a gRNA targeting domain listed in Table 8 or Table 9. In certain embodiments, the dead gRNA can be a modified or unmodified dead gRNA. As shown herein, co-delivery of gRNA-Cpf1-RNP and dead gRNA-Cas9-RNP (i.e., RNP comprising dead gRNA complexed to WT Cas9) or gRNA-Cpf1-RNP and dead Co-delivery with gRNA-nickase-RNP (i.e., RNPs containing dead gRNA complexed to Cas9 nickase (i.e., Cas9 D10A nickase)) reduced overall levels above the levels observed following delivery of gRNA-Cpf1-RNP alone. resulted in increased editing (see, eg, Examples 5, 7, 8). The killing gRNA molecule is directed to a region proximal to or within the target region (e.g., the CCAAT box target region, the 13nt target region, the proximal HBG1/2 promoter target sequence and/or the GATA1 binding motif in BCL11Ae) in the target nucleic acid. Complementary targeting domains may be included. In certain embodiments, "proximal" is 10, 25, 50 of the target region (e.g., the CCAAT box target region, the 13nt target region, the proximal HBG1/2 promoter target sequence and/or the GATA1 binding motif in BCL11Ae). , 100 or 200 nucleotides. In certain embodiments, one or more booster elements are co-delivered with gRNA-Cpf1-RNP, e.g. It may contain one or more dead gRNA-nuclease-RNPs. In certain embodiments, co-delivery of dead gRNA-nuclease-RNP does not alter the indel profile of gRNA-Cpf1-RNP.

In certain embodiments, the booster element may comprise an RNP complex comprising a gRNA molecule complexed to an RNA-guided nuclease nickase molecule (“gRNA-nickase-RNP”). In certain embodiments, the RNA-guided nuclease nickase molecule can be, for example, a Cas9 nickase molecule, such as Cas9 D10A nickase. In certain embodiments, the gRNA of the gRNA-nickase-RNP can comprise any of the gRNAs described herein. For example, a gRNA can comprise a gRNA targeting domain listed in Table 8 or Table 9. In certain embodiments, the gRNA can be modified or unmodified gRNA. As shown herein, co-delivery of gRNA-Cpf1-RNP and gRNA-nickase-RNP complexes (RNPs containing guide RNA complexed to Cas9 D10A nickase molecules) resulted in only gRNA-Cpf1-RNP resulted in an increase in total editing above the levels observed following the delivery of (see, eg, Examples 4, 5). Furthermore, co-delivery of gRNA-nickase-RNP and gRNA-Cpf1-RNP complexes altered the orientation, length and/or position of the indel profile of gRNA-Cpf1-RNP. In certain embodiments, booster enhancers may be used to provide desired editing results, such as increasing the proportion of productive indels. In certain embodiments, co-delivery of gRNA-nickase-RNP and gRNA-Cpf1-RNP complexes can alter the indel profile of gRNA-Cpf1-RNP.

In certain embodiments, booster elements may comprise single-stranded oligodeoxynucleotides (ssODNs) or double-stranded oligodeoxynucleotides (dsODNs). In certain embodiments, the ssODN can be any ssODN disclosed herein. In certain embodiments, the ssODN may comprise a sequence listed in Table 7. For example, in certain embodiments, the ssODN can comprise the sequence set forth in SEQ ID NO:1040.

In one aspect, the present disclosure provides that CRISPRs from Prevotella and Francisella 1 (Cpf1) RNA-inducing nucleases or variants thereof are capable of binding to target sites in the promoter of the HBG gene in cells. It relates to an RNP complex comprising a gRNA capable of In certain embodiments, gRNAs may be modified or unmodified. In certain embodiments, the gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2'-O-methyl modifications, DNA extension, RNA extension, or combinations thereof. can include In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a Cpf1 mutant protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, a Cpf1 mutant protein may contain one or more modifications. In certain embodiments, the one or more modifications include, without limitation, one or more mutations of the wild-type Cpf1 amino acid sequence, one or more of the wild-type Cpf1 nucleic acid sequence, a nuclear localization signal ( NLS), one or more mutations of purification tags (eg, His tags), or combinations thereof. In certain embodiments, the Cpf1 mutant protein is SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110- 17 (Cpf1 polynucleotide sequences).

In one aspect, the present disclosure relates to a method of modifying the promoter of the HBG gene in a cell comprising contacting the cell with an RNP complex disclosed herein. In certain embodiments, alterations may include indels within one or more of the regions listed in Table 11. In certain embodiments, modifications may include indels within the CCAAT box target region of the promoter of the HBG gene. For example, in certain embodiments, the modifications are Chr11 (NC_000011.10): 5,249,955 to 5,249,987 (Table 11, Region 6), Chr11 (NC_000011.10): 5,254,879 to 5,254,909 (Table 11, region 16) or combinations thereof. In certain embodiments, the RNP complex may comprise gRNA and Cpf1 protein. In certain embodiments, gRNAs may comprise RNA targeting domains listed in Table 13. In certain embodiments, the gRNA targeting domain may comprise a sequence selected from the group consisting of SEQ ID NOs: 1002, 1254, 1258, 1260, 1262 and 1264. In certain embodiments, the gRNA may comprise a gRNA sequence listed in Table 13. In certain embodiments, the gRNA may comprise a sequence selected from the group consisting of SEQ ID NOs: 1022, 1023, 1041-1105. In certain embodiments, the gRNA is Chr11:5249973, Chr11:5249977 (HBG1); Chr11:5250042, Chr11:5250046 (HBG1); 179, Chr 11:5250183 ( HBG1); Chr11:5254897, Chr11:5254901 (HBG2); Chr11:5254897, Chr11:5254901 (HBG2); Chr11:5254966, 5254970 (HBG2); 83 (HBG2) (Table 16, Table 17) It can be configured to provide edit events. In certain embodiments, cells may be further contacted with a booster element. In certain embodiments, booster elements may comprise single-stranded oligodeoxynucleotides (ssODNs) or double-stranded oligodeoxynucleotides (dsODNs). In certain embodiments, the ssODN can be any ssODN disclosed herein. In certain embodiments, the ssODN may comprise a sequence listed in Table 7. For example, in certain embodiments, the ssODN can comprise the sequence set forth in SEQ ID NO:1040.

In one aspect, the present disclosure relates to an isolated cell comprising an alteration in the promoter of the HBG gene produced by delivery of an RNP complex to the cell. In certain embodiments, the RNP complex may comprise gRNA and Cpf1 protein. In certain embodiments, gRNAs may be modified or unmodified. In certain embodiments, the gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2'-O-methyl modifications, DNA extension, RNA extension, or combinations thereof. can include In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a Cpf1 mutant protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, a Cpf1 mutant protein may contain one or more modifications. In certain embodiments, the one or more modifications include, without limitation, one or more mutations of the wild-type Cpf1 amino acid sequence, one or more mutations of the wild-type Cpf1 nucleic acid sequence, nuclear localization It may include one or more of the signals (NLS), one or more mutations of the purification tags (eg, His tags), or combinations thereof. In certain embodiments, the Cpf1 mutant protein is SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110- 17 (Cpf1 polynucleotide sequences). In certain embodiments, booster elements may be co-delivered with the RNP complex. In certain embodiments, booster elements may comprise single-stranded oligodeoxynucleotides (ssODNs) or double-stranded oligodeoxynucleotides (dsODNs). In certain embodiments, the ssODN can be any ssODN disclosed herein. In certain embodiments, the ssODN may comprise a sequence listed in Table 7. For example, in certain embodiments, the ssODN can comprise the sequence set forth in SEQ ID NO:1040.

In one aspect, the present disclosure increases levels of fetal hemoglobin (HbF) in human cells by genome editing using an RNP complex comprising gRNA and Cpf1 RNA-inducing nuclease or a variant thereof to increase levels of the HBG gene. It relates to an in vitro method of affecting alterations in the promoter and thereby increasing the expression of HbF. In certain embodiments, gRNAs may be modified or unmodified. In certain embodiments, the gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2'-O-methyl modifications, DNA extension, RNA extension, or combinations thereof. can include In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a Cpf1 mutant protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, a Cpf1 mutant protein may contain one or more modifications. In certain embodiments, the one or more modifications include, without limitation, one or more mutations of the wild-type Cpf1 amino acid sequence, one or more mutations of the wild-type Cpf1 nucleic acid sequence, nuclear localization It may include one or more of the signals (NLS), one or more mutations of the purification tags (eg, His tags), or combinations thereof. In certain embodiments, the Cpf1 mutant protein is SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110- 17 (Cpf1 polynucleotide sequences). In certain embodiments, booster elements may be co-delivered with the RNP complex. In certain embodiments, booster elements may comprise single-stranded oligodeoxynucleotides (ssODNs) or double-stranded oligodeoxynucleotides (dsODNs). In certain embodiments, the ssODN can be any ssODN disclosed herein. In certain embodiments, the ssODN may comprise a sequence listed in Table 7. For example, in certain embodiments, the ssODN can comprise the sequence set forth in SEQ ID NO:1040.

In one aspect, the disclosure provides a population of CD34+ or hematopoietic stem cells, wherein one or more cells of the population comprises an alteration in the promoter of the HBG gene, the alteration comprising gRNA and Cpf1 RNA-induced nucleases or mutations thereof. It relates to a population of CD34+ or hematopoietic stem cells generated by delivering an RNP complex containing the type to the population of CD34+ or hematopoietic stem cells. In certain embodiments, gRNAs may be modified or unmodified. In certain embodiments, the gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2'-O-methyl modifications, DNA extension, RNA extension, or combinations thereof. can include In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a Cpf1 mutant protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15). In certain embodiments, a Cpf1 mutant protein may contain one or more modifications. In certain embodiments, the one or more modifications include, without limitation, one or more mutations of the wild-type Cpf1 amino acid sequence, one or more mutations of the wild-type Cpf1 nucleic acid sequence, nuclear localization It may include one or more of the signals (NLS), one or more mutations of the purification tags (eg, His tags), or combinations thereof. In certain embodiments, the Cpf1 mutant protein is SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110- 17 (Cpf1 polynucleotide sequences). In certain embodiments, booster elements may be co-delivered with the RNP complex. In certain embodiments, booster elements may comprise single-stranded oligodeoxynucleotides (ssODNs) or double-stranded oligodeoxynucleotides (dsODNs). In certain embodiments, the ssODN can be any ssODN disclosed herein. In certain embodiments, the ssODN may comprise a sequence listed in Table 7. For example, in certain embodiments, the ssODN can comprise the sequence set forth in SEQ ID NO:1040.

In one aspect, the present disclosure provides a method of alleviating one or more symptoms of sickle cell disease in a subject in need thereof comprising: a) isolating a population of CD34+ or hematopoietic stem cells from the subject b) modifying the isolated population of cells in vitro by delivering an RNP complex comprising a gRNA and a Cpf1 RNA-inducing nuclease or variant thereof to the population of isolated cells, thereby modifying one of the populations; affecting alteration of the promoter of the HBG gene in one or more cells; a method comprising the step of mitigating. In certain embodiments, the method comprises, for example, at least [1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks after administration, or at least [1, 2, 3, 4 , 5, or 6] months, or at least [1, 2, 3, 4, or 5] years, progeny/daughter cells of the modified cells administered to the subject, e.g., BM-engrafted CD34+ hematopoietic stem cells or derived from them in the form of blood cells (e.g. myeloid progenitor cells or differentiated myeloid cells (e.g. erythrocytes, mast cells, myoblasts); or lymphoid progenitor cells or differentiated lymphocytic cells (e.g. T lymphocytes or B lymphocytes, or NK cells).In certain embodiments, the method detects all hematopoietic cell lineages, e.g., without differentiation bias, e.g., without differentiation bias of erythroid lineage In certain embodiments, the method may comprise administering a plurality of edited cells, wherein the method comprises administering a plurality of [at least 5, 10, 15, 20, 25,...100] long-term engraftment of different HSC clones [e.g. , 2, 3, 4, 5, or 6] months, or at least [1, 2, 3, 4, or 5] years. 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks, or at least [1, 2, 3, 4, 5, or 6] months, or at least [1, 2 , 3, 4, or 5] years, wherein the method comprises detecting the level of total hemoglobin expression in the subject. [at least 50%, at least 60%... at least 99%] of total hemoglobin [e.g. at least [1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks, or at least [1, 2, 3, 4, 5, or 6] months, or at least [1, 2, 3, 4, or 5] years].In certain embodiments, the modification may comprise an indel within the CCAAT box target region of the promoter of the HBG gene.In certain embodiments, the RNP complex uses electroporation to In certain embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% comprise productive indels.

In one aspect, the disclosure provides a gRNA comprising a 5′ end and a 3′ end, and comprising a DNA extension at the 5′ end and a 2′-O-methyl-3′-phosphorothioate modification at the 3′ end, , relates to a gRNA comprising an RNA segment capable of hybridizing to a target site and an RNA segment capable of binding to a Cpf1 RNA-guided nuclease. In certain embodiments, the DNA extension may comprise sequences set forth in SEQ ID NOS:1235-1250. In certain embodiments, gRNAs may be modified or unmodified. In certain embodiments, the gRNA has one or more modifications, including phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2′-O-methyl modifications, DNA extension, RNA extension or combinations thereof. can include In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13.

In one aspect, the disclosure relates to an RNP complex comprising a Cpf1 RNA-inducing nuclease as disclosed herein and a gRNA as disclosed herein.

Provided herein are genome editing systems, guide RNAs and gene editing systems for modifying one or more γ-globin genes (e.g., HBG1, HBG2 or HBG1 and HBG2) and increasing expression of fetal hemoglobin (HbF). A CRISPR-mediated method is also provided. In certain embodiments, one or more gRNAs comprising sequences listed in Table 12 or Table 13 may be used to introduce modifications into the promoter region of the HBG gene. In certain embodiments, genome editing systems, guide RNAs and CRISPR-mediated methods target a 13 nucleotide (nt) target region (“13nt target region”) that is 5′ to the transcription site of the HBG1, HBG2 or HBG1 and HBG2 genes. can be modified. In certain embodiments, the genome editing system, guide RNA and CRISPR-mediated methods modify the CCAAT box target region (“CCAAT box target region”) that is 5′ to the transcription site of the HBG1, HBG2 or HBG1 and HBG2 genes. obtain. In certain embodiments, the CCAAT box target region can be the region of or near the distal CCAAT box, comprising nucleotides of the distal CCAAT box and 25 nucleotides upstream (5′) and downstream of the distal CCAAT box ( 3′) and 25 nucleotides (ie, HBG1/2 c.-86 to -140). In certain embodiments, the CCAAT box target region can be the region of or near the distal CCAAT box, comprising the nucleotides of the distal CCAAT box and 5 nucleotides upstream (5′) and downstream of the distal CCAAT box ( 3′) and 5 nucleotides (ie, HBG1/2 c.-106 to -120). In certain embodiments, the CCAAT box target region is an 18 nt target region, 13 nt target region, 11 nt target region, 4 nt target region, 1 nt target region, -117G>A target region as disclosed herein or It can include combinations. In particular embodiments, the alteration is an 18nt deletion, 13nt deletion, 11nt deletion, 4nt deletion, 1nt deletion, c. It can be a G to A substitution at -117 or a combination thereof. In certain embodiments, modifications may be non-naturally occurring modifications or naturally occurring modifications.

Certain embodiments herein also provide for the use of optional genome editing system components such as template nucleic acids (oligonucleotide donor templates). In certain embodiments, template nucleic acids for use in CCAAT target region targeting can include, without limitation, template nucleic acids that encode modifications of the CCAAT box target region. In certain embodiments, a CCAAT box target region may comprise an 18nt target region, an 11nt target region, a 4nt target region, a 1nt target region, or a combination thereof. In certain embodiments, the template nucleic acid can be a single-stranded oligodeoxynucleotide (ssODN) or a double-stranded oligodeoxynucleotide (dsODN). In certain embodiments, exemplary full-length donor templates encoding modifications in the 5' and 3' homology arms and the CCAAT box target region are also provided below (eg, SEQ ID NOs:974-995, 1040). In certain embodiments, the template nucleic acid can be plus or minus stranded. In certain embodiments, an ssODN can include a 5' homology arm, a replacement sequence and a 3' homology arm. In certain embodiments, the 5' homology arm can be from about 25 to about 200 or more nucleotides, e.g., at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length; A sequence can comprise a length of 0 nucleotides; a 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. It can be more than In certain embodiments, ssODNs can include one or more phosphorothioates.

In certain embodiments, genome editing systems, guide RNAs and CRISPR-mediated methods for modifying one or more γ-globin genes (eg, HBG1, HBG2 or HBG1 and HBG2) may comprise RNA guided nucleases. In certain embodiments, the RNA-guided nuclease can be Cpf1 or modified Cpf1 as disclosed herein.

In one aspect, the present disclosure relates to a composition comprising a plurality of cells produced by the methods disclosed above, wherein at least 20%, 30%, 40%, 50%, 60%, 70% of the cells , 80% or 90% of the cells contain alterations in the sequence of the human HBG1 or HBG2 gene or the 13nt target region of a plurality of cells generated by the methods disclosed above, wherein at least 20%, 30%, 40% of the cells, 50%, 60%, 70%, 80% or 90% contain alterations in the sequence of the 13nt target region of the human HBG1 or HBG2 gene. In certain embodiments, at least a portion of the plurality of cells may be within the erythroid lineage. In certain embodiments, the plurality of cells can be characterized by having increased levels of fetal hemoglobin expression compared to the plurality of unmodified cells. In certain embodiments, the level of fetal hemoglobin may be increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the composition may further comprise a pharmaceutically acceptable carrier.

The present disclosure also relates to a method of modifying a cell comprising contacting the cell with any of the genome editing systems disclosed herein. In certain embodiments, contacting the cell may comprise contacting the cell with a solution comprising the first and second ribonucleoprotein complexes. In certain embodiments, contacting the cell with the solution further comprises electroporating the cell, thereby introducing the first and second ribonucleoprotein complexes into the cell.

A genome editing system or method comprising any of the above features may comprise a target nucleic acid comprising the human HBG1, HBG2 gene or combinations thereof. In certain embodiments, the target region can be the CCAAT box target region of the human HBG1, HBG2 gene, or combinations thereof. In certain embodiments, the first targeting domain sequence can be complementary to a first sequence flanking the CCAAT box target region of the human HBG1, HBG2 gene, or combinations thereof, wherein the first sequence optionally overlaps the CCAAT box target region of the human HBG1, HBG2 gene or combinations thereof. In certain embodiments, the second targeting domain sequence can be complementary to a second sequence flanking the CCAAT box target region of the human HBG1, HBG2 gene, or combinations thereof, wherein the second sequence optionally overlaps the CCAAT box target region of the human HBG1, HBG2 gene or combinations thereof. In certain embodiments, the first targeting domain may comprise a truncated gRNA targeting domain. In certain embodiments, the gRNA targeting domain can comprise a gRNA listed in Table 8 or Table 9, wherein the gRNA targeting domain is truncated from the 5' end of the gRNA targeting domain. In certain embodiments, the first targeting domain is identical to or differs by no more than 3 nucleotides from a dgRNA targeting domain listed in Table 8 or Table 9. In certain embodiments, the second targeting domain differs from a gRNA targeting domain listed in Table 8 or Table 9 by no more than 3 nucleotides. In certain embodiments, the indels may alter the CCAAT box target region indels. In certain embodiments, an indel can be a productive indel that results in increased levels of fetal hemoglobin expression. In certain embodiments, gRNA, dgRNA, or both can be synthesized in vitro or chemically synthesized.

In certain embodiments, the cell can comprise at least one modified allele of the HBG locus produced by any of the methods for modifying a cell disclosed herein, wherein Modified alleles of include modifications of the human HBG1 gene, HBG2 gene, or combinations thereof.

In certain embodiments, the isolated population of cells can be modified by any of the methods for modifying cells disclosed herein, wherein the population of cells has been modified by the method. not, the distribution of indels that may differ from isolated populations of cells or their progeny of the same cell type.

In certain embodiments, a plurality of cells can be produced by any of the methods for modifying cells disclosed herein, wherein at least 20%, 30%, 40%, 50% of the cells , 60%, 70%, 80% or 90% may comprise sequence alterations within the CCAAT box target region of the human HBG1 gene, HBG2 gene, or combinations thereof.

In certain embodiments, the cells disclosed herein can be used for pharmaceuticals. In certain embodiments, the cells may be used in the treatment of β-hemoglobinopathies. In certain embodiments, the β-hemoglobinopathy can be selected from the group consisting of sickle cell disease and β-thalassemia.

In one aspect, the disclosure provides that at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells have a sequence modification of the CCAAT box target region of the human HBG1 or HBG2 gene. at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the cells or cells produced by the above-disclosed dgRNA-containing methods are human HBG1 or HBG2 containing alterations in the sequence of the CCAAT box target region of . In certain embodiments, at least a portion of the plurality of cells may be within the erythroid lineage. In certain embodiments, the plurality of cells can be characterized by having increased levels of fetal hemoglobin expression compared to the plurality of unmodified cells. In certain embodiments, the level of fetal hemoglobin may be increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the composition may further comprise a pharmaceutically acceptable carrier.

In one aspect, the present disclosure relates to a population of cells modified by a genome editing system comprising a dgRNA as described above, wherein the population of cells is higher than a population of cells not modified by the genome editing system Includes percentage productive indels. The present disclosure includes the above dgRNAs, wherein a higher percentage of the population of cells can differentiate into a population of HbF-expressing erythroid lineage cells compared to a population of cells not modified by the genome editing system. It also relates to populations of cells modified by genome editing systems. In certain embodiments, the higher percentage can be at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 40% higher. In certain embodiments, the cells can be hematopoietic stem cells. In certain embodiments, the cells may be capable of differentiating into erythroids, erythrocytes, or progenitors of erythrocytes or erythrocytes. In certain embodiments, indels may be generated by repair mechanisms other than microhomology-mediated end joining (MMEJ) repair.

The present disclosure also relates to the use of any of the cells disclosed herein in the manufacture of a medicament for treating β-hemoglobinopathy in a subject.

In one aspect, the present disclosure relates to a method of treating β-hemoglobinopathy in a subject in need thereof, comprising administering cells disclosed herein to the subject. In certain embodiments, a method of treating beta-hemoglobinopathy in a subject in need thereof may comprise administering to the subject a population of modified hematopoietic cells, wherein one or more are modified according to the methods of modifying cells disclosed herein. In certain embodiments, the method comprises, for example, at least [1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks after administration, or at least [1, 2, 3, 4 , 5, or 6] months, or at least [1, 2, 3, 4, or 5] years, progeny/daughter cells of the modified cells administered to the subject, e.g., BM-engrafted CD34+ hematopoietic stem cells or derived from them in the form of blood cells (e.g. myeloid progenitor cells or differentiated myeloid cells (e.g. erythrocytes, mast cells, myoblasts); or lymphoid progenitor cells or differentiated lymphocytic cells (e.g. T lymphocytes or B lymphocytes, or NK cells).In certain embodiments, the method detects all hematopoietic cell lineages, e.g., without differentiation bias, e.g., without differentiation bias of erythroid lineage In certain embodiments, the method may comprise administering a plurality of edited cells, wherein the method comprises administering a plurality of [at least 5, 10, 15, 20, 25,...100] long-term engraftment of different HSC clones [e.g. , 2, 3, 4, 5, or 6] months, or at least [1, 2, 3, 4, or 5] years. 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks, or at least [1, 2, 3, 4, 5, or 6] months, or at least [1, 2 , 3, 4, or 5] years, wherein the method comprises detecting the level of total hemoglobin expression in the subject. [at least 50%, at least 60%... at least 99%] of total hemoglobin [e.g. at least [1, 2, 3, 4, 5, 6, 7, 8, 12, 16, or 20] weeks, or at least [1, 2, 3, 4, 5, or 6] months, or at least [1, 2, 3, 4, or 5] years] In certain embodiments, the modification may comprise an indel within the CCAAT box target region of the promoter of the HBG gene.

In one aspect, the present disclosure relates to a genome editing system comprising an RNA-guided nuclease and a first guide RNA, wherein the first guide RNA is the CCAAT box target region of the human HBG1, HBG2 gene or combinations thereof. The first sequence optionally overlaps the CCAAT box target region of the human HBG1, HBG2 gene, or combinations thereof. In certain embodiments, the genome editing system may further comprise template nucleic acids encoding modifications of the CCAAT box target regions of the human HBG1, HBG2 genes, or combinations thereof. In certain embodiments, the template nucleic acid can be a single-stranded oligodeoxynucleotide (ssODN) or a double-stranded oligodeoxynucleotide (dsODN). In certain embodiments, an ssODN can include a 5' homology arm, a replacement sequence and a 3' homology arm. In certain embodiments, the homology arms can be symmetrical in length. In certain embodiments, the homology arms may be asymmetric in length. In certain embodiments, ssODNs may include one or more phosphorothioate modifications. In certain embodiments, one or more phosphorothioate modifications can be at the 5' terminus, the 3' terminus, or a combination thereof. In certain embodiments, the ssODN can be plus or minus strand. In certain embodiments, modifications may be non-naturally occurring modifications. In certain embodiments, the modification may include deletion of the CCAAT box target region. In certain embodiments, deletions may comprise 18nt deletions, 11nt deletions, 4nt deletions, 1nt deletions or combinations thereof. In certain embodiments, a CCAAT box target region may comprise an 18nt target region, an 11nt target region, a 4nt target region, a 1nt target region, or a combination thereof. In certain embodiments, the 5' homology arm is from about 25 to about 200 or more nucleotides in length, e.g., at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. the replacement sequence may comprise a length of 0 nucleotides; the 3' homology arm may be at least about 25, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length; can be up to about 200 nucleotides or more. In certain embodiments, the 5' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 18nt target region; The 3' homology arm can comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' to the 18nt target region. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of SEQ ID NO:974 or SEQ ID NO:975. In certain embodiments, the 5' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 11nt target region, The 3' homology arm can comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' to the 11 nt target region. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of SEQ ID NO:976 or SEQ ID NO:978. It may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, on the 5' side, and the 3' homology arm may be 3' of the 4nt target region, for example, 55 bp. It may contain about 50-100 bp of homology, such as ˜95, 60-90, 70-90 or 80-90 bp. In certain embodiments, the ssODN is SEQ ID NO:984, SEQ ID NO:985, SEQ ID NO:986, SEQ ID NO:987, SEQ ID NO:988, SEQ ID NO:989, SEQ ID NO:990, SEQ ID NO:991, SEQ ID NO:992, SEQ ID NO:993, It may comprise, consist essentially of, or consist of a sequence selected from the group consisting of: No. 994 and SEQ ID No. 995. In certain embodiments, the 5' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 1 nt target region; The 3' homology arm can comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 1 nt target region. In certain embodiments, the homology arms can be symmetrical in length. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of SEQ ID NO:982 or SEQ ID NO:983. In certain embodiments, modifications may be naturally occurring modifications. In certain embodiments, alterations may include deletion or mutation of the CCAAT box target region. In certain embodiments, a CCAAT box target region may comprise a 13nt target region, a -117G>A target region, or a combination thereof. In certain embodiments, alterations may include 13nt deletions in 13nt target regions or G to A substitutions in -117G>A target regions, or combinations thereof. In certain embodiments, the 5' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 13nt target region; The 3' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 13nt target region. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of SEQ ID NO:977 or SEQ ID NO:979. In certain embodiments, the 5' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 13nt target region; The 3' homology arm may comprise about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 13nt target region. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of SEQ ID NO:980 or SEQ ID NO:981. In certain embodiments, the RNA-guided nuclease is S . It may be S. pyogenes Cas9. In certain embodiments, the RNA-guided nuclease can be a Cpf1 mutant as disclosed herein. In certain embodiments, the first targeting domain may differ from the targeting domains listed in Table 12, Table 13 or the gRNA of Table 13 by no more than 3 nucleotides. In certain embodiments, the genome editing system may further comprise a second guide RNA, wherein the second guide RNA is a second guide RNA flanking the CCAAT box target region of the human HBG1, HBG2 gene or combinations thereof. The second targeting domain optionally overlaps the CCAAT box target region of the human HBG1, HBG2 gene, or combinations thereof. In certain embodiments, the RNA-guided nuclease can be a nickase, optionally lacking RuvC activity. In certain embodiments, a genome editing system can comprise first and second RNA-guided nucleases. In certain embodiments, the first and second RNA-guided nucleases can complex to the first and second guide RNAs, respectively, to form first and second ribonucleoprotein complexes. In certain embodiments, the genome editing system can further comprise a third guide RNA, and optionally a fourth guide RNA, wherein the third and fourth guide RNAs are of the human BCL11A gene. third and fourth targeting domains complementary to the third and fourth sequences opposite to the location of the GATA1 binding motif in the BCL11A erythroid enhancer (BCL11Ae); optionally overlap with the GATA1 binding motif in BCL11Ae of the human BCL11A gene. In certain embodiments, the genome editing system can further comprise a nucleic acid template encoding a deletion of the GATA1 binding motif in BCL11Ae. In certain embodiments, the RNA-guided nuclease is S . It may be S. pyogenes Cas9. In certain embodiments, the RNA-guided nuclease can be a nickase, optionally lacking RuvC activity. In certain embodiments, the third targeting domain may be complementary to a sequence within 1000 nucleotides upstream of the GATA1 binding motif in BCL11Ae. In certain embodiments, the third targeting domain may be complementary to a sequence within 100 nucleotides upstream of the GATA1 binding motif in BCL11Ae. In certain embodiments, one of the third and fourth targeting domains may be complementary to a sequence within 100 nucleotides downstream of the GATA1 binding motif in BCL11Ae. In certain embodiments, the fourth targeting domain may be complementary to a sequence within 50 nucleotides downstream of the GATA1 binding motif in BCL11Ae. In certain embodiments, a genome editing system can comprise first and second RNA-guided nucleases. In certain embodiments, the first and second RNA-guided nucleases can complex to the third and fourth guide RNAs, respectively, to form third and fourth ribonucleoprotein complexes.

In one aspect, the present disclosure relates to a method of modifying a cell comprising contacting the cell with a genome editing system. In certain embodiments, contacting the cell with the genome editing system can comprise contacting the cell with a solution comprising the first and second ribonucleoprotein complexes. In certain embodiments, contacting the cell with the solution may further comprise electroporating the cell, thereby introducing the first and second ribonucleoprotein complexes into the cell. In certain embodiments, the method of modifying a cell can further comprise contacting the cell with a genome editing system, wherein the step of contacting the cell with the genome editing system comprises: Optionally, contacting with a solution comprising a fourth ribonucleoprotein complex may be included. In certain embodiments, contacting the cell with the solution may further comprise electroporating the cell, whereby the first, second, third and optionally fourth ribonucleoprotein complexes are introduced into cells. In certain embodiments, the cells may be capable of differentiating into erythroids, erythrocytes or progenitors of erythrocytes or erythrocytes. In certain embodiments, the cells can be CD34 ⁺ cells.

In one aspect, the present disclosure provides a CRISPR-mediated method of modifying a cell, wherein the human HBG1 or HBG2 gene is located at c. introducing a first DNA single-strand break (SSB) or double-strand break (DSB) into the genome of cells from -106 to -120; optionally, the location of the human HBG1 or HBG2 gene; c. introducing a second SSB or DSB into the genome of -106 to -120 cells, wherein the first and second SSB or DSB are CCAAT of the human HBG1 or HBG2 gene It can be repaired by cells in a manner that alters the box target region. In certain embodiments, the first and second SSBs or DSBs can be repaired by the cell in a manner that results in alteration of the CCAAT box target region of the human HBG1 or HBG2 gene. In certain embodiments, the CRISPR-mediated method may further comprise template nucleic acids encoding modifications of the CCAAT box target regions of the human HBG1, HBG2 genes, or combinations thereof. In certain embodiments, the template nucleic acid can be a single-stranded oligodeoxynucleotide (ssODN). In certain embodiments, an ssODN can include a 5' homology arm, a replacement sequence and a 3' homology arm. In certain embodiments, the ssODN can be plus or minus strand. In certain embodiments, modifications may be non-naturally occurring modifications. In certain embodiments, the first and second SSBs or DSBs can be repaired by the cell in a manner that results in the formation of at least one of indels, deletions or insertions in the CCAAT box target region of the human HBG1 or HBG2 gene. In certain embodiments, a CCAAT box target region may comprise an 18nt target region, an 11nt target region, a 4nt target region, a 1nt target region, or a combination thereof. In certain embodiments, the 5' homology arm can be from about 25 to about 200 or more nucleotides, e.g., at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length; A sequence can comprise a length of 0 nucleotides; a 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. It can be more than In certain embodiments, the 5' homology arm is 5', such as 55-95, 60-90, 70-90 or 80-90 bp, 5' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region. and the 3' homology arm is 3' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region, such as 55-95, 60-90, 70- It may contain about 50-100 bp of homology, such as 90 or 80-90 bp. In certain embodiments, the ssODN is SEQ ID NO:974, SEQ ID NO:975, SEQ ID NO:976, SEQ ID NO:978, SEQ ID NO:984, SEQ ID NO:985, SEQ ID NO:986, SEQ ID NO:987, SEQ ID NO:988, SEQ ID NO:989, may comprise or consist essentially of a sequence selected from the group consisting of: 990, 991, 992, 993, 994, 995, 982 and 983 , or may consist of In certain embodiments, modifications may be non-naturally occurring modifications. In certain embodiments, the first and second SSBs or DSBs can be repaired by the cell in a manner that results in the formation of at least one of indels, deletions or insertions in the CCAAT box target region of the human HBG1 or HBG2 gene. In certain embodiments, a CCAAT box target region may comprise a 13nt target region, a -117G>A target region, or a combination thereof. In certain embodiments, alterations may include 13nt deletions in 13nt target regions or G to A substitutions in -117G>A target regions, or combinations thereof. In certain embodiments, the 5' homology arm is about 50-100 bp, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 13nt target region or -117G>A target region. and the 3′ homology arm extends from about 50 to It can contain 100 bp of homology. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of a sequence selected from the group consisting of SEQ ID NO:977 or SEQ ID NO:979, SEQ ID NO:980 or SEQ ID NO:981.

In one aspect, the present disclosure relates to compositions that can comprise a plurality of cells produced by the methods of modifying cells disclosed herein, wherein at least 20%, 30%, 40%, 50% of the cells %, 60%, 70%, 80% or 90% may comprise sequence alterations of the CCAAT box target region of the human HBG1 gene, HBG2 gene or combinations thereof. In certain embodiments, the modification comprises an 18 nt deletion, 11 nt deletion, 4 nt deletion, 1 nt deletion, 13 nt deletion, G to A substitution at −117 of the human HBG1 gene, HBG2 gene, or combinations thereof. can contain. In certain embodiments, at least a portion of the plurality of cells may be within the erythroid lineage. In certain embodiments, the plurality of cells can be characterized by an increased level of fetal hemoglobin expression compared to the plurality of unmodified cells. In certain embodiments, the level of fetal hemoglobin may be increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the composition may further comprise a pharmaceutically acceptable carrier.

In one aspect, the disclosure relates to a cell comprising a synthetic genotype produced by the methods of modifying a cell disclosed herein, wherein the cell comprises 18 nt of the human HBG1 gene, HBG2 gene, or combinations thereof. deletion, 11 nt deletion, 4 nt deletion, 1 nt deletion, 13 nt deletion, G to A substitution at -117.

In one aspect, the disclosure relates to a cell comprising at least one allele of the HBG locus produced by the methods of modifying a cell disclosed herein, wherein the cell comprises the human HBG1 gene, the HBG2 gene or a G to A substitution at 18nt deletion, 11nt deletion, 4nt deletion, 1nt deletion, 13nt deletion, -117 of any combination thereof.

In one aspect, the present disclosure relates to AAV vectors that can include template nucleic acids encoding non-naturally occurring modifications of the CCAAT box target regions of human HBG1, HBG2 genes, or combinations thereof. In certain embodiments, the template nucleic acid can be a single-stranded oligodeoxynucleotide (ssODN). In certain embodiments, a CCAAT box target region may comprise an 18nt target region, an 11nt target region, a 4nt target region, a 1nt target region, or a combination thereof. In certain embodiments, an ssODN can include a 5' homology arm, a replacement sequence and a 3' homology arm. In certain embodiments, the 5' homology arm is from about 25 to about 200 or more nucleotides in length, e.g., at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. the replacement sequence may comprise a length of 0 nucleotides; the 3' homology arm may be at least about 25, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length; can be up to about 200 nucleotides or more. In certain embodiments, the 5' homology arm is 5', such as 55-95, 60-90, 70-90 or 80-90 bp, 5' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region. and the 3' homology arm is 3' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region, such as 55-95, 60-90, 70- It may contain about 50-100 bp of homology, such as 90 or 80-90 bp. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of a sequence selected from the group consisting of SEQ ID NOs:974-976, SEQ ID NOs:978, SEQ ID NOs:982-995.

In one aspect, the present disclosure relates to nucleotide sequences comprising template nucleic acids that encode non-naturally occurring modifications of the CCAAT box target regions of human HBG1, HBG2 genes, or combinations thereof. In certain embodiments, the template nucleic acid can be a single-stranded oligodeoxynucleotide (ssODN) or a double-stranded oligodeoxynucleotide (dsODN) containing modifications. In certain embodiments, a CCAAT box target region may comprise an 18nt target region, an 11nt target region, a 4nt target region, a 1nt target region, or a combination thereof. In certain embodiments, an ssODN can include a 5' homology arm, a replacement sequence and a 3' homology arm. In certain embodiments, the 5' homology arm is from about 25 to about 200 or more nucleotides in length, e.g., at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. the replacement sequence may comprise a length of 0 nucleotides; the 3' homology arm may be at least about 25, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length; can be up to about 200 nucleotides or more. In certain embodiments, the 5' homology arm is 5', such as 55-95, 60-90, 70-90 or 80-90 bp, 5' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region. and the 3' homology arm is 3' of the 18 nt target region, 11 nt target region, 4 nt target region or 1 nt target region, such as 55-95, 60-90, 70- It may contain about 50-100 bp of homology, such as 90 or 80-90 bp. In certain embodiments, the ssODN may comprise, consist essentially of, or consist of a sequence selected from the group consisting of SEQ ID NOs:974-976, SEQ ID NOs:978, SEQ ID NOs:982-995.

In one aspect, the disclosure relates to a cell comprising a synthetic genotype, wherein the cell comprises an 18 nt deletion, 11 nt deletion, 4 nt deletion, 1 nt deletion, 13 nt deletion of the human HBG1 gene, HBG2 gene, or combinations thereof. The deletion may include a G to A substitution at -117.

In one aspect, the disclosure relates to a composition comprising a population of cells produced by a method of modifying cells disclosed herein, wherein the cells have the human HBG1 gene, compared to the population of unmodified cells, Including more frequent alterations in the sequence of the CCAAT box target region of the HBG2 gene or combinations thereof. In certain embodiments, the higher frequency is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% higher. In certain embodiments, the modification comprises an 18 nt deletion, 11 nt deletion, 4 nt deletion, 1 nt deletion, 13 nt deletion, G to A substitution at −117 of the human HBG1 gene, HBG2 gene, or combinations thereof. include. In certain embodiments, at least a portion of the population of cells is within the erythroid lineage.

This list is intended to be exemplary and descriptive rather than comprehensive and exclusive. Additional aspects and embodiments may be described or apparent from the remaining portions of the disclosure and claims.

The accompanying drawings are intended to provide illustrative, schematic examples, rather than exhaustive, of certain aspects and embodiments of the present disclosure. The drawings are not intended to be limiting or bound by any particular theory or model, and are not necessarily to scale. Without limiting the above, nucleic acids and polypeptides may be depicted as linear sequences or as schematic two- or three-dimensional structures; these depictions limit any particular model or theory as to their structure. are intended to be illustrative rather than intended to be or be bound by.

FIG. 1 schematically shows the HBG1 and HBG2 genes in relation to the β-globin gene cluster on human chromosome 11. FIG. Figure 1. Each gene within the β-globin gene cluster is transcriptionally regulated by a proximal promoter. While not wishing to be bound by any particular theory, in general, _Aγ and/or _Gγ expression occurs between the proximal promoter and the distal strong cell-specific enhancer locus control region (LCR). It is believed to be activated by engagement. Long-range transactivation by LCR is believed to be mediated by alterations in chromatin arrangement/conformation. The LCR is marked with four erythrocyte-specific Dnase I hypersensitive sites (HS1-4) and two distal enhancer elements (5'HS and 3'HS1). The beta-like gene globin gene expression is regulated in a developmental stage-specific manner, and expression of globin gene alterations coincides with alterations at major sites of blood production. Figures 2A-2B show the HBG1 and HBG2 genes, the coding sequences (CDSs) and small scales within and upstream of the HBG1 and HBG2 proximal promoters identified in patients and associated with elevated fetal hemoglobin (HbF). Deletions and point mutations are indicated. A core element within the proximal promoter (CAAT box, 13nt sequence) that is deleted in some patients with hereditary persistence of fetal hemoglobin (HPFH). The "target sequence" region of each locus screened for gRNA binding target sites has also been identified. Figure 3 shows a mutant form of Acidaminococcus sp. and the HBG gene of mPB CD34+ cells electroporated with His-AsCpf1-sNLS-sNLS (SEQ ID NO: 1001) (“His-AsCpf1-nNLS_HBG1-1 RNP” and “His-AsCpf1-sNLS-sNLS_HBG1-1 RNP”) Indicates editing. RNP was electroporated at 5 μM or 20 μM. Figures 4A-4C show gene editing of HBG in mPB CD34+ cells electroporated with His-AsCpf1-sNLS-sNLS_HBG1-1 RNP alone or in combination with various ssODNs. Figure 4A shows His-AsCpf1-sNLS-sNLS_HBG1-1 RNP alone or OLI164324 ("-4nt-strand"), OLI16430 ("-4nt+strand"), OLI16410 ("-18nt-strand") or OLI16409 (" -18nt+strand") shows the percentage of indels detected by sequencing of HBG PCR products after 72 hours of electroporation. FIG. 4B shows HBG PCR products 72 hours after electroporation with His-AsCpf1-sNLS-sNLS_HBG1-1 RNP alone or in combination with OLI16410 (“−18nt−strand”) or OLI16409 (“−18nt+strand”). The percentage of correct 18-nucleotide deletion indels detected by sequencing is shown (Table 7). FIG. 4C shows HBG PCR products 72 hours after electroporation with His-AsCpf1-sNLS-sNLS_HBG1-1 RNP alone or in combination with OLI16410 (“−18nt−strand”) or OLI16409 (“−18nt+strand”). The percentage of correct 18nt deletions within all indels detected by sequencing is shown. Figures 5A-5F show the HBG1-1 target region and S. cerevisiae used in combination. Schematic representation of pairing with S. Pyogenes Cas9 gRNA. FIG. 5A shows the target region of HBG1-1 gRNA (comprising the RNA targeting domain set forth in SEQ ID NO: 1002 of Table 9). The distal CCAAT box of the HBG promoter (ie HBG1/2 c.-111 to -115) is indicated by a gray box. FIG. 5B shows the target region of HBG1-1, the distal CCAAT box of HBG and the target region SpA gRNA (including SEQ ID NO:941 targeting domain of Table 9). FIG. 5C shows the target region of HBG1-1, the distal CCAAT box of HBG and the target region SpG gRNA (including SEQ ID NO:359 targeting domain of Table 9). FIG. 5D shows the target region of HBG1-1, the distal CCAAT box of HBG, the target region (“dgRNA”) of tSpA-killing gRNA (including the targeting domain of SEQ ID NO:326 in Table 9) and the target region of Sp182 dgRNA ( including the targeting domain of SEQ ID NO: 1028 in Table 15). FIG. 5E shows the target region of HBG1-1, the distal CCAAT box of HBG and the tSpA dgRNA (comprising the targeting domain of SEQ ID NO:326 of Table 9). FIG. 5F shows the target region of HBG1-1, the distal CCAAT box of HBG and the target region Sp182 dgRNA (including SEQ ID NO: 1028 targeting domain of Table 9). Figures 6A-6B show HbF expression achieved by ex vivo editing of mPB CD34+ cells using HBG1-1-AsCpf1-RNP targeting the HBG promoter region. FIG. 6A shows the results of editing in the HBG promoter region in mPB CD34+ cells following delivery of 5 μM or 20 μM HBG1-1-AsCpf1-RNP (“HBG-1-1”) via Amaxa electroporation. . Delivery of 20 μM HBG1-1-AsCpf1-RNP via Amaxa electroporation results in up to approximately 43% editing and 21% HbF induction (above background levels). HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Gray bars indicate the percentage of indels 72 hours after electroporation detected by next generation sequencing (NGS) of HBG PCR products. FIG. 6B shows the results of editing in the HBG promoter region in mPB CD34+ cells following delivery of 5 μM or 20 μM HBG1-1-AsCpf1-RNP (“HBG-1-1”) via MaxCyte electroporation. . Delivery of 20 μM HBG1-1-AsCpf1-RNP via MaxCyte electroporation results in up to approximately 16% editing and 7% HbF induction (above background levels). HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Gray bars indicate the percentage of indels detected by NGS of HBG PCR products at 72 hours post-electroporation. FIG. 7 shows various S. Enhanced editing by HBG1-1-AsCpf1H800A-RNP in the HBG promoter region on the Maxcyte apparatus by co-delivery of S. pyogenes Cas9 WT or Cas9 D10A RNP. "S.Py D10A" refers to the Cas9 D10A nickase protein and "S.Py WT" refers to the Cas9WT protein. The RNPs tested included SpA-D10A-RNP, SpG-D10A-RNP, tSpA-Cas9-RNP, Sp182-Cas9-RNP and tSpA-Cas9-RNP+Sp182-Cas9-RNP (Table 8). HBG1-1-AsCpf1H800A-RNP (“HBG1-1”) alone or S. Shown is total editing in the HBG promoter region (grey bars) and associated HbF protein induction (filled circles) following delivery in combination with S. pyogenes Cas9 RNPs or RNP pairs. HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Gray bars indicate the percentage of indels detected by NGS of HBG PCR products. FIG. 8 shows HBG1-1-AsCpf1H800A-RNP (“HBG1-1”) alone or various S. Figure 2 shows viability of mPB CD34+ cells following MaxCyte delivery in combination with S. Pyogenes Cas9 WT or Cas9 D10A RNP. "S.Py D10A" refers to the Cas9 D10A nickase protein and "S.Py WT" refers to the Cas9WT protein. The RNPs tested included SpA-D10A-RNP, SpG-D10A-RNP, tSpA-Cas9-RNP, Sp182-Cas9-RNP and tSpA-Cas9-RNP+Sp182-Cas9-RNP (Table 8). Twenty-four hours after electroporation, viability was measured by DAPI staining and flow cytometric analysis. 9A-9B show the cleavage sites of HBG1-1-AsCpf1H800A-RNP and D10A-Cas9 RNP in the target region and the editing profile resulting from co-delivery of HBG1-1-AsCpf1H800A-RNP and D10A RNP. FIG. 9A shows the location of the HBG1-1-AsCpf1H800A-RNP cleavage site on each strand of the target region (light gray arrows) and the nicking targeted by SpG-D10A-RNP and SpA-D10A-RNP (Table 8). Locations of sites (dark arrows) are indicated. FIG. 9B shows HBG1-1-AsCpf1H800A-RNP (“HBG1-1 RNP”) and SpG-D10A-RNP (“spG1-1 RNP”) detected by NGS analysis of HBG PCR products 72 hours after electroporation of mPB CD34+ RNP") or SpA-D10A-RNP ("spA RNP") co-delivery profiles are shown (Table 8). The X-axis represents the genomic position of the center of the indel relative to the HBG1-1-AsCpf1H800A-RNP plus-strand break site. The Y-axis represents the length of the indels, deletions are represented as negative values and insertions as positive values. The total frequency of each indel is represented by the area of the symbol. Indels occurring at a frequency of 0.1% or greater are indicated. SpG and SpA target sites are indicated by dashed lines. Figures 10A-10B show a substantial indel profile as co-delivery of HBG1-1-AsCpf1H800A-RNP and Sp182-Cas9-RNP was detected by NGS analysis of HBG PCR products 72 hours after electroporation. We show that total indels and distal CCAAT box-blocking indels are increased without significant alteration. FIG. 10A shows indel profiles following editing with HBG1-1-AsCpf1H800A-RNP (“HBG1-1 RNP”) alone or in combination with Sp182-Cas9-RNP (“sp182 RNP”). The X-axis represents the genomic position of the center of the indel relative to the HBG1-1-AsCpf1H800A-RNP plus-strand break site. The Y-axis represents the length of the indels, deletions are represented as negative values and insertions as positive values. The total frequency of each indel is represented by the area of the symbol. Indels occurring at a frequency of 0.1% or greater are indicated. The Sp182 target site is indicated by a dotted line. FIG. 10B shows the frequency of indels that interfere with either the entire 0nt, 1nt, 2nt, 3nt, 4nt or 5nt of the distal CCAAT box sequence. FIG. 11 shows that optimal doses of HBG1-1-AsCpf1H800A-RNP co-delivered with Sp182-Cas9-RNP result in increased total editing and HbF production (Table 8). Delivery of HBG1-1-AsCpf1H800A-RNP (“HBG1-1”) together with Sp182-Cas9-RNP (“Sp182”) as an RNP pair resulted in up to 34% HbF induction (above background) (filled circles) ) was achieved (grey bars) in the HBG promoter region. No editing was observed when Sp182-Cas9-RNP was delivered alone at 12 μM. HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Gray bars indicate the percentage of indels detected by NGS of HBG PCR products. Figure 12. Measured by UPLC in clonal erythroid progeny of single human mPB CD34+ cells edited in the HBG promoter region with HBG1-1-AsCpf1H800A-RNP combined with Sp182-Cas9-RNP (Table 8). , shows the distribution level of γ-chain expression relative to all β-like chains (γ-chain/[γ-chain + β-chain]). Each filled circle represents the γ-globin protein level detected in clonal erythroid populations derived from single cells isolated by FACS sorting 48 hours after electroporation. Figures 13A-13C show modified HBG1-1 gRNA (SEQ ID NO: 1041 in Table 8) complexed to His-AsCpf1-sNLS-sNLS H800A (SEQ ID NO: 1032 in Table 8) ("His-AsCpf1-sNLS-sNLS H800A_HBG1 -1 RNP", represented as "HBG1-1" in Figures 13A-C) and increasing concentrations of ssODN OLI16431 (SEQ ID NO: 1040 in Table 7) (represented as "OLI16431" in Figures 13A-C). ) (Table 7). FIG. 13A shows distal CAATT box (grey bars) editing and HbF induction (filled circles) after co-delivery of 6 μM His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP with increasing concentrations of ssODN OLI16431. HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Gray bars indicate the percentage of indels detected by NGS of HBG PCR products. FIG. 13B shows viability of mPB CD34+ cells following delivery of His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP alone or in combination with increasing doses of ssODN OLI16431. Viability was measured by DAPI exclusion 72 hours after electroporation. FIG. 13C shows hematopoietic activity of 'HBG1-1' RNP and ssODN OLI16431-treated cells and donor-matched untreated control CD34+ cells in a colony-forming cell (CFC) assay. CFC indicates values per 800 CD34+ cells plated. Colony numbers and subtypes are indicated (GEMM: granulocyte-erythroid-monocyte-macrophage colony (black), GM: granulocyte-macrophage colony (dark grey), E: erythroid colony (light grey)). Figures 14A-14B show His-AsCpf1-sNLS-sNLS co-delivered with various concentrations of ssODN OLI16431 (SEQ ID NO: 1040 in Table 7) (represented as "OLI16431" in Figures 14A-B) (Table 7). Unmodified HBG1-1 gRNA (SEQ ID NO: 1022 in Table 8) complexed to H800A (SEQ ID NO: 1032 in Table 8) (“His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP”, “HBG1 Shown are total editing, HbF production and viability results using different concentrations of RNPs containing RNPs (expressed as "-1"). FIG. 14A shows editing in the distal CAATT box (black bars (48 h) and Light gray bars (day 14 of erythrocyte culture)) and HbF induction (filled circles) are shown. HbF levels are represented by filled circles representing γ-globin chain expression levels relative to total β-like globin chains (γ chain/[γ chain + β chain]) measured by UPLC analysis on erythroid progeny of mPB CD34+ cells. Bars indicate the percentage of indels detected by NGS of HBG PCR products at 48 hours (black) and 14 days (light grey) of erythrocyte culture. FIG. 14B depicts mPB CD34+ cells following delivery of His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP alone (“HBG1-1”), ssODN OLI16431 alone or in combination with various doses of HBG1-1 and OLI16431. Shows viability. Viability was measured by DAPI exclusion 48 hours after electroporation and 14 days after erythrocyte culture. Following editing of mPB CD34+ cells, in vitro differentiation to the erythroid lineage was performed for 18 days (Giarratana 2011). Subsets of cells were isolated on day 14 of culture for viability (DAPI exclusion) and editing (NGS of HB GPCR products) measurements. FIG. 15 shows editing by RNP in mPB CD34+ cells. As described in Table 15, RNPs contained gRNA complexed to Cpf1 protein. Illumina sequencing was performed on the isolated genomic DNA 72 hours after electroporation. Figures 16A-16B show bulk compilation of CD34+ cell populations (black bars), progenitor cells (light gray bars) and HSCs (dark gray bars) as determined by Illumina sequencing 48 hours after electroporation. FIG. 16A shows RNP33 (Table 15) delivered alone or co-delivered with ssODN OLI16431 (SEQ ID NO: 1040 in Table 7). FIG. 16B shows RNP33 delivered alone or co-delivered with Sp182 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)). (Table 15). Figure 17 shows bulk compilation of CD34+ cell populations (black bars), progenitor cells (light gray bars) and HSCs (dark gray bars) as determined by Illumina sequencing 48 hours after electroporation. RNP34, RNP33 and RNP43 (Table 15) alone or Sp182 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)) or ssODN It was delivered in combination with OLI16431 (SEQ ID NO: 1040 in Table 7). Figure 18 shows editing of CD34+ cells as determined by Illumina sequencing 72 hours after electroporation. RNP64, RNP63 and RNP45 (Table 15) were delivered at stoichiometric ratios (gRNA:Cpf1 complexation ratios) of either 2 or 4 with a molar excess of gRNA. Figure 19 shows editing of CD34+ cells as determined by Illumina sequencing 72 hours after electroporation. RNP33, RNP64, RNP63 and RNP45 (Table 15) alone or Sp45 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)) or delivered in combination with ssODN OLI16431 (SEQ ID NO: 1040 in Table 7). Figure 20 shows editing in CD34+ cells as determined by Illumina sequencing. RNPs containing Cpf1 (SEQ ID NO: 1094) complexed to gRNAs with various 5′ DNA extensions (Table 15) were delivered alone or in combination with 8 μM OLI16431 (SEQ ID NO: 1040 in Table 7). . Figure 21 shows editing in CD34+ cells as determined by Illumina sequencing. RNPs containing gRNAs with matching 5' ends (RNP49 vs. RNP58 and RNP59 vs. RNP60, Table 15) were delivered to CD34+ cells and the effects of 3' modifications were assessed. In both comparisons, gRNA with 3'PS-OMe outperformed the unmodified 3' version 24 hours after electroporation. FIG. 22A shows editing in CD34+ cells as determined by Illumina sequencing 24 and 48 hours after electroporation. RNP58 (Table 15) was delivered to CD34+ cells at either a molar ratio of 2:1, 1:1 or 0.5:1 stoichiometry (complexing ratio of gRNA:Cpf1). At all doses tested, editing was highest when RNPs were complexed at a 2:1 ratio. FIG. 22B shows editing in CD34+ cells as determined by Illumina sequencing 24 and 48 hours after electroporation. RNP58 (Table 15) was delivered to CD34+ cells at either a molar ratio of 2:1, 1:1 or 0.5:1 stoichiometric ratio (complexing ratio of gRNA:Cpf1). At all doses tested, editing was highest when RNPs were complexed at a 2:1 ratio. FIG. 23A shows editing in CD34+ cells as determined by Illumina sequencing. Figures 45A and 45B show RNPs containing gRNAs with matching 5' ends but different 3' modifications delivered to CD34+ cells (Table 15) to assess the effect of 3' modifications or extensions. FIG. 23B shows editing in CD34+ cells as determined by Illumina sequencing. Figures 45A and 45B show RNPs containing gRNAs with identical 5' ends but different 3' modifications delivered to CD34+ cells (Table 15) to assess the effect of 3' modifications or extensions. Figures 24A-24C show editing in CD34+ cells and their erythrocyte progeny and HbF levels in erythrocyte progeny following delivery of RNPs targeting various cleavage sites within the HBG locus. FIG. 24A shows RNPs (Table 15) containing guide RNAs containing unmodified 5′ and 3′ terminal 1×PS-Ome. FIG. 24B shows an RNP (Table 15) containing a guide RNA containing a 5′ 2PS+20 DNA extension and a 1×PS-Ome at the 3′ end. FIG. 24C shows RNPs (Table 15) containing guide RNA containing 25 DNA extensions at the 5' end and 1xPS-Ome at the 3' end. FIG. 25 shows editing and HbF levels in erythroid progeny of CD34+ cells following delivery of 1 μM, 2 μM and 4 μM RNP58. FIG. 26 shows editing in CD34+ cells following Maxcyte electroporation of RNPs. RNP58, RNP26, RNP27 and RNP28 (Table 15) containing gRNA SEQ ID NO: 1051 complexed to different Cpf1 proteins (SEQ ID NOs: 1094, 1096, 1107, 1108) were delivered to CD34+ cells. Editing was determined by Illumina sequencing 24 and 48 hours after electroporation. Figure 27 shows editing in CD34+ cells following Maxcyte electroporation of RNPs. RNP58, RNP29, RNP30 and RNP31 (Table 15) containing the Cpf1 protein SEQ ID NO: 1094 complexed to guide RNAs with various 5' extensions were delivered to CD34+ cells. Editing was determined by Illumina sequencing 24 and 48 hours after electroporation. RNP30 was not tested at 1 μM due to limited cell numbers (nt). Figure 28 shows bulk compilation of CD34+ cell populations (black bars), progenitor cells (dark gray bars) and HSCs (light gray bars) as determined by Illumina sequencing 48 hours after electroporation. RNP58, RNP27 and RNP26 (Table 15) were delivered to CD34+ cells at 2 μM or 4 μM. Figure 29 shows bulk compilation of CD34+ cell populations (black bars), progenitor cells (dark gray bars) and HSCs (light gray bars) as determined by Illumina sequencing 48 hours after electroporation. RNP61, RNP62 and RNP34 (Table 15) (8 μM) were co-delivered to CD34+ cells along with ssODN OLI16431 (SEQ ID NO: 1040 in Table 7) (8 μM). Figure 30 shows bulk compilation of CD34+ cell populations (black bars), progenitor cells (dark gray bars) and HSCs (light gray bars) as determined by Illumina sequencing 48 hours after electroporation. RNP58 and RNP32 (Table 15) are delivered to CD34+ cells at 2 μM. Figure 31 shows bulk compilation of CD34+ cell populations (black bars), progenitor cells (dark gray bars) and HSCs (light gray bars) as determined by Illumina sequencing 48 hours after electroporation. RNP58 and RNP1 (Table 15) were delivered to CD34+ cells at 2 μM, 4 μM or 8 μM. Cells edited with 2 μM RNP1 were not sorted (N.S) and thus editing data are not available. Figure 32 shows indels of mPB CD34+ cells transplanted from the BM of 'NBSGW' mice 8 weeks after infusion of electroporated cells. RNP34 and RNP33 (Table 15) (8 μM) were co-delivered to CD34+ cells along with ssODN OLI16431 (SEQ ID NO: 1040 in Table 7) (6 μM). Figures 33A-33B show HbF expression by indels of transplanted mPB CD34+ cells and red blood cells from chimeric BM of "NBSGW" mice 8 weeks after infusion of electroporated cells. RNP33 or RNP34 (Table 15) together with Sp182 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)) (16 μM total RNP) Co-delivered to CD34+ cells. FIG. 33A shows indel frequencies in unfractionated bone marrow or flow-sorted individual populations of CD15+, CD19+, GlyA+ and Lin-CD34+ cells in mock-transfected (no RNP) or RNP-transfected cells. Lin-CD34+ cells were transfected with mock-transfected (no RNP) or RNP-transfected mPB CD34+ cells, non-irradiated NOD, B6. Defined as CD34+ cells that are negative for CD3, CD14, CD15, CD16, CD19, CD20 and CD56) from the bone marrow (BM) of SCID Il2rγ−/−Kit (W41/W41) (“NBSGW”) mice. Indels were determined for each cell population by Illumina sequencing. FIG. 33B shows HbF expression calculated by UPLC as γ/β-like (%) from erythroid cell lysates following 18 days of erythroid differentiation culture from whole chimeric BM. Figures 34A-34B show HbF expression by indels of transplanted mPB CD34+ cells and red blood cells from chimeric BM of "NBSGW" mice 8 weeks after infusion of electroporated cells. RNP61 or RNP62 (Table 15) (8 μM) was co-delivered to CD34+ cells with ssODN OLI16431 (SEQ ID NO: 1040 in Table 7) (8 μM). FIG. 34A shows indels of unfractionated bone marrow or flow-sorted individual populations of CD15+, CD19+, GlyA+ and Lin-CD34+ cells in mock-transfected (no RNP) or RNP-transfected cells. Lin-CD34+ cells (CD3, CD14, CD15, CD16, CD19, CD20 and CD56) from non-irradiated NBSGW mouse bone marrow (BM) infused with mock-transfected (no RNP) or RNP-transfected mPB CD34+ cells defined as CD34+ cells that are negative for Indels were determined for each cell population by Illumina sequencing. FIG. 34B shows HbF expression calculated by UPLC as γ/β-like (%) by erythroid cells following 18 days of erythroid differentiation culture from whole chimeric BM. FIG. 35. Intramedullary cells 8 weeks after infusion of mock-transfected (no RNP) mPB CD34 cells or mPB CD34 cells edited with RNP1 (4 or 8 μM) or RNP58 (2, 4 or 8 μM) (Table 15). human chimerism. Human chimerism and lineage rearrangement in BM (CD45+, CD14+, CD19+, glycophorin A (GlyA, CD235a+), lineage and CD34+ and mouse CD45+ marker expression) were determined by flow cytometry. Figure 36. Unsorted 8 weeks after infusion of mock-transfected (no RNP) mPB CD34+ cells or mPB CD34+ cells edited with RNP1 (4 or 8 μM) or RNP58 (2, 4 or 8 μM) (Table 15). shows indels in the bulk bone marrow of . Indels were determined by Illumina sequencing. FIG. 37 shows indel frequencies in unfractionated bone marrow or flow-sorted individual populations of CD15+, CD19+, GlyA+ and Lin-CD34+ cells in mock-transfected (no RNP) or RNP-transfected cells. Lin-CD34+ cells were transfected with mock-transfected (no RNP) or RNP-transfected mPB CD34+ cells, non-irradiated NOD, B6. Defined as CD34+ cells that are negative for CD3, CD14, CD15, CD16, CD19, CD20 and CD56 from the bone marrow (BM) of SCID Il2rγ−/−Kit (W41/W41) (“NBSGW”) mice. Indels were determined for each cell population by Illumina sequencing. Figure 38 shows HbF from GlyA+ fractions isolated from bone marrow 8 weeks after infusion of mock (no RNP) mPB CD34+ cells or mPB CD34+ cells edited with RNP58 (2, 4 or 8 μM) (Table 15). show. Figure 39 shows the colony-forming ability of cells from bone marrow flushes harvested 8 weeks after infusion of mock or edited human recruited CD34+ cells. Colony numbers and subtypes are indicated (GEMM: granulocyte-erythroid-monocyte-macrophage colony (black), GM: granulocyte-macrophage colony (dark grey), E: erythroid colony (light grey)). FIG. 40 shows the sequences of the Cpf1 protein variants listed in Table 14. Nuclear localization sequences are shown in bold and 6-histidine sequences are underlined. For example, with and without the addition of two or more nNLS sequences or a combination of nNLS and sNLS sequences (or other NLS sequences) to either the N-terminal/C-terminal positions and purification sequences such as 6-histidine sequences. NLS sequence identities, such as sequence additions, and additional permutations of N-terminal/C-terminal positions are within the scope of the presently disclosed subject matter. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Continuation of the sequences of the Cpf1 protein variants listed in Table 14. Figures 41A-41E show editing of the HBG distal CCAAT box region. FIG. 41A shows a schematic representation of Cpf1 (RNP34, Table 15) and SpCas9 (Sp35 RNP) cleavage sites in the HBG distal CCAAT box region. The Sp35 RNP is S. Sp35 gRNA, complexed with S. pyogenes wild-type (Wt) Cas9 protein, SEQ ID NO: 339 (i.e., containing the targeting domain of CUUGUCAAGGCUAUUGGUCA (RNA)); SEQ ID NO: 917 (i.e., CTTGTCAAGGCTATTGGTCA (DNA) ))including. The dark gray jagged line with arrow indicates the expected 4-nucleotide 5′ overhang after cleavage with RNP34. The gray dotted line marks the predicted cleavage site of RNP34. This is the predicted cleavage site of RNPs containing gRNAs containing the gRNA targeting domain sequence UAAUUUCUACUCUUGUAGAUCCUUGUCAAGGCUAUUGGUC (SEQ ID NO: 1022). The predicted cleavage site of Sp35 RNP is indicated by a straight gray line with an arrow. FIG. 41B shows the percentage of indels derived from NHEJ and MMEJ repair in CD34+ cell populations, progenitor cells, and HSCs. MMEJ indels are represented by black striped bars and NHEJ indels by white bars. Figures 41C and 41D show Gγ with indels ≤3 bp in length or greater than 3 bp in their HBG alleles encoding Gγ in erythrocytes from mPB CD34+ cells electroporated with Sp35 RNP or RNP34+Sp182 RNP. Chain expression levels (Gγ chain/[total β-like chain]) are depicted. Only clones with a monoalleleic 4.9 kb deletion (no gγ expression from one side of the chromosome) were analyzed, confirming that gγ expression is driven by a single HBG allele. rice field. Therefore, the expression levels shown represent the level of gγ expression by a single HBG gene in response to promoter-borne indels in cells lacking the gγ gene on the other chromosome. FIG. 41C shows the results grouped by indel lengths less than or equal to 3 bp or greater than 3 bp. FIG. 41D shows γ chain expression levels (gamma chain γ/[total β-like strands]). The position of the deletion is indicated on the X-axis. Xs indicate indels generated by Cas9 (Sp35 RNP) and circles indicate edits generated by Cpf1 (RNP34). FIG. 41E shows a single human mPB edited in the HBG promoter region with spCas9RNP “sp35” or Cpf1 RNP34 (HBG1-1-AsCpf1H800A-RNP) combined with “booster element” Sp182-Cas9-RNP (Table 8). Shown is the distribution level of γ chain expression relative to total β-like chains (γ chain/[γ chain + β chain]) as measured by UPLC in clonal erythroid progeny of CD182+ cells. Each filled circle represents the γ-globin protein level detected in clonal erythroid populations derived from single cells isolated by FACS sorting 48 hours after electroporation. Error bars indicate median and interquartile range. Figures 42A-42J show editing via Cpf1 or SpCas9 enzymatic cleavage. FIG. 42A depicts the percentage of NHEJ-mediated indels at distal CCAAT boxes resulting from editing of RNP58 (Cpf1) (Table 15) or Sp35 RNP (SpCas9). Indel size is indicated by the x-axis. FIG. 42B depicts the percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 3 bp, and indels less than or equal to 3 bp in distal CCAAT boxes resulting from editing with Cpf1 or SpCas9 RNPs. Indels of Sp35 RNP Cas9 in FIG. 42B (left pie chart): NHEJ>3 bp=21.42%; ≤3 bp=48.84%; MMEJ>3 bp=29.74%. BRNP58 Cpf1 indels in Figure 42B (right pie chart): NHEJ > 3 bp = 64.86%; ≤ 3 bp = 11.11%; MMEJ > 3 bp = 24.02%. FIG. 42C shows the wild-type (wt) allele and the top 18 indels along with the average percentage of indels for all samples and the corresponding final sequences after editing. Dashed lines represent deleted bases and vertical lines are cleavage sites (see also Figure 55A). The long dark gray line above the wt allele marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP58 (i.e., CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)) and the short black line marks the distal CCAAT box. (Here it appears to be reverse complemented). Base distances to TSS are marked with arrows. Gray boxes represent regions of homology indicative of MMEJ repair. FIG. 42D shows the wild-type (wt) allele and the top 18 indels along with the average percentage of indels for all samples and the corresponding final sequences after editing. Dashed lines represent deleted bases and vertical lines are cleavage sites (see also Figure 55A). The long dark gray line above the wt allele marks the DNA sequence of the gRNA targeting domain of the Sp35 RNP gRNA (i.e., CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 339)) and the short black line marks the distal CCAAT box. (here, it appears to be reverse complemented). Base distances to TSS are marked with arrows. Gray boxes represent regions of homology indicative of MMEJ repair. FIG. 42E shows the percentage of deleted bases along the target region of samples edited with RNP58 or Sp35 RNP. The black line represents RNP58 editing and the dashed line represents Sp35 RNP editing. Target region DNA sequences are indicated on the x-axis. The gray line marks the DNA sequence of the gRNA targeting domain of the Sp35 RNP gRNA (i.e., CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 339)) and the short black line marks the distal CCAAT box (here reverse-complemented). appear). FIG. 42F shows normalized profiles (maximum value of 1 for each) of deletions detected at each base on the target region of samples edited with RNP58 or Sp35 RNP. The black line represents RNP58 editing and the dashed line represents Sp35 RNP editing. Target region DNA sequences are indicated on the x-axis. The gray line marks the DNA sequence of the gRNA targeting domain of the Sp35 RNP gRNA (i.e., CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 339)) and the short black line marks the distal CCAAT box (here reverse-complemented). appear). FIG. 42G shows the percentage of indels detected as a function of deletion size (negative values on the x-axis) or insertions (positive values on the x-axis). The black line represents RNP58 editing and the dashed line represents Sp35 RNP editing. Figure 42H shows the percentage of indels detected at each position in the target region. Sp35 RNP is represented by a dashed line. RNP58 is represented by a solid line. Target region DNA sequences are indicated on the x-axis. The gray line marks the DNA sequence of the gRNA targeting domain of the Sp35 RNP gRNA (i.e., CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 339)) and the short black line marks the distal CCAAT box (here reverse-complemented). appear). FIG. 42I shows the frequency correlation of shared indels detected above 0.1% in either Sp35 RNP or RNP58 edited cells. FIG. 42J, Frequency correlation of all indels detected in at least one sample. Indels that were not detected in Sp35 RNP-edited cells are displayed to the left of the vertical line at 1E-5. Indels that were not detected in RNP58-edited cells are displayed below the horizontal line at 1E-5. Triangles represent deletions of 3 bp or less, circles represent deletions of more than 3 bp. Figures 43A-43O show editing of RNP32 leading to long-term engraftment, indel maintenance, and high HbF induction in vitro. FIG. 43A depicts the percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 3 bp, and indels less than or equal to 3 bp in distal CCAAT boxes resulting from editing by RNP32 24 h following electroporation. . NHEJ > 3 bp = 66.10%; ≤ 3 bp = 12.60%; MMEJ > 3 bp = 27.10%. FIG. 43B depicts the percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 3 bp, and indels less than or equal to 3 bp in distal CCAAT boxes resulting from editing by RNP32 48 h following electroporation. . NHEJ > 3 bp = 65.30%; ≤ 3 bp = 12.70%; MMEJ > 3 bp = 27.80%. FIG. 43C depicts the percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 3 bp, and indels less than 3 bp in distal CCAAT boxes resulting from editing with RNP32 72 h following electroporation. . NHEJ > 3 bp = 64.70%; ≤ 3 bp = 11.90%; MMEJ > 3 bp = 29.20%. FIG. 43D depicts the percentage of indels at the distal CCAAT box mediated through NHEJ repair resulting from editing by RNP32 at 24, 48, and 72 hours following electroporation and pre-injection. FIG. 43E depicts the percentage of indels at the distal CCAAT box mediated through NHEJ repair resulting from editing by RNP32 24 hours following electroporation and pre-injection. FIG. 43F depicts the percentage of indels at the distal CCAAT box mediated through NHEJ repair resulting from editing by RNP32 48 h following electroporation and pre-injection. FIG. 43G depicts the percentage of indels at the distal CCAAT box mediated through NHEJ repair resulting from editing by RNP32 72 hours following electroporation and pre-injection. FIG. 43H depicts the percentage of all indels in distal CCAAT boxes resulting from editing with RNP32 at 24, 48, and 72 hours following electroporation and pre-injection. FIG. 43I depicts the percentage of all indels in the distal CCAAT box from editing with RNP32 24 hours following electroporation and pre-injection. FIG. 43J depicts the percentage of all indels in the distal CCAAT box resulting from editing with RNP32 48 hours following electroporation and pre-injection. FIG. 43K depicts the percentage of all indels in the distal CCAAT box resulting from editing with RNP32 72 hours following electroporation and pre-injection. FIG. 43L depicts the percentage of indels in long-term repopulating CD34+ cells from NBSGW mice pre-injection of RNP32-edited mPB CD34+ cells (“pre-infusion”) and after 16 weeks engraftment (“BM”). FIG. 43M depicts human chimerism in bone marrow 16 weeks after injection of mock (no RNP added) mPB CD34+ cells or RNP32-edited mPB CD34+ cells (Table 15). Human chimerism and lineage rearrangement in BM (CD19+, CD15+, CD235A+), lineage, and CD34+, and mouse CD45+ marker expression) were determined by flow cytometry. FIG. 43N depicts the percentage of F-positive cells in mock (no RNP added) and CD235a+ (GlyA+) erythrocytes derived from RNP32-edited CD34+ cells. FIG. 43O depicts the percentage of HbF expressed by the expression level of γ-globin chain relative to total β-like globin chains (γ chain/[γ chain + β chain]) as measured by UPLC analysis of CD235a+ (GlyA+) erythrocytes. do. Figures 44A-44B show high polyclonality in NBSGW mice injected with RNP32-edited CD34+ cells. FIG. 44A High polyclonal over 20 weeks in blood (8, 12, 16, and 20 weeks post-injection) in mice injected with RNP32-edited CD34+ cells (mouse A, mouse B, mouse C, mouse D). show gender. "0" represents data from pre-infused RNP32-edited mPB CD34+ cells. In Figures 44A and 44B, each shade of gray within the graphical representation represents a different idenl signature, with the most frequent indel types near the x-axis and the least frequent indel types near the top of the plot. ing. FIG. 44B shows high polyclonality in the bone marrow (BM) of NBSGW mice injected with RNP32-edited mPB CD34+ cells 20 weeks after engraftment. FIG. 45 shows a schematic representation of the β-globin locus. "CD34+ cells ^a " means CD34+ hematopoietic stem and progenitor cells, "HS" means hypersensitive site, "LCR" means locus control region, and "TSS" means transcription start site. FIG. 46A shows viability of normal and SCD CD34+ cells 1-3 days after electroporation with 6 μM RNP32 compiled from two independent experiments. Individual data points and mean values for each treatment group are shown. N=4 for normal donors; N=5 for sickle donors (CEL238-001 [normal CD34+ cells] and CEL211-001 [SCD CD34+ cells] were each tested twice). FIG. 46B shows viability of CD34 cells after electroporation with RNP32. ^a , applicable only to RNP32-electroporated cells. ^b , Viability was not assessed due to insufficient cell numbers available. FIG. 47A shows indel levels in normal and SCD CD34+ cells 1-3 days after electroporation with 6 μM RNP32 compiled from two independent experiments. Individual data points and mean values for each treatment group are shown. N=4 for normal donors; N=5 for sickle donors. (CEL238-001 [normal CD34+ cells] and CEL211-001 [SCD CD34+ cells] were each tested twice. Indels = insertions and/or deletions. FIG. 47B shows comparable and efficient editing in RNP32-edited CD34+ cells from normal donors and SCD patients. The percentage of indels was assessed on unedited and RNP32-edited CD34+ cells from normal donors (n=3) and unedited and RNP32-edited CD34+ cells from patients with SCD (n=4). Unedited cells did not undergo electroporation. FIG. 47C shows indel levels of CD34+ cells after electroporation with RNP32. ND=not determined (insufficient sample to perform sequence analysis). Figures 48A-48D depict PCR primers and mixes for digital droplet polymerase chain reaction (ddPCR). FIG. 48A is a schematic showing the positions of the ddPCR primers and probes relative to the RNP32 cleavage site. Figure 48B shows primer and probe sequences for 4.9 kb fragment deletion assessment. FIG. 48C shows the composition of master mixes 1 and 2 for ddPCR assays 6 and 9. FIG. 48D shows the composition of Master Mix 3. FIG. 49A shows the frequency of 4.9 kb fragment deletion in normal and SCD CD34+ cells 1 day after electroporation with 6 μM RNP32. Data are from one study (SCD014). Each dot represents one sample. Untreated cells did not undergo electroporation. FIG. 49B shows the frequency of the 4.9 kb deletion in CD34+ cells after electroporation with RNP32. NA = data not available due to digital droplet polymerase chain reaction assay failure. Untreated cells did not undergo electroporation. FIG. 50A shows fold expansion of erythroid progeny of CD34+ cells. Normal and SCD CD34+ cells, untreated or electroporated with 6 μM RNP32, were placed in erythrocyte-inducing conditions for 18 days. Data were compiled from two independent experiments. Each dot represents one sample. FIG. 50B shows the nuclear elimination frequency of erythroid progeny of CD34+ cells. Normal and SCD CD34+ cells, untreated or electroporated with 6 μM RNP32, were placed in erythrocyte-inducing conditions for 18 days. Data were compiled from two independent experiments. Each dot represents one sample. FIG. 51A shows assessment of HbF induction in erythroid progeny in two experiments. Normal and SCD CD34+ cells, untreated or electroporated with 6 μM RNP32, were placed in erythrocyte-inducing conditions for 18 days. The relative abundance of globin chains in erythrocyte lysates was analyzed by RP-UPLC and HbF levels were calculated as HbF (%) = (Aγ + Gγ)/(Aγ + Gγ + β) (%). FIG. 51B shows the frequency of HbF+ RBCs assessed by flow cytometry in one independent experiment. In Figures 51A and 51B, each dot represents one sample. A paired T-test was performed to determine if the difference between RNP32-treated and untreated samples was statistically significant. ^* p<0.05, ^** p<0.01, ^*** p<0.0001. RP-UPLC = reversed phase ultra high performance liquid chromatography. Untreated cells did not undergo electroporation. FIG. 51C shows comparable and robust ex vivo HbF expression in RNP32-edited CD34+ cells from normal donors and patients with SCD. Percentage of HbF relative to total β-like globin chains in unedited and RNP32-edited CD34+ cells from normal donors (n=3), unedited and RNP32-edited CD34+ cells from patients with SCD (n=4). It is indicated by the expression level of globin chains (γ chain/[γ chain + β chain]). Unedited cells did not undergo electroporation. Figure 52 shows that RNP32-edited CD34+-derived red blood cells (RBCs) from SCD patients were exposed to sodium metabisulfite and examined under a microscope showed reduced sickle cell disease compared to unedited RBCs from SCD patients. indicate that The percentage of sickled RBCs is shown for unedited SCD-derived RBCs and RNP32-edited SCD-derived RBCs. Mean HbF percentages are also shown for unedited SCD-derived RBCs and RNP32-edited SCD-derived RBCs. FIG. 53A Measured loss of deformability as a result of oxygen depletion over time, the sickling point of SCD RBCs, followed by deformation of RBCs during subsequent reoxygenation visualized with Oxygenscan. Figure 2 shows a graphical representation of the acquisition of ability. EImax represents the RBC deformability under normoxia and EImin represents the deformability during deoxygenation. The sickling point (PoS) reflects the pO2 at which sickling begins and a >5% decrease in EI is observed during deoxygenation. EI = elongation index. Figures 53B-53E show the assessment of RBC deformability. Cultured RBCs from 3 batches of untreated normal CD34+ cells (Fig. 53B) and 4 batches of untreated or RNP32-edited SCD CD34+ cells (Fig. 53C) were analyzed on the Lorrca ektacytomete and under shear stress when oxygen concentration was reduced. is measured and expressed as the elongation index. FIG. 53C depicts RBCs cultured from CD34+ cells that have not been electroporated with RNP32 (untreated) as black lines (lower lines in all plots to at least 25 mmHg), RBCs cultured from RNP32 edited. RBCs cultured from SCD CD34+ cells are represented by the dark gray line (upper line in all plots up to at least 25 mmHg). FIG. 53D plots for each SCD RBC sample the sickling point, which represents the relative oxygen tension when the SCD RBCs begin to sickle during deoxygenation. FIG. 53E shows the minimum elongation index for each SCD RBC sample, which approximates the flexibility of RBCs when deoxygenated. Each line connects RBCs cultured from untreated and RNP32 edited cells from the same donor in the same experiment. CEL211-001 was tested twice in two independent experiments. A paired T-test was performed to determine if the difference between cultured RBCs from RNP32-treated and untreated samples was statistically significant. ^** p<0.01, ^*** p<0.001. FIG. 54A shows rheological evaluation of cultured RBCs. Untreated CD34+ cells and untreated or RNP32 electroporated SCD CD34+ cells were placed in erythrocyte-inducing conditions for 18 days to generate red blood cells. The rheological behavior of RBCs cultured under different concentrations of oxygen was evaluated using a microfluidic platform. The percentage speed drop was calculated based on the difference in speed at the specified oxygen concentration and atmospheric oxygen level (21%). Data shown are mean ± standard deviation. N=5: untreated SCD samples, N=5: RNP32 treated SCD samples, N=4: untreated normal samples. Mean values were compared between cultured RBCs from untreated and corresponding RNP32-edited SCD samples using a paired T-test. ^* p<0.05, ^*** p<0.001, ^*** p<0.0001. FIG. 54B shows a summary of the percentage speed reduction by varying the oxygen concentration. ^b indicates that the sample clogged during evaluation and the experiment was aborted. Only data collected before the occlusion was formed are reported. The data are plotted in Figure 54A. FIG. 54C shows unedited CD34+ cells from SCD patients when cultured from RNP32-edited CD34+ cells from SCD patients were placed in microfluidic channels mimicking capillary blood flow at a range of oxygen levels. Compared to RBCs cultured from cells, RBCs from normal donors are shown to have improved rheological properties more similar. Percentages of normalized velocities are shown for RBCs cultured from CD34+ cells from unedited normal donors (diamonds), from CD34+ cells from unedited SCD− donors (triangles), and at various oxygen percentages. RBCs (circles) cultured from RNP32-edited SCD-induced CD34+ cells were evaluated. Typical oxygen levels observed in the venous circulation are approximately 4% to 6% oxygen. FIG. 54D shows the correlation between HbF induction and rheological behavior. The level of HbF represented by the SCD samples (x-axis) was plotted against the corresponding percent rate reduction (y-axis) at oxygen concentrations of 0%, 2%, 4% and 6%. A simple linear regression analysis was performed and the coefficient of determination is shown in each panel. FIG. 54E RBCs cultured from CD34+ cells from unedited SCD (triangles) and from CD34+ cells from RNP32-edited SCD (circles) mimic capillary blood flow at 4% oxygen level. Figure 2 shows the correlation between HbF levels and velocities when placed in microfluidic channels. The percentage of HbF is indicated by the expression level of γ-globin chain relative to total β-like globin chains (γ chain/[γ chain + β chain]). FIG. 55A is distal with 0-based hg38 coordinates chr11: 5,249,949-5,249,987 (+) in HBG1 and chr11: 5,254,873-5,254,911 (+) in HBG2 A schematic representation of the CCAAT box region is shown. The black line marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP32 (ie, CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)). A black box marks the distal CCAAT box. The dark gray jagged line with arrow indicates the expected 4-nucleotide 5′ overhang after cleavage with RNP32. The gray dotted line marks the predicted cleavage site of RNP32. The base before the cleavage site is marked with a black arrow at base −118 relative to the TSS. Base distances flanking the TSS are marked with black arrows at the end of the sequence (TSS: -93 and -130). FIG. 55B shows oligonucleotides used to sequence the indel profile generated by RNP32. FIG. 55C shows amplicons used for analysis of RNP32 editing. FIG. 55D shows an example of indel_id used to characterize the indel profile of RNP32. Indel_id is a string that identifies an indel and is indicated as indel_start_position+_+indel_length+_+ID. ID is NA for deletions and inserted sequence for insertions. FIG. 56 depicts the percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 32 bp, and indels less than or equal to 3 bp in distal CCAAT boxes resulting from editing with RNP32: NHEJ greater than 3 bp=65. ≤3 bp = 8.6%; MMEJ >3 bp = 25.5%. Figure 57 shows the wild-type (wt) allele and the top 20 indels along with the average percentage in indels for all samples and the corresponding final sequences after editing. Dashed lines represent deleted bases and vertical lines are cleavage sites (see also Figure 55A). The black line marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP32 (i.e., CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)) and the short black line marks the distal CCAAT box (here seen reverse-complemented). . Base distances to TSS are marked with arrows. Figure 58 shows the percentage of deletions detected as a function of the distance from the deletion center to the cleavage site, represented by the vertical gray dashed line taken as the middle of the 5' overhang. The highest peak is located -6 bp relative to and from the cleavage site (TSS: -113) towards the TSS (see also Figure 55A). Profiles are shown as black for normal donor samples (Normal, N=4), light gray for sickle cell donor samples (SCD, N=5), and dark gray for an additional set of normal donor samples (Normal=5, SCD1). shown. A second set of normal samples was from the SCD1 study, mobilized using G-CSF and plelixafor, and generated using a large scale process. The black line marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP32 (i.e., CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)) and the short black line marks the distal CCAAT box (here seen reverse-complemented). . Figure 59 shows the percentage of indels detected as a function of deletion size (negative values on the x-axis) or insertions (positive values on the x-axis). Vertical lines separate insertions and deletions. The highest peak is a size 18 deletion corresponding to indel 159_-18_NA. Profiles are shown as black for normal donor samples (Normal, N=4), light gray for sickle cell donor samples (SCD, N=5), and dark gray for an additional set of normal donor samples (Normal=5, SCD1). shown. A second set of normal samples was from the SCD1 study, mobilized using G-CSF and plelixafor, and generated using a large scale process. Figure 60 shows the average percentage of indel length between 50 and -50. A positive value indicates an insert. Negative values indicate deletions. A series of mean percentages of indel lengths between 50 and -50 are shown. A series of mean percentages of indel lengths between 50 and -50 are shown. Figure 61 shows the percentage of deleted bases along the target region of RNP32 edited samples. Profiles are shown as black for normal donor samples (Normal, N=4), light gray for sickle cell donor samples (SCD, N=5), and dark gray for an additional set of normal donor samples (Normal=5, SCD1). shown. A second set of normal samples was from the SCD1 study, mobilized using G-CSF and plelixafor, and generated using a large scale process. Target region DNA sequences are indicated on the x-axis. The black line marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP32 (i.e., CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)) and the short black line marks the distal CCAAT box (here seen reverse-complemented). . The gray vertical dashed line represents the cut point, taken as the middle of the 5' overhang (see also Figure 55A). FIG. 62 shows normalized profiles (maximum value of 1 for each) of deletions detected at each base on the target region of samples edited with RNP32. Profiles are shown in black for normal donor samples (Normal, N=4) and light gray for sickle cell donor samples (SC). A second set of normal samples was from the SCD1 study, mobilized using G-CSF and plelixafor, and generated using a large scale process. The sequence of the region target regions is indicated on the x-axis. The black line marks the DNA sequence of the gRNA targeting domain of the gRNA of RNP32 (i.e., CCUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1254)) and the short black line marks the distal CCAAT box (here seen reverse-complemented). . The gray vertical dashed line represents the cut point, taken as the middle of the 5' overhang (see also Figure 55A). FIG. 63 shows the number of indels detected across multiple samples. The count of indels as a function of the number of samples in which indels were detected is shown. Figure 64 shows the reproducibility of indels across all 14 samples as a function of their average percentage of indels. Shown is the average percentage in indels (y-axis) grouped by the number of samples in which indels were detected (x-axis). The 0.22% gray horizontal line marks the lowest percentage of indels among the top 55 indels. Figures 65A-65B show on-target indel levels of CD34+ cells based on RNP32 concentration upon electroporation. Total on-target editing indel levels were determined via Illumina sequencing on days 1 (FIG. 65A) and 2 (FIG. 65B) after electroporation of CD34+ cells with the indicated concentrations of RNP32. was done. Data were compiled from eight independent experiments. A total of 5 RNP batches and 4 lots of normal donor CD34+ cells were tested with 11 concentrations of RNP32 ranging from 0.125 μM to 8 μM. Each dot represents an individual electroporation. Indels = insertions and/or deletions. FIG. 66 shows a summary of cell viability on day 1 electroporation with RNP32. Data marked with ^* indicate that data were excluded because differential scanning fluorescence measurements revealed insufficient complexation of RNP32. FIG. 67 shows a summary of on-target indel levels 1 day after electroporation with RNP32. Data marked with ^* indicate that data were excluded because differential scanning fluorescence measurements revealed insufficient complexation of RNP32. FIG. 68 shows a summary of on-target indels 2 days after electroporation. Data marked with ^* indicate that data were excluded because differential scanning fluorescence measurements revealed insufficient complexation of RNP32. Figures 69A-69B show the frequency of the 4.9 kb fragment deletion in edited CD34+ cells 1 and 2 days after electroporation. FIG. 69A. The frequency of 4.9 kb fragment deletions between the two RNP32 cleavage sites in CD34+ cells was assessed by ddPCR assays 1 and 2 days after electroporation with the indicated concentrations of RNPs. . FIG. 69B shows the ratio of 4.9 kb deletions to indels was calculated by dividing the level of deletions by the level of indels for each sample. Data were compiled from three independent experiments. A total of two RNP batches and two lots of normal donor CD34+ cells were tested with eight concentrations of RNP32 ranging from 0.125 μM to 8 μM. Each dot represents an individual electroporation. Indels = insertions and/or deletions. Figure 70 shows a summary of the 4.9 kb fragment deletion frequencies 1 and 2 days after electroporation with RNP32. Figures 71A-71B show on-target indel levels of hematopoietic stem and progenitor cell subpopulations. FIG. 71A. CD34+ cells were sorted into CMP, MPP, and LT-HSC subpopulations based on surface immunophenotype 2 days after electroporation. Shows on-target indel levels as determined by Illumina sequencing. FIG. 71B shows the ratio of on-target indel levels to total CD34+ cells within the phenotypic LT-HSC population. A ratio of 1 (dotted line) indicates that LT-HSCs have the same on-target indel levels as total CD34+ cells. CMP = common myeloid progenitor; indels = insertions and/or deletions; LT-HSC = long-term hematopoietic stem cells; MPP = multipotent progenitor cells. FIG. 72 shows a summary of on-target indel levels in total CD34+ cells and sorted subpopulations 2 days after electroporation. CMP = common myeloid progenitors; indels = insertions and/or deletions; LT HSCs = long-term hematopoietic stem cells; MPP = multipotent progenitor cells. Figures 73A-73B show the frequency of deletion of the 4.9 kb fragment in total CD34+ cells and subpopulations of sorted hematopoietic stem and progenitor cells. FIG. 73A CD34+ cells were sorted into CMP, MPP, and LT HSC subpopulations based on surface immunophenotype 2 days after electroporation. Frequency of 4.9 kb deletions determined by ddPCR is shown. FIG. 73B shows the ratio of 4.9 kb deletions to indels was calculated by dividing the level of deletions by the level of indels for each sample. A lower ratio indicates a lower propensity for the 4.9 kb deletion to occur within the population. Multiple comparisons were performed using the Friedman test. ^* p=0.01, ^*** p<0.0001. CMP = common myeloid progenitors; indels = insertions and/or deletions; LT HSCs = long-term hematopoietic stem cells; MPP = multipotent progenitor cells. Figure 74 shows a summary of the 4.9 kb fragment deletion frequencies in total CD34+ cells and sorted subpopulations 2 days after electroporation with RNP32. CMP = common myeloid progenitors; indels = insertions and/or deletions; LT-HSC = long-term hematopoietic stem cells; MPP = multipotent progenitor cells; RNP = ribonucleoprotein.

Definitions and Abbreviations Unless otherwise specified, each of the following terms has the meaning associated with it in this section.

The indefinite articles "a" and "an" refer to at least one of the associated nouns and are used interchangeably with the terms "at least one" and "one or more" . For example, "module" means at least one module or one or more modules.

The conjunctions "or" and "and/or" are used synonymously as non-exclusive disjunctives.

A "domain" is used to denote a segment of a protein or nucleic acid. Domains need not have any particular functional property unless otherwise specified.

The term "exogenous trans-acting factor" includes (a) interacting with an RNA-guided nuclease or gRNA by means of modification such as insertion or fusion of peptides or nucleotides into the RNA-guided nuclease or gRNA; Refers to any peptide or nucleotide component of a genome editing system that interacts to alter its helical structure. Peptide or nucleotide insertions or fusions include, without limitation, direct covalent linkages between RNA-guided nucleases or gRNAs and exogenous trans-acting factors and/or RNA/protein interaction domains such as the MS2 loop and DZ, Lim or Non-covalent binding mediated by insertion or fusion of protein/protein interaction domains such as SH1, 2 or 3 domains can be included. Other specific RNA and amino acid interaction motifs will be familiar to those of skill in the art. Trans-acting factors generally can include transcriptional activators.

The term "booster element" refers to the amount of target nucleic acid without a booster element when co-delivered with a ribonucleoprotein (RNP) complex comprising gRNA complexed to an RNA-guided nuclease ("gRNA-nuclease-RNP"). Refers to an element that increases editing of a target nucleic acid relative to editing. In certain embodiments, co-delivery may be sequential or simultaneous. In certain embodiments, the booster element is a RNP complex comprising a death guide RNA complexed to a WT Cas9 protein, a Cas9 nickase protein (e.g., Cas9 D10A protein) or an enzymatically inactive Cas9 (eiCas9) protein can be In certain embodiments, the booster element can be an RNP complex comprising a guide RNA complexed to a Cas9 nickase protein (eg, Cas9 D10A protein) or an enzymatically inactive Cas9 (eiCas9) protein. In certain embodiments, the booster element can be single-stranded or double-stranded donor template DNA. In certain embodiments, one or more booster elements may be co-delivered with gRNA-nuclease-RNP to increase editing of the target nucleic acid. In certain embodiments, booster elements may be co-delivered with RNPs, including gRNA complexed to Cpf1 molecules (“gRNA-Cpf1-RNP”), to increase editing of the target nucleic acid.

A "productive indel" refers to an indel (deletion and/or insertion) that results in HbF expression. In certain embodiments, productive indels can induce HbF expression. In certain embodiments, productive indels may result in increased levels of HbF expression.

An "indel" is an insertion and/or deletion in a nucleic acid sequence. Indels can be the product of repair of DNA double-strand breaks, such as the double-strand breaks created by the genome editing system of the present disclosure. Indels are most commonly formed when a break is repaired by a "faulty" repair pathway, such as the NHEJ pathway described below.

"Gene conversion" refers to the alteration of a DNA sequence by incorporation of endogenous homologous sequences (eg, homologous sequences within gene arrays). "Gene correction" refers to alteration of a DNA sequence by incorporation of exogenous homologous sequences, such as exogenous single- or double-stranded donor template DNA. Gene conversion and gene correction are products of the repair of DNA double-strand breaks by the HDR pathway, such as those described below.

Indels, gene conversions, gene corrections and other genome editing results are typically analyzed by sequencing (most commonly by "next generation" or "sequencing-by-synthesis" methods, although Sanger sequencing is still used). obtained) and quantified by the relative frequency of numerical changes (eg, ±1, ±2 or more bases) in all sequencing reads. DNA samples for sequencing can be prepared by a variety of methods known in the art, including amplification of sites of interest by the polymerase chain reaction (PCR), Tsai 2016, incorporated herein by reference. can involve the capture of DNA ends caused by double-strand breaks as in the GUIDEseq process described in , or can be prepared by other means well known in the art. Genome editing results may also be assessed by in situ hybridization methods such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France) and any other suitable method known in the art.

"Alt-HDR", "alternative homology-directed repair" or "alternative HDR" are used interchangeably to refer to a homologous nucleic acid (e.g., an endogenous homologous sequence such as a sister chromatid or an exogenous nucleic acid such as a template nucleic acid). ) to repair DNA damage. Alt-HDR differs from canonical HDR in that the process utilizes different pathways than canonical HDR and can be inhibited by the canonical HDR mediators RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid templates, whereas standard HDR generally involves double-stranded homologous templates.

"Canonical HDR," "canonical homology-directed repair," or "cHDR" repairs NA damage using homologous nucleic acids (e.g., endogenous homologous sequences such as sister chromatids or exogenous nucleic acids such as template nucleic acids). refers to the process. Standard HDR typically functions when there is a significant excision at the double-strand break, forming at least one single-stranded portion of DNA. In normal cells, cHDR typically undergoes a series of actions including recognition of breaks, stabilization of breaks, excision, stabilization of single-stranded DNA, formation of DNA crossover intermediates, degradation of crossover intermediates and ligation. Accompanied by a process. The process requires RAD51 and BRCA2, and homologous nucleic acids are typically double-stranded.

Unless otherwise specified, the term "HDR" as used herein includes both standard HDR and alt-HDR.

"Non-homologous end joining" or "NHEJ" refers to ligation-mediated repair and/or non-template-mediated repair, such as canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn is followed by microhomology-mediated end joining ( MMEJ), single-strand annealing (SSA) and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

"Substitution" or "substituted" when used in reference to modification of a molecule (eg, nucleic acid or protein) does not require process limitations, merely indicating the presence of a replacement entity.

"Subject" means a human, mouse or non-human primate. A human subject can be of any age (eg, infant, child, young adult or adult) and can be afflicted with a disease or in need of a genetic modification.

"treat", "treating" and "treatment" means to suppress a disease, i.e. arrest or prevent its onset or progression; treating a disease in a subject (eg, a human subject), including one or more of alleviating one or more symptoms of; curing the disease.

"Prevent", "preventing" and "prevention" means (a) avoiding or eliminating a disease; (b) affecting a predisposition to a disease; or (c) preventing at least one symptom of a disease. Refers to the prevention of disease in a subject, including preventing or delaying onset.

"Kit" refers to any collection of two or more components that together constitute a functional unit that can be used for a particular purpose. By way of illustration (and not limitation), one kit according to the present disclosure may include a guide RNA complexed or capable of complexing with an RNA-guided nuclease (e.g., suspended therein or suspendable) with a pharmaceutically acceptable carrier. In certain embodiments, the kit may include booster elements. For example, a kit can be used to introduce a complex into a cell or subject for the purpose of causing a desired genomic modification in such a cell or subject. The components of the kit can be packaged together or they can be packaged separately. Kits according to the disclosure optionally also include instructions for use (DFU) that, for example, describe the use of the kit according to the methods of the disclosure. A DFU may be physically packaged with the kit, or it may be provided to the kit user, eg, by electronic means.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" refer to sequences of nucleotide bases (also referred to as "nucleotides") in DNA and RNA. refers to any chain of two or more nucleotides. The polynucleotides, nucleotide sequences, nucleic acids, etc. can be single-stranded or double-stranded, chimeric mixtures or derivatives or modified versions thereof. They can be modified, for example, at base moieties, sugar moieties or phosphate backbones to improve the stability of the molecule, its hybridization parameters and the like. Nucleotide sequences typically carry genetic information, including, but not limited to, information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotides and both sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases.

Conventional IUPAC notation is used in the nucleotide sequences presented herein, as shown in Table 1 below (Cornish-Bowden A, Nucleic Acids Res. 1985 May 10;13(9):3021-30). However, it should be noted that "T" denotes "thymine or uracil" when the sequence can be encoded by either DNA or RNA, eg, in a gRNA targeting domain.

The terms "protein", "peptide" and "polypeptide" are used interchangeably and refer to a continuous chain of amino acids linked together via peptide bonds. The term includes individual proteins, groups or complexes of proteins bound together as well as fragments or portions, variants, derivatives and analogs of such proteins. Peptide sequences are presented herein using conventional notation, starting at the amino or N-terminus on the left and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.

A designation such as "CCAAT box target region" refers to sequences located 5' to the transcription start site (TSS) of the HBG1 and/or HBG2 genes. The CCAAT box is a highly conserved motif within the promoter regions of α-like and β-like globin genes. Regions within or near the CCAAT box play an important role in the regulation of globin genes. For example, the γ-globin distal CCAAT box is associated with heritable persistence of fetal hemoglobin. Several transcription factors have been reported to bind overlapping CCAAT box regions of the γ-globin promoter such as NF-Y, COUP-TFII (NF-E3), CDP, GATA1/NF-E1 and DRED. (Martyn 2017). Without wishing to be bound by theory, it is believed that the binding site of the transcriptional activator NF-Y overlaps with the transcriptional repressor of the γ-globin promoter. HPFH mutations present in the distal γ-globin promoter region, for example in or near the CCAAT box, alter the competitive binding of these factors, thus contributing to increased γ-globin expression and elevated HbF levels. can. The genomic locations provided herein for HBG1 and HBG2 are based on the coordinates provided in NCBI Reference Sequence NC_000011 "Homo sapiens chromosome 11, GRCh38.p12 Primary Assembly, (Version NC_000011.10)". The distal CCAAT boxes of HBG1 and HBG2 are HBG1 and HBG2 c. -111 to -115 (genomic positions are Hg38 Chr11:5,249,968 to Chr11:5,249,972 and Hg38 Chr11:5,254,892 to Chr11:5,254,896, respectively) . HBG1 c. The -111 to -115 region is exemplified at positions 2823 to 2827 in SEQ ID NO:902 (HBG1), HBG2 c. The -111 to -115 region is exemplified at positions 2747 to 2751 in SEQ ID NO:903 (HBG2). In certain embodiments, "CCAAT box target region" refers to the region of or near the distal CCAAT box, comprising nucleotides of the distal CCAAT box and 25 nucleotides upstream (5') and downstream of the distal CCAAT box. (3') 25 nucleotides (i.e., HBG1/2 c. -86 to -140) (genomic locations are Hg38 Chr11:5249943 to Hg38 Chr11:5249997 and Hg38 Chr11:5254867 to Hg38 Chr11:5254921, respectively) ). HBG1 c. The −86 to −140 region is exemplified at positions 2798 to 2852 in SEQ ID NO:902 (HBG1), HBG2 c. The -86 to -140 region is exemplified at positions 2723 to 2776 in SEQ ID NO:903 (HBG2). In other embodiments, "CCAAT box target region" refers to the region of or near the distal CCAAT box, comprising the nucleotides of the distal CCAAT box and 5 nucleotides upstream (5') and downstream of the distal CCAAT box. (3′) 5 nucleotides (i.e., HBG1/2c.-106 to -120 (genomic locations are Hg38 Chr11:5249963 to Hg38 Chr11:5249977 (HGB1 and Hg38 Chr11:5254887 to Hg38 Chr11:5254901, respectively)) and The HBG1 c.-106 to -120 region is exemplified in SEQ ID NO:902 (HBG1) from positions 2818 to 2832, and the HBG2 c.-106 to -120 region is illustrated in SEQ ID NO:903 (HBG2) from positions 2742 to 2756. The term "CCAAT box target site modification" refers to modification (eg, deletion, insertion, mutation) of one or more nucleotides in the CCAAT box target region, etc. Exemplary CCAAT box targets Examples of region alterations include, without limitation, 1 nt deletion, 4 nt deletion, 11 nt deletion, 13 nt deletion and 18 nt deletion and −117 G>A alteration. The terms "box" and "CAAT box" may be used interchangeably.

Notations such as "c. -114 to -102 region", "c. -102 to -114 region", "-102:-114", "13nt target region" refer to genomic locations Hg38 Chr11:5,249, respectively. 959 to Hg38 Chr11:5,249,971 and Hg38 Chr11:5,254,883 to Hg38 Chr11:5,254,895 refer to sequences 5′ of the transcription start sites (TSS) of the HBG1 and/or HBG2 genes. . HBG1 c. The -102 to -114 region is exemplified at positions 2824 to 2836 in SEQ ID NO:902 (HBG1), HBG2 c. The -102 to -114 region is exemplified at positions 2748 to 2760 in SEQ ID NO:903 (HBG2). Terms such as "13nt deletion" refer to deletion of the target region of 13nt.

Notations such as "c. -121 to -104 region", "c. -104 to -121 region", "-104:-121", "18nt target region" refer to genomic locations Hg38 Chr11:5,249, respectively. 961 to Hg38 Chr11:5,249,978 and Hg38 Chr11:5,254,885 to Hg38 Chr11:5,254,902 refer to sequences 5′ of the transcription start sites (TSS) of the HBG1 and/or HBG2 genes. . HBG1 c. The -104 to -121 region is exemplified at positions 2817 to 2834 in SEQ ID NO:902 (HBG1), HBG2 c. The -104 to -121 region is exemplified at positions 2741 to 2758 in SEQ ID NO:903 (HBG2). Terms such as "18nt deletion" refer to deletion of the target region of 18nt.

Notations such as "c. -105 to -115 region", "c. -115 to -105 region", "-105:-115", "11nt target region" refer to genomic locations Hg38 Chr11:5,249, respectively. 962 to Hg38 Chr11:5,249,972 and Hg38 Chr11:5,254,886 to Hg38 Chr11:5,254,896 refer to sequences 5′ of the transcription start sites (TSS) of the HBG1 and/or HBG2 genes. . HBG1 c. The -105 to -115 region is exemplified at positions 2823 to 2833 in SEQ ID NO:902 (HBG1), HBG2 c. The -105 to -115 region is exemplified at positions 2747 to 2757 in SEQ ID NO:903 (HBG2). Terms such as "11nt deletion" refer to deletion of the target region of 11nt.

Notations such as "c. -115 to -112 region", "c. -112 to -115 region", "-112:-115", "4nt target region" refer to genomic locations Hg38 Chr11:5,249, respectively. 969-Hg38 Chr11:5,249,972 and Hg38 Chr11:5,254,893-Hg38 Chr11:5,254,896 refer to sequences 5′ of the transcription start sites (TSS) of the HBG1 and/or HBG2 genes. . HBG1 c. The -112 to -115 region is exemplified at positions 2823 to 2826 in SEQ ID NO:902, HBG2 c. The -112 to -115 region is exemplified at positions 2747 to 2750 in SEQ ID NO:903 (HBG2). Terms such as "4nt deletion" refer to deletion of the target region of 4nt.

References such as "c.-116 region", "HBG-116", "1nt target region" refer to HBG1 and/or HBG2 at genomic locations Hg38 Chr11:5,249,973 and Hg38 Chr11:5,254,897, respectively. It refers to the sequence 5' of the transcription start site (TSS) of a gene. HBG1 c. The -116 region is exemplified at position 2822 in SEQ ID NO:902 and HBG2c. The -116 region is exemplified at position 2746 in SEQ ID NO:903 (HBG2). Terms such as "1 nt deletion" refer to the deletion of 1 nt of the target region.

Notations such as "c.-117G>A region", "HBG-117G>A", "-117G>A target region" refer to genomic locations Hg38 Chr11:5,249,974 to Hg38 Chr11:5,249, respectively. 974 and Hg38 Chr11:5,254,898 to Hg38 Chr11:5,254,898 5′ of the transcription start site (TSS) of the HBG1 and/or HBG2 gene. HBG1 c. The −117G>A region is exemplified by a guanine (G) to adenine (A) substitution at position 2821 in SEQ ID NO:902, HBG2 c. The −117G>A region is exemplified by a G to A substitution at position 2745 in SEQ ID NO:903 (HBG2). Terms such as "-117G>A modification" refer to a G to A substitution in the -117G>A target region.

The term "proximal HBG1/2 promoter target sequence" refers to a region within 50, 100, 200, 300, 400 or 500 bp proximal to the HBG1/2 promoter sequence containing the 13nt target region. Modifications by the genome editing system according to the present disclosure facilitate (eg, cause, promote, or tend to increase the likelihood of) upregulation of HbF production in erythroid progeny.

The term "GATA1 binding motif in BCL11Ae" refers to the sequence that is the GATA1 binding motif in the erythrocyte-specific enhancer of BCL11A (BCL11Ae) in the +58 DNase I hypersensitive site (DHS) region of intron 2 of the BCL11A gene. . The genomic coordinates of the GATA1 binding motif in BCL11Ae are chr2: 60,495,265-60,495,270. The +58 DHS site contains the 115 base pair (bp) sequence set forth in SEQ ID NO:968. The +58 DHS site sequence, including approximately 500 bp upstream and approximately 200 bp downstream, is set forth in SEQ ID NO:969.

Where ranges are provided herein, the endpoints are included. Further, unless otherwise indicated to the contrary or otherwise clear from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges are subject to contextual exceptions in different embodiments of the invention. Unless otherwise specified, it is understood that it can take any particular value within the stated range, down to tenths of the unit of the lower range limit. Also, unless stated otherwise or otherwise clear from the context and/or the understanding of one of ordinary skill in the art, the values expressed in ranges can take any subrange within the given range. , the endpoints of the subranges are understood to be expressed with the same precision of tenths of the units of the lower range limit.

Various embodiments of the present disclosure generally involve alterations (e.g., deletions or insertions or other Mutation of) relates to a genome editing system configured to introduce. In certain embodiments, increasing expression of one or more γ-globin genes (e.g., HBG1, HBG2) using the methods provided herein results in preferential formation of HbF over HbA. and/or increased HbF levels as a percentage of total hemoglobin. In certain embodiments, the disclosure generally relates to the use of RNP complexes comprising gRNA complexed to Cpf1 molecules. In certain embodiments, the gRNA can be unmodified or modified and the Cpf1 molecule can be a wild-type Cpf1 protein or a modified Cpf1 protein. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the modified Cpf1 comprises SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110-17 ( Cpf1 polynucleotide sequences). In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15).

Patients with hereditary persistent pathology of fetal hemoglobin (HPFH) have mutations in the γ-globin regulatory elements that are not repressed before and after birth, leading to lifelong expression of fetal γ-globin. has been previously shown (Martyn 2017). This results in increased expression of fetal hemoglobin (HbF). HPFH mutations can be deletional or non-deletion (eg, point mutations). Subjects with HPFH exhibit lifelong expression of HbF, ie, do not undergo globin switching or undergo only partial without symptoms of anemia.

HbF expression is, for example, HBG1 c. −114 C>T; c. −117 G>A; c. −158 C>T; c. −167 C>T; c. −170 G>A; c. -175 T>G; c. −175 T>C; c. -195 C>G; c. -196 C>T; c. -197 C>T; c. -198 T>C; c. -201 C>T; c. -202 C>T; c. −211 C>T, c. -251 T>C; or c. -499 T>A; or HBG2 c. −109 G>T; c. −110 A>C; c. -114 C>A; c. −114 C>T; c. -114 C>G; c. −157 C>T; c. −158 C>T; c. −167 C>T; c. -167 C>A; c. −175 T>C; c. -197 C>T; c. −200+C; c. -202 C>G; c. −211 C>T; c. −228 T>C; c. -255 C>G; c. -309 A>G; c. -369 C>G; or c. It can be induced through point mutations in the γ-globin regulatory elements associated with naturally occurring HPFH variants, including −567 T>G.

Spontaneous mutations in the distal CCAAT box motif, found within the promoters of the HBG1 and/or HBG2 genes (ie, HBG1/2 c.-111 to -115) also lead to continued expression of γ-globin and pathology of HPFH. is shown. Modification (mutation or deletion) of the CCAAT box could disrupt binding of one or more transcriptional repressors, leading to continued expression of the γ-globin gene and elevated HbF expression (Martyn 2017). For example, the naturally occurring 13 base pair del c. −114 to −102 (“13nt deletion”) has been shown to be associated with elevated HbF levels (Martyn 2017). The distal CCAAT box likely overlaps with binding motifs within and around the CCAAT box of negative regulatory transcription factors that are expressed in adulthood and repress HBG (Martyn 2017).

A gene editing strategy disclosed herein is to disrupt one or more nucleotides within and/or around the distal CCAAT box to increase HbF expression. In certain embodiments, the "CCAAT box target region" can be the region of or near the distal CCAAT box, comprising the nucleotides of the distal CCAAT box and the 25 nucleotides upstream (5') of the distal CCAAT box and the downstream (3') 25 nucleotides (ie, HBG1/2 c.-86 to -140). In other embodiments, the "CCAAT box target region" can be the region of or near the distal CCAAT box, comprising the nucleotides of the distal CCAAT box and the 5 nucleotides upstream (5') of the distal CCAAT box and the downstream (3') 5 nucleotides (ie, HBG1/2 c.-106 to -120). Without limitation, HBG del c. -104to-121 (“18nt deletion”), HBG del c. -105to-115 (“11nt deletion”), HBG del c. -112to-115 (“4nt deletion”) and HBG del c. Disclosed herein are unique, non-naturally occurring modifications of the CCAAT box target region that induce HBG expression, including -116 (a "1nt deletion"). In certain embodiments, modifications can be introduced into the CCAAT box target regions of HBG1 and/or HBG2 using the genome editing system disclosed herein. In certain embodiments, the genome editing system may comprise one or more DNA donor templates that encode modifications (such as deletions, insertions or mutations) of the CCAAT box target region. In certain embodiments, modifications may be non-naturally occurring modifications or naturally occurring modifications. In certain embodiments, the donor template is a 1 nt deletion, 4 nt deletion, 11 nt deletion, 13 nt deletion, 18 nt deletion or c. -117 G>A modification may be encoded. In certain embodiments, the genome editing system may comprise an RNA-guided nuclease including Cas9, modified Cas9, Cpf1 or modified Cpf1. In certain embodiments, the genome editing system may comprise RNPs comprising gRNAs and Cpf1 molecules. In certain embodiments, the gRNA can be unmodified or modified, and the Cpf1 molecule can be wild-type Cpf1 protein or modified Cpf1 protein, or a combination thereof. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. In certain embodiments, the modified Cpf1 comprises SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110-17 ( Cpf1 polynucleotide sequences). In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32 in Table 15).

The genome editing system of the present disclosure comprises an RNA-guided nuclease, such as Cpf1, and one or more gRNAs having targeting domains complementary to sequences in or near the target region, and optionally One or more of the DNA donor templates encoding nearby specific mutations (such as deletions or insertions), and/or, without limitation, random oligonucleotides, small amounts of gene products involved in DNA repair or DNA damage response. Agents that increase the efficiency with which such mutations occur may be included, including molecular agonists or antagonists, or peptide agents.

Various approaches for introducing mutations into the CCAAT box target region, 13nt target region, and/or proximal HBG1/2 promoter target sequences can be used in embodiments of the present disclosure. In one approach, single modifications such as double-strand breaks are generated within the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence, e.g., formation of indels or deletion of regions. Incorporation of the encoding donor template sequence repairs the region in a manner that interferes with its function. In the second approach, two or more modifications are made on either side of the region, resulting in deletion of intervening sequences including the CCAAT box target region and/or the 13nt target region.

Treatment of hemoglobinopathies by gene therapy and/or genome editing, in which red blood cells or RBCs, cells phenotypically affected by the disease, have been enucleated and targeted in the exemplary genome editing approaches described above , is complicated by the fact that it does not contain genetic material encoding abnormal hemoglobin protein (Hb) subunits or either Aγ or Gγ subunits. This complication is addressed in certain embodiments of the present disclosure by modifying cells that are capable of differentiating into or otherwise giving rise to red blood cells. Cells within the erythroid lineage that are modified according to various embodiments of the present disclosure include, but are not limited to, hematopoietic stem and progenitor cells (HSC), erythroblasts (basophilic, polychromatic and/or normochromatic). erythroblasts), proerythroblasts, polychromatic or reticulocytes, embryonic stem (ES) cells and/or induced pluripotent stem (iPSC) cells. These cells can be modified in situ (eg, within a subject's tissue) or ex vivo. Implementations of genome editing systems for in situ and ex vivo cell modification are described below under the heading "Implements of Genome Editing Systems: Delivery, Formulations and Routes of Administration."

In certain embodiments, modifications that result in induction of Aγ and/or Gγ expression include RNA-guided nucleases and sequences within or adjacent to the CCAAT box target region of HBG1 and/or HBG2 (e.g., 10 of the CCAAT box target region, and at least one gRNA having a complementary targeting domain within 20, 30, 40 or 50, 100, 200, 300, 400 or 500 bases). As discussed in more detail below, the RNA-guided nuclease and gRNA form a complex that can bind and modify the CCAAT box target region or regions adjacent thereto. Examples of suitable gRNAs and gRNA targeting domains directed to or adjacent to the CCAAT box target region of HBG1 and/or HBG2 for use in the embodiments disclosed herein include: Those described in are included.

In certain embodiments, modifications that result in induction of Aγ and/or Gγ expression include RNA-guided nucleases and sequences within or adjacent to the 13nt target regions of HBG1 and/or HBG2 (e.g., 10, 20, 13nt target regions, and at least one gRNA having a complementary targeting domain within 30, 40 or 50, 100, 200, 300, 400 or 500 bases). As discussed in more detail below, RNA-guided nucleases and gRNAs form complexes that can bind and modify the 13nt target region or regions adjacent thereto. Examples of suitable gRNAs and gRNA targeting domains directed to the 13nt target region of HBG1 and/or HBG2 or regions proximate thereto for use in the embodiments disclosed herein include: Those mentioned are included.

Genome editing systems can be implemented in a variety of ways, as discussed in detail below. As an example, the genome editing system of the present disclosure can be implemented as a ribonucleoprotein complex or multiple complexes in which multiple gRNAs are used. This ribonucleoprotein complex is disclosed in co-assigned WO 2016/182959 by Jennifer Gori (“Gori”), published Nov. 17, 2016, which is incorporated herein by reference in its entirety. can be introduced into target cells using methods known in the art, including electroporation, as described in US Pat.

Ribonucleoprotein complexes within these compositions may be used, without limitation, by electroporation (e.g., Nucleofection™, a technology commercialized by Lonza, Basel, Switzerland, or e.g., Maxcyte Inc. Gaithersburg, Maryland). into target cells by methods known in the art, including using similar techniques commercialized by Thermo Fisher Scientific, Waltham Massachusetts) and lipofection (e.g., using the Lipofectamine™ reagent commercialized by Thermo Fisher Scientific, Waltham Massachusetts). introduced into Alternatively or additionally, a ribonucleoprotein complex is formed within the target cell itself following introduction of a nucleic acid encoding the RNA-guided nuclease and/or gRNA. These and other modes of delivery are described in general terms and Gori below.

Cells that have been modified in vitro according to the present disclosure can be manipulated (eg, expanded, passaged, frozen, differentiated, dedifferentiated, transduced with a transgene, etc.) prior to their delivery to a subject. Cells are variously delivered to the subject from which they were obtained (“autologous” transplantation) or to a recipient immunologically distinct from the donor of the cells (“allogeneic” transplantation). ).

Optionally, autologous transplantation involves obtaining from a subject a plurality of cells circulating in peripheral blood or in bone marrow or other tissues (e.g., spleen, skin, etc.); Cells may be manipulated (e.g., induced to generate iPSCs, purified for cells expressing specific cell surface markers such as CD34, CD90, CD49f and/or characterized for non-erythroid lineages such as CD10, CD14, CD38). enriching for cells within the erythroid lineage (by purifying cells that do not express a specific surface marker). The cells are optionally or additionally grown prior to transduction with a genome editing system targeting the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence, and transgene Transduced, exposed to cytokines or other peptide or small molecule agents, and/or freeze/thawed. The genome editing system is implemented or delivered to the cell in any suitable form, such as as a ribonucleoprotein complex, as separate protein and nucleic acid components, and/or as nucleic acids encoding the components of the genome editing system. obtain.

In certain embodiments, CD34+ hematopoietic stem and progenitor cells (HSPC) that have been edited using the genome editing methods disclosed herein are used for the treatment of hemoglobinopathies in a subject in need thereof. can be used. In certain embodiments, the hemoglobinopathy can be severe sickle cell disease (SCD) or thalassemia, such as beta thalassemia, delta thalassemia or beta/delta-thalassemia. In certain embodiments, an exemplary protocol for the treatment of hemoglobinopathies is to obtain CD34+ HSPCs from a subject in need thereof, edit autologous CD34+ HSPCs ex vivo using the genome editing methods disclosed herein. followed by reinfusion of the edited autologous CD34+ HSPCs into the subject. In certain embodiments, treatment with edited autologous CD34+ HSPCs can result in increased HbF induction.

In certain embodiments, prior to collection of CD34+ HSPCs, the subject may discontinue treatment with hydroxyurea, if applicable, and receive blood transfusions to maintain adequate hemoglobin (Hb) levels. In certain embodiments, a subject may be administered plelixafor (eg, 0.24 mg/kg) intravenously to mobilize CD34+ HSPCs from bone marrow into peripheral blood. In certain embodiments, the subject may undergo one or more leukoapheresis cycles (e.g., two plelixafor-recruiting leukoapheresis cycles performed on consecutive days, with approximately one month between cycles). harvesting). In certain embodiments, the number of leukoapheresis cycles administered to the subject is the same as the dose of unedited autologous CD34+ HSPCs/kg for backup storage for re-infusion of the subject (e.g., ≧1.5× 10 ⁶ cells/kg), doses of edited autologous CD34+ HSPCs (e.g., ≧2×10 ⁶ cells/kg, ≧3×10 ⁶ cells/kg, ≧4×10 ⁶ cells/kg, ≧5×10 6 cells/ ^kg ). cells/kg, 2×10 ⁶ cells/kg to 3×10 ⁶ cells/kg, 3×10 ⁶ cells/kg to 4×10 6 cells/kg, 4× ^{10 6 cells} /kg to 5×10 ⁶ cells/kg kg). In certain embodiments, CD34+ HSPCs obtained from a subject can be edited using any of the genome editing methods discussed herein. In certain embodiments, any one or more of the gRNAs and one or more of the RNA-guided nucleases disclosed herein may be used in genome editing methods.

In certain embodiments, treatment may include autologous stem cell transplantation. In certain embodiments, the subject may undergo myeloablative conditioning by busulfan conditioning (eg, titrated based on initial dose pharmacokinetic analysis at a test dose of 1 mg/kg). In certain embodiments, habituation may be performed for 4 consecutive days. In certain embodiments, edited autologous CD34+ HSPCs (e.g., ≧2×10 ⁶ cells/kg, ≧3×10 ⁶ cells/kg, ≧4×10 ⁶ cells/kg) after a busulfan washout period of 3 days , ≧5×10 ⁶ cells/kg, 2×10 ⁶ cells/kg to 3×10 ⁶ cells/kg, 3×10 ⁶ cells/kg to 4×10 ⁶ cells/kg, 4×10 ⁶ cells/kg to 5×10 ⁶ cells/kg) can be reinfused into the subject (eg, into peripheral blood). In certain embodiments, edited autologous CD34+ HSPCs can be manufactured and cryopreserved for a particular subject. In certain embodiments, the subject may achieve a neutrophil engraftment following a continuous myeloablative conditioning regimen and infusion of edited autologous CD34+ cells. A neutrophil engraftment may be defined as 3 consecutive measurements of ANC ≧0.5×10 ⁹ /L.

However implemented, the genome editing system may be an aryl hydrocarbon receptor antagonist such as, without limitation, StemRegenin-1 (SR1), UM171, LGC0006, α-naphthoflavone and CH-223191 and/or Innate immune response antagonists such as cyclosporin A, dexamethasone, resveratrol, MyD88 inhibitory peptides, RNAi agents targeting Myd88, B18R recombinant proteins, glucocorticoids, OxPAPC, TLR antagonists, rapamycin, BX795 and RLR shRNAs. may include, or be co-delivered with, one or more factors that improve cell viability during and after editing. These and other factors that improve cell viability during and after editing are described under the heading "I. Optimization of Stem Cells" at pages 36-61 in Gori, which is incorporated herein by reference. be done.

Following delivery of the genome editing system, the cells are optionally manipulated, e.g., enriched for HSCs and/or cells within the erythroid lineage and/or the edited cells, which are expanded, frozen/thawed. The cells are otherwise prepared for use or return to the subject. The edited cells are then returned to the subject into the circulatory system or solid tissue such as bone marrow, for example, by means of intravenous delivery or delivery.

Functionally, modification of the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence using the compositions, methods and genome editing systems of the present disclosure can be performed between hemoglobin-expressing cells, e.g. such as an induction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of Aγ and/or Gγ subunit expression compared to a modified control; Resulting in significant induction of Aγ and/or Gγ subunits (also referred to interchangeably as HbF expression). This induction of protein expression generally includes, for example, without limitation, indels, insertions or deletions within or near the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence. at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc. of a plurality of cells comprising at least one allele comprising a sequence alteration that CCAAT box target regions, 13nt target regions, and/or proximal HBG1/2 promoter target sequences in some or all of the treated cells (e.g., expressed as a percentage of the total genome containing indel mutations in the cells). is the result of modification of

The functional effects of modifications caused or facilitated by the genome editing systems and methods of the present disclosure can be assessed in any number of suitable ways. For example, the effect of alterations on fetal hemoglobin expression can be assessed at the protein or mRNA level. HBG1 and HBG2 mRNA expression can be assessed by digital droplet PCR (ddPCR) performed on cDNA samples obtained by reverse transcription of mRNA taken from treated or untreated samples. Primers for HBG1, HBG2, HBB and/or HBA can be used individually or multiplexed using methods known in the art. For example, ddPCR analysis of a sample can be performed using the QX200™ ddPCR system manufactured by BioRad (Hercules, Calif.) and associated protocols published by Bio-Rad. Fetal hemoglobin protein can be obtained by high pressure liquid chromatography (HPLC) or fast protein liquid chromatography (FPLC), for example according to the methods discussed on pages 143-44 of Chang 2017, incorporated herein by reference. As known in the art, it can be evaluated using ion exchange and/or reverse phase columns to separate HbF, HbB and HbA and/or Aγ and Gγ globin chains.

Rate that CCAAT box target regions (eg, 18nt, 11nt, 4nt, 1nt, c.-117 G>A target regions), 13nt target regions, and/or proximal HBG1/2 promoter target sequences are modified in target cells. It should be noted that can be modified by the use of optional genome editing system components such as oligonucleotide donor templates. Donor template design is described in general terms below under the heading "Donor Template Design". Donor templates for use in targeting the 13nt target region include, but are not limited to, HBG1 c. -114 to -102 (corresponding to nucleotides 2824 to 2836 of SEQ ID NO:902), HBG1 c. -225 to -222 (corresponding to nucleotides 2716 to 2719 of SEQ ID NO:902)) and/or HBG2 c. Donor templates that encode modifications (eg, deletions) from -114 to -102 (corresponding to nucleotides 2748-2760 of SEQ ID NO:903) can be included. Exemplary 5' and 3' homology arms and c. Exemplary full-length donor templates encoding deletions such as -114 to -102 are also provided below. In certain embodiments, donor templates for use in targeting 18nt target regions include, but are not limited to, HBG1 c. -104 to -121, HBG2 c. Donor templates that encode alterations (eg, deletions) from -104 to -121 or combinations thereof can be included. c. Exemplary full-length donor templates encoding deletions such as -104 to -121 include SEQ ID NOS:974 and 975. In certain embodiments, donor templates for use in targeting 11nt target regions include, but are not limited to, HBG1 c. -105 to -115, HBG2 c. Donor templates encoding modifications (eg, deletions) of -105 to -115 or combinations thereof can be included. c. Exemplary full-length donor templates encoding deletions such as -105 to -115 include SEQ ID NOS:976 and 978. In certain embodiments, donor templates for use in targeting 4nt target regions include, but are not limited to, HBG1 c. −112 to −115, HBG2 c. Donor templates encoding modifications (eg, deletions) of -112 to -115 or combinations thereof can be included. c. Exemplary full-length donor templates encoding deletions such as -112 to -115 include SEQ ID NOs:984-995. In certain embodiments, donor templates for use in targeting 1nt target regions include, but are not limited to, HBG1 c. -116, HBG2 c. Donor templates encoding modifications (eg, deletions) of -116 or combinations thereof can be included. c. Exemplary full-length donor templates encoding deletions such as -116 include SEQ ID NOs:982 and 983. In certain embodiments, c. Donor templates for use in targeting −117 G>A target regions include, but are not limited to, HBG1 c. −117 G>A, HBG2 c. Donor templates that encode modifications (eg, deletions) of −117 G>A or combinations thereof can be included. c. Exemplary full-length donor templates encoding deletions such as −117 G>A include SEQ ID NOS:980 and 981. In certain embodiments, the donor template can be plus or minus strand.

A donor template as used herein can be a non-specific template that is non-homologous to regions of DNA within or near the target sequence. In certain embodiments, donor templates for use in targeting the 13nt target region include, but are not limited to, non-target-specific templates that are non-homologous to regions of DNA within or near the 13nt target region. can be For example, a non-specific donor template for use in targeting the 13nt target region may be heterologous to DNA regions within or near the 13nt target region, such as HBG1 c. A donor template that encodes a deletion from -225 to -222 (corresponding to nucleotides 2716 to 2719 of SEQ ID NO:902) can be included.

The embodiments described herein can be used with all classes of vertebrates including, but not limited to, primates, mice, rats, rabbits, pigs, dogs and cats.

This overview focuses on a few exemplary embodiments that depict the principles of genome editing systems and CRISPR-mediated methods of modifying cells. However, for the sake of clarity, this disclosure encompasses modifications and variations not expressly addressed above, but which may be apparent to those skilled in the art. With this in mind, the following disclosure is intended to more generally illustrate the principles of operation of genome editing systems. The following should not be understood as limiting, but rather exemplifies certain principles of genome editing systems and CRISPR-mediated methods utilizing these systems, which are combined with the present disclosure. will inform those skilled in the art about additional implementations and modifications within its scope.

RNA guided helicase, guide RNA and death guide RNA
In certain embodiments of the above approaches and methods, modification of the DNA helix structure is accomplished through the action of an "RNA-guided helicase," which term generally refers to: (a) interacting with (e.g., complexing) gRNAs; do), (b) used to refer to a molecule, typically a peptide, that binds and unwinds with a gRNA at a target site. In certain embodiments, an RNA-guided helicase can comprise an RNA-guided nuclease that has been configured to lack nuclease activity. However, the present inventors demonstrated that even a cleaving-capable RNA-guided nuclease can be used as an RNA-guided helicase by complexing it to a dead gRNA with a truncated targeting domain of 15 nucleotides or less in length. Observing that it can be adapted for use. Complexes of dead gRNA and wild-type RNA-guided nuclease show reduced or no RNA cleavage activity, but appear to retain helicase activity. RNA-guided helicases and dead gRNAs are described in more detail below.

With respect to RNA-guided helicases, according to the present disclosure, RNA-guided helicases include, but are not limited to, Cas9 or Cpf1 RNA-guided nucleases herein and hereinafter under the heading "RNA-guided nucleases." any of the RNA-guided nucleases disclosed in The helicase activity of these RNA-guided nucleases allows the unwinding of DNA and the desired target region to be edited (e.g., the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence). provide increased access to genome editing system components (eg, without limitation, catalytically active RNA-guided nucleases and gRNAs). In certain embodiments, the RNA-guided nuclease can be a catalytically active RNA-guided nuclease having nuclease activity. In certain embodiments, the RNA-guided helicase can be configured to lack nuclease activity. For example, in certain embodiments, the RNA-guided helicase can be a non-catalytic RNA-guided nuclease that lacks nuclease activity, such as a catalytically inactive Cas9 molecule, that still provides helicase activity. In certain embodiments, an RNA-guided helicase can complex to a dead gRNA to form a dead RNP that is incapable of cleaving nucleic acids. In other embodiments, the RNA-guided helicase can be a catalytically active RNA-guided nuclease that complexes to dead gRNAs to form dead RNPs that are incapable of cleaving nucleic acids. In certain embodiments, the RNA-guided nuclease directs an exogenous transacting factor to the desired target region to be edited (e.g., the CCAAT box target region, the 13nt target region, and/or the proximal HBG1/2 promoter target sequence). Not configured to mobilize.

When referring to dead gRNAs, these include any of the dead gRNAs discussed under the heading "Dead gRNA Molecules" herein and below. Dead gRNAs (also referred to herein as "dgRNAs") can be generated by truncating the 5' ends of gRNA targeting domain sequences, resulting in targeting domain sequences of 15 nucleotides or less in length. Death guide RNA molecules according to the present disclosure include death guide RNA molecules with reduced, low or undetectable cleavage activity. The killing guide RNA targeting domain sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides shorter in length compared to the targeting domain sequence of the active guide RNA. The killing gRNA molecule is a region proximal to or within the target region (e.g., the CCAAT box target region, the 13nt target region, the proximal HBG1/2 promoter target sequence and/or the GATA1 binding motif in BCL11Ae) in the target nucleic acid. can include a targeting domain complementary to In certain embodiments, "proximal" is 10, 25, 50 of the target region (e.g., the CCAAT box target region, the 13nt target region, the proximal HBG1/2 promoter target sequence and/or the GATA1 binding motif in BCL11Ae). , 100 or 200 nucleotides. In certain embodiments, the dead gRNA comprises a targeting domain complementary to the transcribed or non-transcribed strand of DNA. In certain embodiments, the death guide RNA directs an exogenous transacting factor to the target region (e.g., the CCAAT box target region, the 13nt target region, the proximal HBG1/2 promoter target sequence and/or the GATA1 binding motif in BCL11Ae). Not configured to mobilize.

This overview focuses on a few exemplary embodiments that depict the principles of genome editing systems and CRISPR-mediated methods of modifying cells. However, for the sake of clarity, this disclosure encompasses modifications and variations not expressly addressed above, but which may be apparent to those skilled in the art. With this in mind, the following disclosure is intended to more generally illustrate the principles of operation of genome editing systems. The following should not be understood as limiting, but rather exemplifies certain principles of genome editing systems and CRISPR-mediated methods utilizing these systems, which are combined with the present disclosure. will inform those skilled in the art about additional implementations and modifications within its scope.

Genome Editing System The term "genome editing system" refers to any system that has RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components adapted from the naturally occurring CRISPR system: a guide RNA (gRNA) and an RNA-guided nuclease. These two components combine with specific nucleic acid sequences to generate, for example, one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSBs) and/or point mutations. , form complexes that can edit DNA in or around nucleic acid sequences.

Genome editing systems may be implemented (eg, administered or delivered to cells or subjects) in a variety of ways, and different implementations may be suitable for different uses. For example, genome editing systems are implemented in certain embodiments as protein/RNA complexes (ribonucleoproteins or RNPs), which include pharmaceutical agents such as, but not limited to, lipid or polymer microparticles or nanoparticles, micelles or liposomes. It can be included in a pharmaceutical composition optionally comprising a pharmaceutically acceptable carrier and/or an encapsulating agent. In certain embodiments, the genome editing system is implemented as one or more nucleic acids (optionally with one or more additional components) encoding the RNA-guided nuclease and guide RNA components described above. in certain embodiments, the genome-editing system is implemented as one or more vectors comprising such nucleic acids, e.g. See section below under the heading "Routes of Administration"); in certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate in accordance with the principles described herein will be apparent to those skilled in the art and are within the scope of the disclosure.

The genome editing system of the present disclosure may or may not target a single specific nucleotide sequence, and through the use of two or more guide RNAs, can edit two or more specific nucleotide sequences in parallel. It should be noted. The use of multiple gRNAs, referred to throughout this disclosure as "multiplexing", to target multiple unrelated target sequences of interest or to form multiple SSBs or DSBs within a single target domain; It can optionally be used to make specific edits within such target domains. For example, Maeder et al. WO2015/138510 by (“Maeder”) corrects a point mutation in the human CEP290 gene that results in the creation of a cryptic splice junction, thereby reducing or eliminating the function of the gene (C. 2991+1655A to G). Maeder's genome editing system utilizes two guide RNAs that target sequences on either side of (ie, flanking) a point mutation to form DSBs that flank the mutation. This in turn facilitates deletion of the intervening sequence containing the mutation, thereby removing the cryptic splice site and restoring normal gene function.

As another example, WO 2016/073990 by Cotta-Ramusino et al. (“Cotta-Ramusino”), which is incorporated herein by reference, is an arrangement referred to as the “dual nickase system.” , describe a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (Cas9 that produces single-stranded nicks such as S. pyogenes D10A). The Cotta-Ramusino double nickase system is configured to generate two nicks on the opposite strand of the sequence of interest that are offset by one or more nucleotides, the nicks combined to have an overhang. Creates a double-stranded break (5' in the case of Cotta-Ramusino, but 3' overhangs are also possible). Overhangs, in turn, can facilitate homology-directed repair events in some situations. Also, as another example, WO 2015/070083 by Palestrant et al. (incorporated herein by reference) describes a gRNA (referred to as "governing RNA") that targets a nucleotide sequence encoding Cas9. ), which includes adding one or more additional gRNAs to allow transient expression of Cas9, which may otherwise be constitutively expressed, e.g., in some virally transduced cells. It can be included in a genome editing system including. These multiplexing applications are intended to be illustrative rather than limiting, and one skilled in the art will appreciate that other multiplexing applications are generally compatible with the genome editing systems described herein. you will understand.

As disclosed herein, in certain embodiments, the genome editing system comprises multiple gRNAs that can be used to introduce mutations into the GATA1 binding motif in BCL11Ae or the 13nt target region of HBG1 and/or HBG2. can include In certain embodiments, the genome editing system disclosed herein can comprise multiple gRNAs used to introduce mutations in the GATA1 binding motif in BCL11Ae and the 13nt target regions of HBG1 and/or HBG2. .

Genome editing systems can optionally create double-strand breaks that are repaired by cellular DNA double-strand break breaks such as NHEJ or HDR. These mechanisms have been described throughout the literature (e.g. Davis & Maizels 2014 (describing Alt-HDR); Frit 2014 (describing Alt-NHEJ); Iyama & Wilson 2013 (describing canonical HDR and NHEJ pathways in general). do)).

Where genome editing systems function by forming DSBs, such systems optionally include one or Contains multiple components. For example, Cotta-Ramusino also describes a genome editing system into which a single-stranded oligonucleotide "donor template" has been added; can integrate and result in changes in the target sequence.

In certain embodiments, the genome editing system alters the target sequence or alters the expression of genes in or near the target sequence without causing single- or double-strand breaks. For example, genome editing systems can include RNA-directed nucleases fused to functional domains that act on DNA, thereby altering target sequences or their expression. As an example, an RNA-guided nuclease can bind (eg, fuse) to a cytidine deaminase functional domain and function by generating targeted C to A substitutions. Exemplary nuclease/deaminase fusions are described in Komor 2016, incorporated herein by reference. Alternatively, genome editing systems may utilize cleaving inactivating (i.e., "dead") nucleases, such as dead Cas9 (dCas9), to form stable complexes on one or more targeted regions of cellular DNA. and thereby function by interfering with functions involving the target region, including but not limited to mRNA transcription, chromatin remodeling, and the like. In certain embodiments, a genome editing system can include an RNA-guided helicase that unwinds DNA within or proximal to a target sequence without causing single- or double-strand breaks. For example, a genome editing system can include an RNA-guided helicase configured to bind within or near a target sequence to unwind DNA and induce accessibility to the target sequence. In certain embodiments, an RNA-guided helicase may complex to a death guide RNA configured to lack cleavage activity, allowing it to unwind without causing DNA cleavage.

Guide RNA (gRNA) Molecules The terms "guide RNA" and "gRNA" facilitate the specific binding (or "targeting") of an RNA-guided nuclease, such as Cpf1, to a target sequence, such as a genomic or episomal sequence within a cell. refers to any nucleic acid that gRNAs may be unimolecular (comprising a single RNA molecule or also referred to as chimeras) or modular (e.g., two or more, typically two separate RNA molecules, such as crRNA and tracrRNA, usually linked together by duplexing). (including RNA molecules of ). gRNAs and their constituent parts have been described throughout the literature (see, eg, Briner 2014; Cotta-Ramusino, incorporated by reference). Examples of modular and unimolecular gRNAs that can be used according to embodiments herein include, without limitation, the sequences set forth in SEQ ID NOS:29-31 and 38-51. Examples of gRNA proximal and tail domains that may be used according to embodiments herein include, without limitation, the sequences set forth in SEQ ID NOs:32-37.

In bacteria and archaea, the type II CRISPR system is generally an RNA-guided nuclease protein such as Cas9; a CRISPR RNA (crRNA) that includes a 5' region complementary to a foreign sequence; and a 3' region of the crRNA that is complementary to , which contains a transactivating crRNA (tracrRNA) containing a 5' region, forming a duplex with it. Without intending to be bound by any theory, it is believed that this duplex promotes formation of the Cas9/gRNA complex and is required for its activity. While adapting the Type II CRISPR system for use in gene editing, in one non-limiting example, 4 nucleotides (e.g., GAAA ) It was discovered that crRNA and tracrRNA can be joined into a single unimolecular or chimeric guide RNA by a "tetraloop" or "linker" sequence. (Mali 2013; Jiang 2013; Jinek 2012; all incorporated herein by reference).

A guide RNA, whether unicellular or modular, includes a "targeting domain" that is fully or partially complementary to a target domain in a target sequence, such as a DNA sequence in the genome of a cell in which editing is desired. Targeting domains include, but are not limited to, “guide sequences” (Hsu 2013, incorporated herein by reference), “complementary regions” (Cotta-Ramusino), “spacers” (Briner 2014). and generically referred to by various names in the literature, including "crRNA" (Jiang). Regardless of the name given to them, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20 , 21, 22, 23 or 24 nucleotides in length) and is at or near the 5′ end for Cas9 gRNAs and at or near the 3′ end for Cpf1 gRNAs.

In addition to the targeting domain, gRNAs typically (but not necessarily, as discussed below) contain multiple domains that can influence gRNA/Cas9 complex formation or activity. . For example, as described above, the duplex structure formed by the first and second complementary domains of the gRNA (repeat: also called anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9. Acting, it can mediate the formation of the Cas9/gRNA complex (Nishimasu 2014; Nishimasu 2015; both incorporated herein by reference). It should be noted that the first and/or second complementary domain may contain one or more poly A strands that can be recognized as termination signals by RNA polymerase. As such, the sequences of the first and second complementary domains are optionally modified, for example through the use of an AG swap or an AU swap as described in Briner 2014, such that these areas are removed to promote full in vitro transcription of the gRNA. These and other similar modifications to the first and second complementary domains are within the scope of this disclosure.

Along with the first and second complementary domains, the Cas9g RNA typically contains two or more additional double-stranded regions that are involved in nuclease activity in vivo, but not necessarily in vitro. (Nishimasu 2015). The first stem loop 1 near the 3′ portion of the second complementary domain is the “proximal domain” (Cotta-Ramusino), the “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner 2014), etc. One or more additional stem-loop structures are generally present near the 3' end of the gRNA, and the number varies by species, S. cerevisiae. S. pyogenes gRNAs typically contain two 3' stem-loops (a total of four stem-loop structures, including a repeat: anti-repeat duplex: anti-repeat duplex), whereas S. pyogenes gRNAs typically contain two 3' stem-loops (a total of four stem-loop structures, including a repeat: anti-repeat duplex: anti-repeat duplex). S. aureus and other species have only one (three stem-loop structures total). A description of conserved stem-loop structures (and gRNA structures more generally) organized by species is provided in Briner 2014.

Although the foregoing description has focused on gRNAs for use with Cas9, it should be understood that there are other RNA-guided nucleases that utilize gRNAs that differ in some respects from those described so far. . For example, Cpf1 (CRISPR from "Prevotella and Francicella 1") is a recently discovered RNA-guided nuclease that does not require tracrRNA to function. (Zetsche 2015, incorporated herein by reference). gRNAs for use in the Cpf1 genome editing system generally include a targeting domain and a complementary domain (alternatively referred to as "handles"). In gRNAs for use with Cpf1, the targeting domain is usually at or near the 3' end rather than at the 5' end, as described above for Cas9 gRNAs (the handle is the 5' end of the Cpf1 gRNA or near it). Exemplary targeting domains of Cpf1 gRNA are listed in Tables 6 and 12.

However, those skilled in the art will appreciate that although there may be structural differences between gRNAs from different prokaryotic species or between Cpf1 and Cas9 gRNAs, the principles by which gRNAs work are generally consistent. . Because of this consistency of operation, gRNAs can be broadly defined by their targeting domain sequences, and one skilled in the art will appreciate that a given targeting domain sequence may be a single molecular or chimeric gRNA or one or more chemical It will be appreciated that modifications and/or sequence modifications (substitutions, additional nucleotides, truncations, etc.) can be incorporated into any suitable gRNA, including gRNAs. Thus, for economy of presentation in this disclosure, gRNAs can be described only in terms of their targeting domain sequences.

More generally, those skilled in the art will appreciate that some aspects of this disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA encompasses not only specific species-compatible gRNAs of Cas9 or Cpf1, but also any suitable gRNA that can be used with any RNA-guided nuclease. should be understood. By way of example, the term gRNA, in certain embodiments, is used with or with or derived from or adapted from a class 2 CRISPR system, such as type II or type V, or any RNA-guiding nuclease present in a CRISPR system. It can include gRNAs for use with nucleases.

gRNA Design Methods for target sequence selection and validation and off-target analysis have been previously described (see, eg, Mali 2013; Hsu 2013; Fu 2014; Heigwer 2014; Bae 2014; Xiao 2014). Each of these references is incorporated herein by reference. As a non-limiting example, gRNA design is a software tool for optimizing the selection of potential target sequences corresponding to a user's target sequence, e.g., to minimize total genome-wide off-target activity. may involve use. Off-target activity is not limited to cleavage, although cleavage efficiency at each off-target sequence can be predicted using, for example, an experimentally derived weighting scheme. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

Targeting domain sequences of gRNAs designed to target interference with the CCAAT box target region include, but are not limited to, SEQ ID NO:1002. In certain embodiments, a gRNA comprising the sequence set forth in SEQ ID NO: 1002 can be conjugated to a Cpf1 protein or modified Cpf1 protein to generate alterations in the CCAAT box target region. In certain embodiments, gRNAs comprising any of the Cpf1 gRNAs listed in Table 9, Table 12, or Table 13 are complexed to Cpf1 protein or modified Cpf1 protein to form RNP ("gRNA-Cpf1-RNP"). , generating modifications in the CCAAT box target region. In certain embodiments, the modified Cpf1 protein can be His-AsCpf1-nNLS (SEQ ID NO:1000) or His-AsCpf1-sNLS-sNLS (SEQ ID NO:1001). In certain embodiments, the Cpf1 molecule of gRNA-Cpf1-RNP is according to SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021 (Cpf1 polynucleotide sequences). It can be encoded by the described sequences.

gRNA Modifications The activity, stability or other characteristics of gRNAs can be altered by incorporating certain modifications. As an example, transiently expressed or delivered nucleic acids may be susceptible to degradation, eg, by cellular nucleases. Accordingly, the gRNAs described herein may contain one or more modified nucleosides or nucleotides that introduce stability to nucleases. While not wishing to be bound by theory, it is also believed that certain modified gRNAs described herein may exhibit reduced innate immune responses when introduced into cells. Those skilled in the art are aware of certain cellular responses commonly observed in cells, such as mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which can include induction of cytokine expression and release and cell death, can be reduced or completely eliminated by the modifications presented herein.

Certain exemplary modifications discussed in this section include, but are not limited to: or at or near the 3' end (eg, within 1-10, 1-5, or 1-2 nucleotides of the 3' end) at any position in the gRNA sequence. Optionally, the modifications are located within functional motifs such as the repeat:anti-repeat duplex of Cas9 gRNAs, the stem-loop structure of Cas9 or Cpf1 gRNAs and/or the targeting domain of gRNAs.

キャップ又はキャップ類似体は、ｇＲＮＡの化学合成又はインビトロ転写中に含まれ得る。 As an example, the 5′ end of a gRNA may be attached to a eukaryotic mRNA cap structure or a G cap analogue (e.g., G(5′)ppp(5′)G cap analogue, m7G(5′) as shown below. ) ppp(5′)G cap analogue or 3′-O-Me-m7G(5′)ppp(5′)G anticap analogue (ARCA)).

A cap or cap analog may be included during chemical synthesis or in vitro transcription of the gRNA.

In a similar line, the 5' end of gRNAs can lack a 5' triphosphate group. For example, in vitro transcribed gRNA can be phosphatase treated (eg, using calf intestinal alkaline phosphatase) to remove the 5' triphosphate group.

Another common modification adds multiple (e.g., 1-10, 10-20, or 25-200) adenine (A) residues to the 3' end of the gRNA, referred to as poly-A tracts. accompanied by The polyA sequence is transferred to the gRNA by a polyadenylation sequence during chemical synthesis following in vitro transcription using a polyadenosine polymerase (e.g., E. coli poly(A) polymerase) or in vivo as described in Maeder. can be added.

The modifications described herein can be combined in any suitable manner, e.g., transcribed from a DNA vector in vivo, or gRNA transcribed in vitro, either with a 5' cap structure or with a cap analogue. or both and a 3' poly A sequence.

式中、「Ｕ」は、未修飾又は修飾ウリジンであり得る。 Guide RNAs may be modified with a 3' terminal U-ribose. For example, the two terminal hydroxyl groups of U ribose can be oxidized to aldehyde groups, with simultaneous opening of the ribose ring resulting in the modified nucleoside shown below,

wherein "U" can be unmodified or modified uridine.

式中、「Ｕ」は、未修飾又は修飾ウリジンであり得る。 The 3' terminal U-ribose can be modified with a 2'3' cyclic phosphate as shown below,

wherein "U" can be unmodified or modified uridine.

The guide RNA may contain 3' nucleotides that can be stabilized against degradation by, for example, incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines may be substituted with modified uridines such as, for example, 5-(2-amino)propyluridine and 5-bromouridine or any modified uridine described herein; adenosine and guanosine may be replaced by modified adenosines and guanosines, eg modified at position 8, such as 8-bromoguanosine, or any modified adenosine or guanosine described herein.

In certain embodiments, sugar-modified ribonucleotides can be incorporated into gRNAs, for example, the 2'OH group is H, -OR, -R, where R is, for example, alkyl, cycloalkyl, aryl, aralkyl, can be heteroaryl or sugar), halo, -SH, -SR (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be which may be _alkylamino , dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); or cyano (--CN). . In certain embodiments, the phosphate backbone can be modified, eg, with phosphorothioate (PhTx) groups, as described herein. In certain embodiments, one or more of the nucleotides of the gRNA are each independently 2'-sugar modifications, such as 2'-O-methyl, 2'-O-methoxyethyl or, for example, 2'-F or 2 '-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2′-fluoro modifications such as 2′-O-methyl, thymidine (T) 1,2′-F or 2′-O-methyl, guanosine (G), 2′-O-methoxyethyl-5-methyl including but not limited to uridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5CeO) and any combination thereof may be modified or unmodified nucleotides that are not

A guide gRNA can also include a “locked” nucleic acid (LNA), where the 2′ OH group can be attached, for example, by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4′ carbon of the same ribose sugar. . To provide such bridges, but are not limited to methylene, propylene, ether or amino bridges; O-amino (wherein amino is, for example, NH ₂ ; alkylamino, dialkylamino, heterocyclyl arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino) and aminoalkoxy or O(CH ₂ ) _n -amino (wherein amino is for example NH ₂ ; alkylamino, dialkyl Any suitable moiety may be used, including amino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino.

In certain embodiments, the gRNA comprises modified nucleotides that are polycyclic (e.g., tricyclo; and "unlocked" forms such as glycol nucleic acids (GNA) (e.g., glycol units in which the ribose is attached to a phosphodiester bond). substituted, R-GNA or S-GNA) or threose nucleic acids (TNA in which ribose is substituted with α-L-threofuranosyl-(3′→2′)).

In general, gRNAs contain ribose, which is a five-membered sugar ring with oxygen. Exemplary modified gRNAs include, but are not limited to, replacement of oxygen in ribose (e.g. by sulfur (S), selenium (Se) or alkylene such as methylene or ethylene); addition of double bonds (e.g. replacing ribose with cyclopentenyl or cyclohexenyl); ribose ring contraction (e.g. forming a 4-membered ring of cyclobutane or oxetane); ribose ring expansion (e.g. anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl and morpholinos which also have phosphoramidate backbones, forming a 6- or 7-membered ring with additional carbon or heteroatoms. The majority of sugar analogue modifications are located at the 2' position, but other sites, including the 4' position, are also susceptible to modification. In certain embodiments, the gRNA comprises 4'-S, 4'-Se or 4'-C-aminomethyl-2'-O-Me modifications.

In certain embodiments, deaza nucleotides such as, for example, 7-deaza-adenosine can be incorporated into gRNAs. In certain embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into gRNAs. In certain embodiments, one or more or all nucleotides in the gRNA are deoxyribonucleotides.

In certain embodiments, a gRNA as used herein can be a modified or unmodified gRNA. In certain embodiments, a gRNA may contain one or more modifications. In certain embodiments, the one or more modifications may include phosphorothioate linkage modifications, phosphorodithioate (PS2) linkage modifications, 2'-O-methyl modifications, or combinations thereof. In certain embodiments, one or more modifications may be at the 5' end of the gRNA, the 3' end of the gRNA, or combinations thereof.

In certain embodiments, gRNA modifications may include one or more phosphorodithioate (PS2) linkage modifications.

In some embodiments, a gRNA as used herein comprises one or more or a stretch of deoxyribonucleic acid (DNA) bases, also referred to herein as "DNA extensions." In some embodiments, a gRNA as used herein comprises a DNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof. In certain embodiments, the DNA extension is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, It can be 96, 97, 98, 99 or 100 DNA bases long. For example, in certain embodiments, the DNA extension can be 1, 2, 3, 4, 5, 10, 15, 20 or 25 DNA bases long. In certain embodiments, the DNA extension may comprise one or more DNA bases selected from adenine (A), guanine (G), cytosine (C) or thymine (T). In certain embodiments, the DNA extensions contain identical DNA bases. For example, a DNA extension can include a stretch of adenine (A) bases. In certain embodiments, the DNA extension can include stretches of thymine (T) bases. In certain embodiments, the DNA extension comprises a combination of different DNA bases. In certain embodiments, the DNA extension may comprise a sequence listed in Table 18. For example, a DNA extension can include sequences set forth in SEQ ID NOS:1235-1250. In certain embodiments, gRNAs used herein have DNA extension and one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2' -O-methyl modifications or combinations thereof. In certain embodiments, one or more modifications may be at the 5' end of the gRNA, the 3' end of the gRNA, or combinations thereof. In certain embodiments, a gRNA comprising a DNA extension can comprise a sequence listed in Table 13 comprising a DNA extension. In certain embodiments, a gRNA comprising a DNA extension can comprise the sequence set forth in SEQ ID NO:1051. In certain embodiments, the gRNA comprising the DNA extension is SEQ ID NOS: 1046-1060, 1067, 1068, 1074, 1075, 1078, 1081-1084, 1086-1087, 1089-1090, 1092-1093, 1098-1102 and 1106. Without wishing to be bound by theory, the present invention is provided so long as it does not hybridize to the target nucleic acid targeted by the gRNA and exhibits increased editing at the target nucleic acid site compared to a gRNA that does not contain such a DNA extension. It is envisioned herein that any DNA extension may be used.

In some embodiments, a gRNA as used herein comprises one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an "RNA extension." In some embodiments, a gRNA as used herein comprises an RNA extension at the 5' end of the gRNA, the 3' end of the gRNA, or a combination thereof. In certain embodiments, the RNA elongation is 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, It can be 96, 97, 98, 99 or 100 RNA bases long. For example, in certain embodiments, an RNA extension can be 1, 2, 3, 4, 5, 10, 15, 20 or 25 RNA bases long. In certain embodiments, the RNA extension may comprise one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC) or uracil (rU), where "r" represents RNA, 2'-hydroxy. In certain embodiments, the RNA extensions contain identical RNA bases. For example, an RNA extension can include a stretch of adenine (rA) bases. In certain embodiments, the RNA extension comprises different combinations of RNA bases. In certain embodiments, an RNA extension may comprise a sequence listed in Table 18. For example, RNA extensions can include sequences described in 1231-1234, 1251-1253. In certain embodiments, a gRNA as used herein has an RNA extension and one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2′ linkage modifications. -O-methyl modifications or combinations thereof. In certain embodiments, one or more modifications may be at the 5' end of the gRNA, the 3' end of the gRNA, or combinations thereof. In certain embodiments, a gRNA comprising an RNA extension can comprise a sequence listed in Table 13 comprising an RNA extension. A gRNA comprising an RNA extension at the 5' end of the gRNA can comprise a sequence selected from the group consisting of SEQ ID NOs: 1042-1045, 1103-1105. A gRNA comprising an RNA extension at the 3' end of the gRNA can comprise a sequence selected from the group consisting of SEQ ID NOs: 1070-1075, 1079, 1081, 1098-1100.

It is envisioned that gRNA as used herein may also include RNA extensions and DNA extensions. In certain embodiments, both RNA extensions and DNA extensions can be at the 5' end of the gRNA, the 3' end of the gRNA, or combinations thereof. In certain embodiments, the RNA extension is at the 5' end of the gRNA and the DNA extension is at the 3' end of the gRNA. In certain embodiments, the RNA extension is at the 3' end of the gRNA and the DNA extension is at the 5' end of the gRNA.

In some embodiments, a gRNA comprising both a 3′-terminal phosphorothioate modification and a 5′-terminal DNA extension is conjugated to an RNA-guided nuclease, such as, for example, Cpf1, to form an RNP, which in turn , is used to edit hematopoietic stem cells (HSC) or CD34+ cells ex vivo (ie, ex vivo from the subject from which such cells are derived) at the HBG locus.

As used herein, an example gRNA comprises the sequence set forth in SEQ ID NO:1051.

Death gRNA Molecules Death guide RNA (dgRNA) molecules according to the present disclosure include death guide RNA molecules with reduced, low or undetectable cleavage activity. The killing guide RNA targeting domain sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides shorter in length compared to the targeting domain sequence of the active guide RNA. In certain embodiments, the killing guide RNA molecule comprises a targeting RNA molecule comprising a length of 15 nucleotides or less, a length of 14 nucleotides or less, a length of 13 nucleotides or less, a length of 12 nucleotides or less, or a length of 11 nucleotides or less. May contain domains. In some embodiments, death guide RNAs are configured such that they do not provide for RNA-induced nuclease cleavage events. Killing guide RNA can be generated by removal of the 5' end of the gRNA targeting domain sequence, resulting in a truncated targeting domain sequence. For example, if a gRNA sequence configured to provide a cleavage event (i.e., 17 nucleotides or more in length) has a targeting domain sequence that is 20 nucleotides in length, then 5 nucleotides from the 5' end of the gRNA sequence Removal may generate a death guide RNA. For example, a dgRNA as used herein can comprise a targeting domain listed in Tables 8, 9 or 13 comprising a length of 15 nucleotides or less truncated from the 5' end of the gRNA sequence. In certain embodiments, the dgRNA binds (or associates with) nucleic acid sequences within or proximal to the target region to be edited (e.g., CCAAT box target region, 13nt target region, proximal HBG1/2 promoter target sequence). can be configured to In certain embodiments, proximal refers to a region within 10, 25, 50, 100 or 200 nucleotides of the target region (e.g., CCAAT box target region, 13nt target region, proximal HBG1/2 promoter target sequence). obtain. In certain embodiments, the death guide RNA is not configured to recruit exogenous transacting factors to the target region. In certain embodiments, the dgRNA is configured such that it does not provide a DNA cleavage event when complexed to an RNA-guided nuclease. Those skilled in the art will appreciate that the death guide RNA molecule can be designed to include a targeting domain complementary to a region proximal to or within the target region in the target nucleic acid. In certain embodiments, the death guide RNA comprises a targeting domain sequence complementary to the transcribed or non-transcribed strand of double-stranded DNA. The dgRNA herein can include modifications at the 5' and 3' ends of the gRNA, as described for guide RNAs in the "gRNA Modifications" section herein. For example, in certain embodiments, a death guide RNA can include an anti-reverse cap analog (ARCA) at the 5' end of the RNA. In certain embodiments, the dgRNA may contain a poly-A tail at the 3' end.

In certain embodiments, the use of dead guide RNAs by the genome editing systems and methods disclosed herein can increase the total editing level of active guide RNAs. In certain embodiments, use of death guide RNAs by the genome editing systems and methods disclosed herein can increase the frequency of deletions. In certain embodiments, the deletion may extend from the cleavage site of the active guide RNA towards the death guide RNA binding site. In this way, the dying guide RNA can change the orientation of the active guide RNA, directing editing toward the desired target region.

As used herein, the terms "killed gRNA" and "truncated gRNA" are used interchangeably.

RNA-Guided Nucleases RNA-Guided Nucleases according to the present disclosure include, but are not limited to, naturally occurring class 2 CRISPR nucleases such as Cpf1 and Cas9 and other nucleases derived or obtained therefrom. Certain RNA-guided nucleases, such as Cas9, have also been shown to have helicase activity that allows them to unwind nucleic acids. In certain embodiments, an RNA-guided helicase according to the present disclosure can be any of the RNA-nucleases described herein and in the section entitled "RNA-guided nucleases" above. In certain embodiments, the RNA-guided nuclease is not configured to recruit exogenous trans-acting factors to the target region. In certain embodiments, the RNA-guided helicase can be an RNA-guided nuclease that has been configured to lack nuclease activity. For example, in certain embodiments, the RNA-guided helicase can be a non-catalytic RNA-guided nuclease that lacks nuclease activity but still maintains its helicase activity. In certain embodiments, the RNA-guided nuclease can be mutated to abolish its nuclease activity (e.g., dead Cas9), a non-catalytic RNA that is incapable of cleaving nucleic acids but still capable of unwinding DNA. An inducible nuclease is produced. In certain embodiments, it may be an RNA-guided helicase complexed to any of the death guide RNAs as described herein. For example, a catalytically active RNA-guided helicase (eg, Cas9 or Cpf1) can form an RNP complex with a death guide RNA, resulting in a non-catalytic dead RNP (dRNP). In certain embodiments, non-catalytic RNA-guided helicases (eg, death Cas9) and death guide RNAs may form dRNPs. These dRNPs are incapable of providing a cleavage event, but still maintain a helicase activity important for nucleic acid unwinding.

Functionally, the RNA-guided nuclease (a) interacts with (e.g., forms a complex with) the gRNA; and (b) along with the gRNA, (i) a sequence complementary to the gRNA's targeting domain, and optionally (ii) binding and optionally cleaving a target region of DNA comprising an additional sequence called a "protospacer adjacent motif" or "PAM", described in more detail below or a modifying nuclease. As the examples below illustrate, RNA-guided nucleases broadly define their PAM specificity and cleavage, even though there may be variation between individual RNA-guided nucleases sharing the same PAM specificity or cleavage activity. It can be defined by activity. Those skilled in the art will appreciate that some aspects of this disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease with specific PAM specificity and/or cleavage activity. Will. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood generically and refers to any particular type of RNA-guided nuclease (e.g. Cpf1 versus Cas9), species (e.g. S S. aureus versus S. pyogenes) or variations (e.g., truncations or splits versus full length; engineered PAM specificities versus native PAM specificities, etc.). is also not limited. For example, in certain embodiments the RNA-guided nuclease can be Cas-Φ (Pausch 2020).

Various RNA-guided nucleases may require different sequence relationships between PAM and protospacer. Generally, Cas9 recognizes the PAM sequence 3' of the protospacer. On the other hand, Cpf1 recognizes PAM sequences that are generally 5' of the protospacer.

In addition to recognizing specific sequence orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. For example, S. S. aureus Cas9 recognizes the PAM sequence of NNGRRT or NNGRRV, where the N residues flank 3' of the region recognized by the gRNA targeting domain. S. S. pyogenes Cas9 recognizes the NGG PAM sequence. Also, F. F. novicida Cpf1 recognizes the TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and strategies for identifying novel PAM sequences are described by Shmakov 2015. It should also be noted that the engineered RNA-guided nuclease may have a PAM-specificity that differs from that of the reference molecule (e.g., in the case of an engineered RNA-guided nuclease, the reference molecule is the RNA-guided nuclease may be a naturally occurring variant derived from it, or a naturally occurring variant having maximum amino acid sequence homology with the engineered RNA-guided nuclease). Examples of PAMs that can be used according to embodiments herein include, without limitation, the sequences set forth in SEQ ID NOS: 199-205.

In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA-cleaving activity, with native RNA-guided nucleases typically forming DSBs in target nucleic acids, whereas do they produce only SSBs? Or engineered variants have been generated that do not cut at all (discussed above and in Ran & Hsu 2013, incorporated herein by reference).

Cas9
The crystal structure shows S. cerevisiae complexed with unimolecular guide RNA and target DNA. S. pyogenes Cas9 (Jinek 2014) and S. pyogenes Cas9 (Jinek 2014). has been determined for S. aureus Cas9 (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

The naturally occurring Cas9 protein contains two lobes, the recognition (REC) lobe and the nuclease (NUC) lobe, each of which contains specific structural and/or functional domains. The REC lobe comprises an arginine-rich bridging helix (BH) domain and at least one REC domain (eg, a REC1 domain, optionally a REC2 domain). The REC lobe shares no structural similarity with other known proteins, suggesting it is a unique functional domain. Without wishing to be bound by any theory, mutational analysis suggests specific functional roles for the BH and REC domains. The BH domain appears to play a role in gRNA:DNA recognition, while the REC domain interacts with the gRNA repeat:anti-repeat duplex and is thought to mediate Cas9/gRNA complex formation.

The NUC lobe contains the RuvC domain, the HNH domain and the PAM interaction (PI) domain. The RuvC domain shares structural similarities with retroviral integrase superfamily members and cleaves the non-complementary (ie, bottom) strand of target nucleic acids. It can be formed from two or more split RuvC motifs, such as RuvCI, RuvCII and RuvCIII in S. pyogenes and S. aureus. HNH domains, on the other hand, are structurally similar to HNN endonuclease motifs and cleave the complementary (ie, top) strand of target nucleic acids. As its name suggests, the PI domain contributes to PAM specificity. Examples of polypeptide sequences encoding Cas9 RuvC-like and Cas9 HNH-like domains that may be used according to embodiments herein are SEQ ID NOs: 15-23, 52-123 (RuvC-like domains) and SEQ ID NOs: 24-28 , 124-198 (HNH-like domains).

While certain functions of Cas9 have been linked (although not necessarily completely determined by) to the above specific domains, these and other functions may be linked to other Cas9 domains or any lobe. It can be mediated or influenced by multiple domains above. For example, as described in Nishimasu 2014, S. In S. pyogenes Cas9, the gRNA repeat:anti-repeat duplex enters the groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI and REC domains. do. Some nucleotides in the first stem-loop structure are multiple domains (PI, BH and It also interacts with amino acids within REC1). Examples of polypeptide sequences encoding Cas9 molecules that may be used in accordance with embodiments herein are set forth in SEQ ID NOs: 1-2, 4-6, 12 and 14.

Cpf1
The crystal structure of Acidaminococcus sp. Cpf1 complexed with crRNA and a double-stranded (ds) DNA target such as the TTTN PAM sequence was solved by Yamano 2016 (incorporated herein by reference). It is Similar to Cas9, Cpf1 has two lobes, a REC (recognition) lobe and a NUC (nuclease) lobe. The REC lobe contains the REC1 and REC2 domains, which have no similarity to any known protein structure. The NUC lobe, on the other hand, contains three RuvC domains (RuvC-I, -II and -III) and one BH domain. However, in contrast to Cas9, the Cpf1 REC lobe lacks an HNH domain, a structurally unique PI domain, other domains that lack similarity to known protein structures, and three wedges ( WED) domains (WED-I, -II and -III) and a nuclease (Nuc) domain.

While Cas9 and Cpf1 share similarities in structure and function, it should be understood that certain Cpf1 activities are mediated by structural domains that are not similar to any Cas9 domain. For example, cleavage of the complementary strand of target DNA appears to be mediated by the Nuc domain, which differs in sequence and spatially from the HNH domain of Cas9. Furthermore, the non-targeting portion (handle) of Cpf1 gRNA adopts a pseudoknot structure rather than the stem-loop structure formed by the repeat:anti-repeat duplex in Cas9 gRNA.

In certain embodiments, the Cpf1 protein can be a modified Cpf1 protein. In certain embodiments, a modified Cpf1 protein may contain one or more modifications. In certain embodiments, the modifications include, but are not limited to, one or more mutations in the Cpf1 nucleotide sequence or Cpf1 amino acid sequence, one or more additional sequences or combinations thereof. In certain embodiments, a modified Cpf1 may also be referred to herein as a Cpf1 variant.

In certain embodiments, the Cpf1 protein is Acidaminococcus sp. strain BV3L6 Cpf1 protein (AsCpf1), Lachnospiraceae bacterium ND2006 Cpf1 protein (LbCpf1) and Lachnospirac eae bacterium) from MA2020 (Lb2Cpf1) derived from a Cpf1 protein selected from the group consisting of In certain embodiments, the Cpf1 protein may comprise a sequence selected from the group consisting of SEQ ID NOs: 1016-1018, with codon-optimized nucleic acid sequences of SEQ ID NOs: 1019-1021, respectively.

In certain embodiments, a modified Cpf1 protein may contain a nuclear localization signal (NLS). For example, but not by way of limitation, NLS sequences useful in connection with the methods and compositions disclosed herein would include amino acid sequences that can facilitate protein import into the cell nucleus. NLS sequences useful in connection with the methods and compositions disclosed herein are known in the art. Examples of such NLS sequences include the Nucleoplasmin NLS having the amino acid sequence: KRPAATKKAGQAKKKK (SEQ ID NO: 1006) and the Simian Virus 40 "SV40" NLS having the amino acid sequence PKKKRKV (SEQ ID NO: 1007).

In certain embodiments, the NLS sequence of the modified Cpf1 protein is positioned at or near the C-terminus of the Cpf1 protein sequence. For example, without limitation, modified Cpf1 proteins include His-AsCpf1-nNLS (SEQ ID NO: 1000); His-AsCpf1-sNLS (SEQ ID NO: 1008); and His-AsCpf1-sNLS-sNLS (SEQ ID NO: 1001). wherein “His” refers to the 6-histidine purified sequence, “AsCpf1” refers to the Acidaminococcus sp. Cpf1 protein sequence, and “nNLS” refers to the nucleoplasmin NLS. , “sNLS” refers to the SV40 NLS. Identity of NLS sequences, for example addition of two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) and addition of sequences with or without purification sequences such as 6-histidine sequences Additional permutations of gender and C-terminal position are within the scope of the presently disclosed subject matter.

In certain embodiments, the NLS sequence of the modified Cpf1 protein may be placed at or near the N-terminus of the Cpf1 protein sequence. For example, without limitation, modified Cpf1 proteins are from His-sNLS-AsCpf1 (SEQ ID NO: 1009), His-sNLS-sNLS-AsCpf1 (SEQ ID NO: 1010) and sNLS-sNLS-AsCpf1 (SEQ ID NO: 1011). can be selected. Identity of NLS sequences, for example addition of two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) and addition of sequences with or without purification sequences such as 6-histidine sequences Additional permutations of gender and N-terminal position are within the scope of the presently disclosed subject matter.

In certain embodiments, a modified Cpf1 protein may comprise NLS sequences located at or near both the N-terminus and C-terminus of the Cpf1 protein sequence. For example, without limitation, the modified Cpf1 protein can be selected from His-sNLS-AsCpf1-sNLS (SEQ ID NO: 1012) and His-sNLS-sNLS-AsCpf1-sNLS-sNLS (SEQ ID NO: 1013). For example, with or without the addition of two or more nNLS sequences or a combination of nNLS and sNLS sequences (or other NLS sequences) to either the N-terminal/C-terminal positions and purification sequences such as 6-histidine sequences. NLS sequence identities, such as sequence additions, and additional permutations of N-terminal/C-terminal positions are within the scope of the presently disclosed subject matter.

In certain embodiments, modified Cpf1 proteins may include alterations (eg, deletions or substitutions) to one or more cysteine residues in the Cpf1 protein sequence. For example, without limitation, a modified Cpf1 protein may comprise modifications at positions selected from the group consisting of C65, C205, C334, C379, C608, C674, C1025 and C1248. In certain embodiments, modified Cpf1 proteins may include substitution of one or more cysteine residues for serine or alanine. In certain embodiments, the modified Cpf1 protein may comprise modifications selected from the group consisting of C65S, C205S, C334S, C379S, C608S, C674S, C1025S and C1248S. In certain embodiments, the modified Cpf1 protein may comprise modifications selected from the group consisting of C65A, C205A, C334A, C379A, C608A, C674A, C1025A and C1248A. In certain embodiments, a modified Cpf1 protein may contain alterations at positions C334 and C674 or C334, C379 and C674. In certain embodiments, modified Cpf1 proteins may comprise modifications of C334S and C674S or C334S, C379S and C674S. In certain embodiments, modified Cpf1 proteins may comprise modifications of C334A and C674A or C334A, C379A and C674A. In certain embodiments, the modified Cpf1 protein has one or more cysteine residues, e.g., His-AsCpf1-nNLS Cys-less (SEQ ID NO: 1014) or His-AsCpf1-nNLS Cys-low (SEQ ID NO: 1015) It can involve both modification as well as introduction of one or more NLS sequences. In various embodiments, Cpf1 proteins comprising deletions or substitutions of one or more cysteine residues exhibit reduced aggregation.

In certain embodiments, other modified Cpf1 proteins known in the art can be used with the methods and systems described herein. For example, in certain embodiments, a modified Cpf1 can be a Cpf1 containing mutations S542R/K548V/N552R (“Cpf1 RVR”). Cpf1 RVR has been shown to cleave target sites with the TATV PAM. In certain embodiments, the modified Cpf1 can be Cpf1 containing the mutations S542R/K607R (“Cpf1 RR”). Cpf1 RR has been shown to cleave target sites with TYCV/CCCC PAM.

In some embodiments, Cpf1 variants are used herein, wherein the Cpf1 variants are 11, 12, 13, 14, 15, 16, 17, 34, 36, 39, 40, 43, 46, 47, 50, 54, 57, 58, 111, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 676, 679, 680, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 707, 711, 714, 715, 716, 717, 718, 719, 720, 721, 722, 739, 765, 768, 769, 773, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 870, 871, AsCpf1 (Acida containing mutations in one or more residues of Acidaminococcus sp. BV3L6).

In certain embodiments, the Cpf1 variant, as used herein, is a variant of Zhang et al. any of the Cpf1 proteins described in WO 2017/184768 A1 (the "'768 publication") by

In certain embodiments, modified Cpf1 proteins (also referred to as Cpf1 variants) used herein have the (Cpf1 polypeptide sequences) or by any of the sequences set forth in SEQ ID NOs: 1019-1021, 1110-17 (Cpf1 polynucleotide sequences). Table 14 lists exemplary Cpf1 variant amino acid and nucleotide sequences. These sequences are described in FIG. 40 detailing the positions of the 6-histidine sequences (underlined letters) and the NLS sequences (bold). For example, addition of two or more nNLS sequences or combinations of nNLS and sNLS sequences (or other NLS sequences) to either the N-terminal/C-terminal positions and sequences with or without purification sequences such as 6-histidine sequences. Additional permutations of NLS sequence identities and N-terminal/C-terminal positions such as the addition of are within the scope of the presently disclosed subject matter.

In certain embodiments, any of the Cpf1 proteins or modified Cpf1 proteins disclosed herein are conjugated to one or more gRNAs comprising a targeting domain set forth in SEQ ID NOs: 1002 and/or 1004. can be used to modify the CCAAT box target region. In certain embodiments, any of the Cpf1 proteins or modified Cpf1 proteins disclosed herein can be conjugated to one or more gRNAs comprising the sequences listed in Table 12 or Table 13. In certain embodiments, the modified Cpf1 protein can be His-AsCpf1-nNLS (SEQ ID NO:1000) or His-AsCpf1-sNLS-sNLS (SEQ ID NO:1001). In certain embodiments, the modified Cpf1 protein used herein has SEQ ID NOS: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOS: 1019-1021, 1110-17 (Cpf1 polynucleotide sequences). In certain embodiments, a modified Cpf1 protein may comprise the sequence set forth in SEQ ID NO:1097.

In certain embodiments, modified Cpf1 proteins may comprise Cpf1 variants as described in Kleinstiver 2019. For example, without limitation, in certain embodiments the modified Cpf1 protein may be enAsCasl2a. In certain embodiments, a modified Cpf1 protein may cleave a target site with a TTTV PAM. In certain embodiments, the modified Cpf1 protein may cleave target sites with NWYN PAM.

Modifications of RNA-guided nucleases Although the RNA-guided nucleases described above have activities and properties that may be useful in a variety of applications, it will be appreciated by those skilled in the art that RNA-guided nucleases may also be optionally modified to enhance cleavage activity, PAM specificity, or other will recognize that the structural or functional characteristics of may be modified.

Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe are described above. Exemplary mutations that can be made in the RuvC domain, Cas9 HNH domain or Cpf1 Nuc domain are described in Ran & Hsu 2013 and Yamano 2016 and Cotta-Ramusino. In general, mutations that reduce or eliminate the activity of one of the two nuclease domains will result in an RNA-guided nuclease with nickase activity, although the type of nickase activity will vary depending on which domain is inactivated. It should be noted. As an example, inactivation of the RuvC domain of Cas9 results in a nickase that cleaves the complementary strand or top strand, as shown below (where C indicates the site of cleavage).

Inactivation of the Cas9 HNH domain, on the other hand, results in a nickase that cleaves the bottom strand or the non-complementary strand.

Modification of PAM specificity relative to naturally occurring Cas9 reference molecules is described by Kleinstiver et al. According to S. S. pyogenes (Kleinstiver 2015a) and S. pyogenes (Kleinstiver 2015a). both S. aureus (Kleinstiver 2015b). Kleinstiver et al. also described modifications that improve the targeting fidelity of Cas9 (Kleinstiver 2016). Kleinstiver et al. also describe modifications of Cpf1 that provide increased activity and improved targeting coverage (Kleinstiver 2019). Each of these references is incorporated herein by reference.

RNA-guided nucleases are divided into two or more parts, as described by Zetsche 2015 and Fine 2015 (both incorporated herein by reference).

RNA-guided nucleases may, in certain embodiments, be size-optimized or shortened, for example, through one or more deletions that reduce the size of the nuclease while still maintaining gRNA binding, target and PAM recognition and cleavage activity. . In certain embodiments, the RNA-guided nuclease is covalently or non-covalently attached to another polypeptide, nucleotide or other structure, optionally by a linker. Exemplary conjugated nucleases and linkers are described by Guilinger 2014, which is incorporated herein by reference for all purposes.

The RNA-guided nuclease optionally includes a label, including but not limited to a nuclear localization signal, to also facilitate translocation of the RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease may incorporate C-terminal and/or N-terminal nuclear localization signals. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.

The foregoing list of modifications is intended to be exemplary in nature, and it will be apparent to those skilled in the art that other modifications may be possible or desirable in particular applications in light of this disclosure. you will understand. Thus, for the sake of brevity, the exemplary systems, methods and compositions of this disclosure are presented with reference to particular RNA-guided nucleases, although the RNA-guided nucleases used vary in their principle of operation. It should be understood that it may be modified in a non-intrusive manner. Such modifications are within the scope of this disclosure.

Nucleic Acids Encoding RNA-Guided Nucleases Provided herein are nucleic acids that encode RNA-guided nucleases, such as, for example, Cas9, Cpf1, or functional fragments thereof. Exemplary nucleic acids encoding RNA-guided nucleases have been previously described (see, eg, Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

Optionally, the nucleic acid encoding the RNA-guided nuclease can be a synthetic nucleic acid sequence. For example, synthetic nucleic acid molecules can be chemically modified. In certain embodiments, an mRNA encoding an RNA-guided nuclease will have one or more (eg, all) of the following properties: it may be capped; it may be polyadenylated; 5-methylcytidine and/or pseudouridine.

A synthetic nucleic acid sequence can also be codon-optimized, eg, at least one uncommon or less-common codon is replaced by a common codon. For example, synthetic nucleic acids can direct the synthesis of optimized messenger mRNAs optimized for expression, eg, within the mammalian expression systems described herein. An example of a codon-optimized Cas9 coding sequence is presented in Cotta-Ramusino.

Additionally or alternatively, a nucleic acid encoding an RNA-guided nuclease can include a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

Functional Analysis of Candidate Molecules Candidate RNA-guided nucleases, gRNAs and complexes thereof can be evaluated by standard methods known in the art. See, for example, Cotta-Ramusino. The stability of RNP complexes can be assessed by differential scanning fluorimetry as described below.

Differential scanning fluorimetry (DSF)
Thermostability of ribonucleoprotein (RNP) complexes containing gRNA and RNA-guided nucleases can be measured by DSF. DSF technology measures protein thermostability, which can increase under favorable conditions, eg, the addition of binding RNA molecules such as gRNA.

The DSF assay can be performed according to any suitable protocol, including but not limited to: (a) testing different conditions (e.g., different stoichiometric ratios of gRNA:RNA-derived nuclease protein, different buffers, etc.); and (b) testing RNA-guided nucleases and/or gRNA modifications (e.g., chemical modifications, sequence modifications, etc.) to improve RNP formation or stability. can be used in any suitable setting, including identifying modifications that One readout of the DSF assay is the change in melting temperature of the RNP complex; a relatively high change indicates that the RNP complex is more stable compared to standard RNP complexes characterized by a lower change. (thus may have greater activity or more favorable formation kinetics, degradation kinetics or other functional properties). If the DSF assay is deployed as a screening tool, a threshold melting temperature change can be identified, whereby the result is one or more RNPs with a melting temperature change greater than or equal to the threshold. For example, the threshold can be 5-10°C (eg, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C) or more, and the result is a melting temperature change greater than or equal to the threshold. can be one or more RNPs characterized by

Two non-limiting examples of DSF assay conditions are as follows.

To determine the best solution for forming RNP complexes, a fixed concentration (e.g., 2 μM) of Cas9+10×SYPRO Orange® (Life Technologies, Catalog No. S-6650) in water was added in 384-well plates. dispense into Equimolar amounts of gRNA diluted in solutions with varying pH and salt are then added. After incubating for 10 minutes at room temperature and briefly centrifuging to remove any air bubbles, 20 minutes were cycled using a Bio-Rad CFX384™ Real Time System C1000 Touch™ Thermal Cycler with Bio-Rad CFX Manager software. A ramp is run from °C to 90 °C in 1 °C increments every 10 seconds.

The second assay mixed various concentrations of gRNA with a fixed concentration (e.g., 2 μM) of Cas9i in optimal buffer from Assay 1 above and incubated in a 384-well plate (e.g., 10 minutes at room temperature). consists of doing An equal volume of Optimal Buffer plus 10× SYPRO Orange® (Life Technologies, Catalog No. S-6650) is added and the plate is sealed with Microseal® B adhesive (MSB-1001). After a brief centrifugation to remove any air bubbles, a Bio-Rad CFX384™ real-time system C1000 Touch™ thermal cycler with Bio-Rad CFX manager software was used to cycle from 20°C to 90°C for 10 minutes. Run the ramp in 1° C. increments every second.

Genome Editing Strategies The genome editing systems described above are used, in various embodiments of the present disclosure, to edit (ie, modify) within a target region of DNA within a cell or obtained from a cell. Various strategies for making specific edits are described herein, and these strategies generally describe the desired repair outcome, the number and location of individual edits (e.g., SSBs or DSBs), and the targets of such edits. Described by parts.

Genome editing strategies involving the formation of SSBs or DSBs include (a) deletion of all or part of the target region; (b) insertion into or replacement of all or part of the target region; Characterized by repair results including full or partial interruption. This grouping is not intended to be limiting or bound by any particular theory or model, and is provided solely for the sake of economy of presentation. Those skilled in the art will appreciate that the results listed are not mutually exclusive and that some repairs may lead to other results. A description of a particular editing strategy or method should not be understood to require a particular repair result unless specified otherwise.

Replacement of the target region generally involves replacement of all or part of a sequence present within the target region by a homologous sequence through, for example, gene correction or gene conversion, two repair outcomes mediated by the HDR pathway. . HDR is facilitated by the use of donor templates, which can be single-stranded or double-stranded, as described in more detail below. Single- or double-stranded templates can be exogenous, where they facilitate gene correction, or they can be endogenous (e.g., homologous sequences in the cell genome), where gene conversion is performed. promote The exogenous template can have asymmetric overhangs (i.e., the portion of the template that is complementary to the site of the DSB is It can be offset in the 3' or 5' direction rather than being centered within the mold). If the template is single-stranded, it can correspond to either the complementary (top) or non-complementary (bottom) strand of the target region.

As described in Ran & Hsu 2013 and Cotta-Ramusino, gene conversion and gene correction are optionally facilitated by forming one or more nicks in or around the target region. Optionally, a double nickase strategy is used to form two offset SSBs and then a single DSB with an overhang (eg, a 5' overhang).

Interruption and/or deletion of all or part of the target sequence can be achieved through a variety of repair outcomes. As an example, as described in Maeder for the LCA10 mutation, sequences can be deleted by simultaneously generating two or more DSBs flanking the target region, which are then excised as the DSBs are repaired. be done. As another example, a sequence can be interrupted by deletions generated by formation of a double-stranded break with a single-stranded overhang, followed by nucleotide-terminal hydrolytic processing of the overhang prior to repair.

One particular subset of target sequence interruptions is mediated by the formation of indels in the target sequence, where repair outcomes are typically mediated by the NHEJ pathway (including Alt-NHEJ). NHEJ is referred to as a "failure-prone" repair pathway because of its association with indel mutations. In some cases, however, DSBs are repaired by NHEJ without alteration of the sequences surrounding them (so-called 'perfect' or 'scarless' repair); this generally requires that both ends of the DSB be fully ligated. and Indels, on the other hand, are thought to arise from the enzymatic processing of free DNA ends before they are ligated, which adds and/or removes nucleotides from one or both strands of one or both free ends. .

Since enzymatic processing of free DSB ends can be stochastic in nature, indel mutations tend to be variable and occur along a distribution, depending on the specific target site, cell type used, It can be influenced by a variety of factors, including genome editing strategies. Nonetheless, a limited generalization about indel formation is possible: deletions made by repair of a single DSB range most commonly from 1 to 50 bp, although 100 to 200 bp may exceed. Insertions formed by repair of a single DSB tend to be shorter and often contain a short duplication of sequence immediately surrounding the cut site. However, it is possible to obtain large inserts, in which case the insert sequences are often traced to other regions of the genome or to plasmid DNA present within the cell.

Genome editing systems configured to generate indel mutations and indels are useful for interrupting target sequences, for example, when generation of a particular final sequence is not desired and/or when frameshift mutations are tolerated. be. They may also be useful in situations where a particular sequence is preferred, as long as the particular sequence desired tends to preferentially result from SSB or DSB repair at a given site. Indel mutations are also useful tools for evaluating or screening the activity of specific genome editing systems and their components. In these and other settings, indels are defined as (a) their relative and absolute frequencies in the genome of cells contacted with the genome editing system, and (b) ±1, ±2, e.g. It can be characterized by a distribution of numerical differences such as ±3. As an example, in a lead discovery setting, multiple gRNAs can be screened to identify those that most efficiently drive cleavage at the target site based on indel reads under controlled conditions. Guides that generate indels above a threshold frequency or guides that generate a particular distribution of indels can be selected for further research and development. The frequency and distribution of indels can also be used as a readout to evaluate different genome editing system implementations or formulation and delivery methods, e.g., by keeping the gRNA constant and varying other specific reaction conditions or delivery methods. can be useful.

Multiplex Strategy Genome editing systems according to the present disclosure can also be used for multiplex gene editing, generating two or more DSBs either at the same locus or at different loci. Any of the RNA-guided nucleases and gRNAs disclosed herein can be used in genome editing systems for multiplex gene editing. Editing strategies involving the formation of multiple DSBs or SSBs are described, for example, in Cotta-Ramusino. In certain embodiments, multiple gRNAs and RNA-directed nucleases may be used in a genome editing system to introduce modifications (eg, deletions, insertions) into the CCAAT box target regions of HBG1 and/or HBG2. In certain embodiments, the RNA-guided nuclease may be Cpf1 or modified Cpf1.

Donor Template Design Donor template design is described in detail in, for example, Cotta-Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single-stranded (ssODN) or double-stranded (dsODN), facilitate HDR-based repair of DSBs or improve overall editing efficiency It is particularly useful for introducing alterations into a target DNA sequence, inserting new sequences into the target sequence, or completely replacing the target sequence.

Whether single-stranded or double-stranded, the donor template is homologous to DNA regions in or near (e.g., flanking or flanking) the target sequence to be cleaved. Generally includes regions. These regions of homology are referred to herein as "homology arms" and are shown schematically below.
[5' homology arm]--[replacement sequence]--[3' homology arm]

The homology arms can have any suitable length (including none if only one homology arm is used), and the 3' and 5' homology arms have the same length. or may differ in length. The selection of appropriate homology arm lengths can be influenced by a variety of factors, such as the desire to avoid homology or micro-homology with particular sequences such as Alu repeats or other very common elements. . For example, the 5' homology arm can be shortened to avoid sequence repeat elements. In other embodiments, the 3' homology arms may be shortened to avoid sequence repeat elements. In some embodiments, both the 5' and 3' homology arms may be shortened to avoid inclusion of certain sequence repeat elements. In addition, some homology arm designs may improve editing efficiency or increase the frequency of desired repair outcomes. For example, Richardson 2016, incorporated herein by reference, found that the relative asymmetry of the 3' and 5' homology arms of a single-stranded donor template affects repair rate and/or outcome.

Replacement sequences in donor templates have been described elsewhere, including Cotta-Ramusino. A replacement sequence can be of any suitable length (including no nucleotides if the desired result of repair is a deletion) and will typically be contains 1, 2, 3 or more sequence modifications. One common sequence modification involves altering the native sequence to repair mutations associated with the disease or condition for which treatment is desired. Another common sequence modification is one or more sequences that are complementary to the PAM sequence of the RNA-guided nuclease or the targeting domain of the gRNA used to generate the SSB or DSB or thus the PAM sequence or target modification domain to reduce or eliminate repetitive cleavage of the target site after the replacement sequence has been incorporated into the target site.

When a linear ssODN is used, it is (i) annealed to the nicked strand of the target nucleic acid, (ii) annealed to the intact target nucleic acid strand, (iii) annealed to the plus strand of the target nucleic acid, and/or (iv) be configured to anneal to the minus strand of the target nucleic acid. The ssODN can be any nucleotide, such as, for example, about or at least 80-200 nucleotides or less (eg, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 nucleotides). can have a suitable length of

It should be noted that the template nucleic acid can also be a nucleic acid vector such as a viral genome or a circular double-stranded DNA such as a plasmid. A nucleic acid vector containing a donor template may contain other coding or non-coding elements. For example, the template nucleic acid can include specific genomic backbone elements (e.g., inverted terminal repeats in the case of the AAV genome), and optionally additional sequences encoding gRNAs and/or RNA-guided nucleases of the viral genome. It can be delivered as part (eg in an AAV or lentiviral genome). In certain embodiments, the donor template may be flanked by or flanked by target sites recognized by one or more gRNAs, facilitating the formation of free DSBs at one or both ends of the donor template, which are identical It may be involved in the repair of corresponding SSBs or DSBs formed in cellular DNA using gRNAs. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino, incorporated by reference.

Whatever form is used, the template nucleic acid can be designed to avoid unwanted sequences. In certain embodiments, one or both homology arms may be shortened to avoid overlapping with certain sequence repetitive elements, eg Alu repeats, LINE elements.

In certain embodiments, silent, nonpathogenic SNPs may be included in the ssODN donor template to allow identification of gene editing events.

In certain embodiments, the donor template can be a non-specific template that is non-homologous to regions of DNA within or near the target sequence to be cleaved. In certain embodiments, donor templates for use in targeting the GATA1 binding motif in BCL11Ae include, but are not limited to, non-homologous regions of DNA within or near the GATA1 binding motif in BCL11Ae. A target-specific template may be included. In certain embodiments, donor templates for use in targeting the 13nt target region include, but are not limited to, non-target-specific templates that are non-homologous to regions of DNA within or near the 13nt target region. may be mentioned.

A donor template or template nucleic acid, as used herein, is a nucleic acid sequence that can be used in conjunction with an RNA nuclease molecule and one or more gRNA molecules to alter (e.g., delete, disrupt or modify) a target DNA sequence. point to In certain embodiments, the template nucleic acid provides modifications (eg, deletions) to the CCAAT box target regions of HBG1 and/or HBG2. In certain embodiments, modifications are non-naturally occurring modifications. In certain embodiments, non-naturally occurring alterations in the CCAAT box target regions of HBG1 and/or HBG2 may comprise 18 nt target regions, 11 nt target regions, 4 nt target regions or 1 nt target regions or combinations thereof. In certain embodiments, modifications are naturally occurring modifications. In certain embodiments, naturally occurring alterations in the CCAAT box target region of HBG1 and/or HBG2 are 13nt target regions, c. -117G>A target region or combinations thereof. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODNs are plus or minus strand.

For example, a template nucleic acid for introducing an 18nt deletion in an 18nt target region (HBG1 c.-104 to -121, HBG2 c.-104 to -121, or combinations thereof) includes a 5' homology arm, a replacement sequence and The replacement sequence is 0 nucleotides or 0 bp, which may include a 3' homology arm. In certain embodiments, the 5' homology arm can be from about 25 to about 200 nucleotides or more, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 18 nt target region. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides or more, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 3' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 18 nt target region. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN comprises, consists essentially of, or consists of SEQ ID NO:974 (OLI16409) or SEQ ID NO:975 (OLI16410).

In certain embodiments, the template nucleic acid for introducing 11nt deletions in the 11nt target region (HBG1 c.-105 to -115, HBG2 c.-105 to -115, or combinations thereof) comprises the 5' homology arm , a replacement sequence and a 3' homology arm, wherein the replacement sequence is 0 nucleotides or 0 bp. In certain embodiments, the 5' homology arm can be from about 25 to about 200 nucleotides, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the target region of 11 nt. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides. In certain embodiments, the 3' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the target region of 11 nt. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN comprises, consists essentially of, or consists of SEQ ID NO:976 (OLI16411) or SEQ ID NO:978 (OLI16413).

In certain embodiments, the template nucleic acid for introducing a 4nt deletion in the 4nt target region (HBG1 c.-112 to -115, HBG2 c.-112 to -115, or combinations thereof) comprises the 5' homology arm , a replacement sequence and a 3' homology arm, wherein the replacement sequence is 0 nucleotides or 0 bp. In certain embodiments, the 5' homology arm can be from about 25 to about 200 nucleotides, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 4nt target region. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides. In certain embodiments, the 3' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 4nt target region. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN is SEQ ID NO:984 (OLI16419), SEQ ID NO:985 (OLI16420), SEQ ID NO:986 (OLI16421), SEQ ID NO:987 (OLI16422), SEQ ID NO:988 (OLI16423), SEQ ID NO:989 (OLI16424) , SEQ ID NO:990 (OLI16425), SEQ ID NO:991 (OLI16426), SEQ ID NO:992 (OLI16427), SEQ ID NO:993 (OLI16428), SEQ ID NO:994 (OLI16429) or SEQ ID NO:995 (OLI16430), or essentially consists of or consists of

In certain embodiments, the template nucleic acid for introducing a 1-nt deletion in a 1-nt target region (HBG1 c.-116, HBG2 c.-116, or a combination thereof) comprises a 5' homology arm, a replacement sequence and a 3' The replacement sequence, which may contain homology arms, is 0 nucleotides or 0 bp. In certain embodiments, the 5' homology arm can be from about 25 to about 200 nucleotides, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 1 nt target region. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides. In certain embodiments, the 3' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' of the 1 nt target region. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN comprises, consists essentially of, or consists of SEQ ID NO:982 (OLI16417) or SEQ ID NO:983 (OLI16418).

In certain embodiments, alterations in the CCAAT box target region reproduce or mimic naturally occurring alterations such as 13nt deletions. In certain embodiments, the template nucleic acid for introducing a 13nt deletion into a 13nt target region (HBG1 c.-116, HBG2 c.-116 or a combination thereof) comprises a 5' homology arm, a replacement sequence and a 3' The replacement sequence, which may contain homology arms, is 0 nucleotides or 0 bp. In certain embodiments, the 5' homology arm can be from about 25 to about 200 nucleotides, eg, at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the 13nt target region. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides. In certain embodiments, the 3' homology arm comprises about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' to the 13nt target region. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN comprises, consists essentially of, or consists of SEQ ID NO:979 (OLI16414) or SEQ ID NO:977 (OLI16412).

In certain embodiments, alterations in the CCAAT box target region reproduce or mimic naturally occurring alterations such as G to A substitutions in the -117G>A target region. In certain embodiments, the template nucleic acid for introducing a −117G>A substitution into the −117G>A target region (HBG1 c.-117G>A, HBG2 c.-117G>A, or combinations thereof) is 5′ It may include a homology arm, a replacement sequence and a 3' homology arm, where the replacement sequence is 0 nucleotides or 0 bp. In certain embodiments, the 5' homology arm can be from about 100 to about 200 nucleotides, eg, at least about 100, 125, 150, 175 or 200 nucleotides in length. In certain embodiments, the 5' homology arm has about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 5' to the target region of 117G>A. include. In certain embodiments, the 3' homology arm can be from about 25 to about 200 nucleotides in length, such as at least about 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides. In certain embodiments, the 3' homology arm has about 50-100 bp of homology, such as 55-95, 60-90, 70-90 or 80-90 bp, 3' to the target region of 117G>A. include. In certain embodiments, the 5' and 3' homology arms are symmetrical in length. In certain embodiments, the 5' and 3' homology arms are asymmetric in length. In certain embodiments, the template nucleic acid is an ssODN. In certain embodiments, the ssODN is the positive strand. In certain embodiments, the ssODN is the minus strand. In certain embodiments, the ssODN comprises, consists essentially of, or consists of SEQ ID NO:980 (OLI16415) or SEQ ID NO:981 (OLI16416).

In certain embodiments, the 5' homology arm comprises a 5' phosphorothioate (PhTx) modification. In certain embodiments, the 3' homology arm comprises a 3' PhTx modification. In certain embodiments, the template nucleic acid includes 5' and 3' PhTx modifications.

In certain embodiments, the ssODN for introducing modifications (e.g., deletions) into the CCAAT box target region is an RNA nuclease and a CCAAT target region, such as the gRNAs disclosed in Table 6, Table 12, Table 13. It can be used in combination with one or more gRNAs to target.

Target Cells Using the genome editing system according to the present disclosure, cells can be manipulated or modified, eg, target nucleic acids can be edited or modified. Manipulation may be performed in vivo or ex vivo in various embodiments.

Various cell types may be engineered or engineered according to embodiments of the present disclosure, such as for in vivo applications, where multiple cell types are engineered or engineered, e.g., by delivering a genome editing system according to the present disclosure to multiple cell types. obtain. However, in other cases it may be desirable to limit the manipulation or modification to particular cell types. For example, in some instances, editing cells with limited differentiation potential or terminally differentiated cells, such as photoreceptor cells in Maeder's example, where genotypic modifications are expected to result in changes in cell phenotype. may be desirable. However, in other cases it may be desirable to edit poorly differentiated, multipotent or pluripotent, stem or progenitor cells. By way of example, the cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem/progenitor cells (HSPCs) or other stem or progenitor cell types that differentiate into cell types relevant to a given use or indication. can be

Of course, the cells to be modified or manipulated are variously dividing or non-dividing cells, depending on the targeted cell type and/or the desired editing result.

When cells are manipulated or modified ex vivo, the cells can be used immediately (eg, administered to a control) or they can be maintained or stored for later use. One skilled in the art will appreciate that cells can be maintained or preserved in culture (eg, frozen in liquid nitrogen) using any suitable method known in the art.

Implementation of Genome Editing Systems: Delivery, Formulation and Routes of Administration As discussed above, the genome editing system of the present disclosure may be implemented in any suitable manner, including, but not limited to, RNA Components of the system, including inducible nucleases, gRNAs, any donor template nucleic acids, are designed to effect transduction, expression or introduction of the genome editing system and/or to cause a desired repair outcome in a cell, tissue or subject. can be delivered, formulated or administered in any suitable form or combination of forms. Some non-limiting examples of genome editing system implementations are listed in Tables 2 and 3. However, those skilled in the art will appreciate that these lists are not exhaustive and that other implementations are possible. With particular reference to Table 2, this table lists several exemplary implementations of genome editing systems comprising a single gRNA and optional donor template. However, genome editing systems according to the present disclosure may incorporate other components such as multiple gRNAs, multiple RNA-guided nucleases and proteins, and a variety of implementations will occur to those of skill in the art based on the principles shown in the table. will become clear. In the table, [N/A] indicates that the genome editing system does not contain the indicated component.

Table 3 summarizes various delivery methods for the components of the genome editing system as described herein. Again, the list is intended to be illustrative rather than limiting.

Nucleic Acid-Based Delivery of Genome Editing Systems Nucleic acids encoding the various elements of a genome editing system according to the present disclosure are administered to a subject by methods known in the art or as described herein. or delivered intracellularly. For example, the DNA encoding the RNA-guided nuclease and/or the DNA encoding the gRNA and the donor template nucleic acid can be used, for example, in vectors (e.g., viral vectors or non-viral vectors), non-vector-based methods (e.g., naked DNA or DNA complex) or a combination thereof.

Nucleic acids encoding genome editing systems or components thereof can be delivered directly to cells as naked DNA or RNA, e.g., by transfection or electroporation, or facilitate uptake by target cells (e.g., erythrocytes, HSCs). It can be conjugated to a molecule such as N-acetylgalactosamine. Nucleic acid vectors such as those summarized in Table 3 can also be used.

Nucleic acid vectors can include one or more sequences encoding genome editing system components such as RNA-guided nucleases, gRNAs and/or donor templates. The vector includes a signal peptide (e.g., for nuclear, nucleolar, or mitochondrial localization) that is linked to (e.g., inserted into or fused to) a protein-encoding sequence. ) may also be included. As an example, a nucleic acid vector can include a Cas9 coding sequence that includes one or more nuclear localization sequences (eg, nuclear localization sequences from SV40).

Nucleic acid vectors can also include any suitable number of regulatory/control elements such as, for example, promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences or internal ribosome entry sites (IRES). These elements are well known in the art and described in Cotta-Ramusino.

Nucleic acid vectors according to the present disclosure include recombinant viral vectors. Exemplary viral vectors are described in Table 3, and additional suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art may also be used. Additionally, viral particles can be used to deliver the components of the genome editing system in the form of nucleic acids and/or peptides. For example, an "empty" virus particle can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For example, organic (eg, lipid and/or polymeric) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of the present disclosure. Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 4, and Table 5 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.

Non-viral vectors optionally contain targeting modifications to improve uptake and/or selectively target particular cell types. These targeted modifications include, for example, cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars (eg, N-acetylgalactosamine (GalNAc)) and cell-penetrating peptides. Such vectors also optionally employ fusogenic and endosomal destabilizing peptides/polymers, undergo acid-induced conformational changes (e.g., to facilitate endosomal leakage of cargo), and/ or incorporate stimuli-cleavable polymers for release into cellular compartments, for example. For example, disulfide-based cationic polymers that are cleaved in a reducing cellular environment can be used.

In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than components of the genome editing system, e.g., the RNA-guided nuclease components and/or gRNA components described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered simultaneously with one or more of the components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered by one or more of the components of the genome editing system (e.g., about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks or less than 4 weeks) before or after. In certain embodiments, the nucleic acid molecule is delivered by a different means than by which one or more components of the genome editing system are delivered, eg, an RNA-guided nuclease component and/or a gRNA component. Nucleic acid molecules can be delivered by any of the delivery methods described herein. For example, nucleic acid molecules can be delivered by viral vectors, such as, for example, integration-deficient lentiviruses, and can be conjugated to RNA-guided nuclease molecule components and/or gRNA constructs, such that toxicity caused by nucleic acids (e.g., DNA) can be reduced. Elements can be delivered by electroporation. In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, eg, a protein described herein. In certain embodiments, a nucleic acid molecule encodes an RNA molecule, eg, an RNA molecule described herein.

Delivery of Genome Editing System Components Encoding RNPs and/or RNAs RNAs encoding RNPs (complexes of gRNAs and RNA-guided nucleases) and/or RNA-guided nucleases and/or gRNAs, some of which are cotta-Ramusino It can be delivered to cells or administered to a subject by methods known in the art as described. In vitro, RNAs encoding RNA-guided nucleases and/or gRNAs can be delivered by, for example, microinjection, electroporation, transient cell compaction or squeezing (see, eg, Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc or other conjugate-mediated delivery and combinations thereof can also be used for in vitro and in vivo delivery. A protective interactive non-condensing (PINC) system can be used for delivery.

In vitro delivery via electroporation involves mixing cells with RNA encoding an RNA-guided nuclease and/or gRNA in the presence or absence of a donor template nucleic acid molecule in a cartridge, chamber or cuvette, once or and applying a plurality of electrical impulses of defined length and amplitude. Electroporation systems and protocols are known in the art, and any suitable electroporation tool and/or protocol may be used in connection with various embodiments of the present disclosure.

Routes of Administration Genome editing systems or cells modified or engineered using such systems may be administered to a subject by any suitable manner or route, whether locally or systemically. Systemic administration methods include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intramedullary, intraarterial, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Systemically administered components can be modified or formulated to target, for example, HSCs, hematopoietic stem/progenitor cells or erythroid progenitor or progenitor cells.

Local modes of administration include, by way of example, intramedullary injection into the trabecular bone or intrafemoral injection into the medullary cavity and injection into the portal vein. In certain embodiments, significantly less components (compared to systemic approaches) are administered locally (e.g., directly into the bone marrow) compared to systemic administration (e.g., intravenously). can be effective. Local administration methods can reduce or eliminate the occurrence of potentially toxic side effects that can occur when a therapeutically effective amount of a component is administered systemically.

Administration can be provided as a periodic bolus (eg, intravenously) or as a continuous infusion from an internal or external reservoir (eg, from an IV bag or implantable pump). Components can be administered locally by continuous release, eg, from a sustained release drug delivery device.

Additionally, the components can be formulated to be released over an extended period of time. The release system may comprise a matrix of biodegradable material or material that releases incorporated components by diffusion. Components may be uniformly or non-uniformly distributed within the release system. A variety of release systems may be useful, but selection of an appropriate system will depend on the release rate required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include inorganic and organic excipients and diluents such as, but not limited to, polymers and polymeric matrices, non-polymeric matrices or calcium carbonate and sugars such as trehalose. . Release systems can be natural or synthetic. However, synthetic release systems are preferred, as they are generally more reliable, more reproducible and produce more defined release profiles. Release system materials can be selected such that components with different molecular weights are released by diffusion through the material or by decomposition of the material.

Exemplary synthetic biodegradable polymers include, for example, polyamides such as poly(amino acids) and poly(peptides); poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) and poly(caprolactone poly(anhydrides); polyorthoesters; polycarbonates; modifications), copolymers and mixtures thereof. Typical synthetic non-degradable polymers include, for example, polyethers such as poly(ethylene oxide), poly(ethylene glycol) and poly(tetramethylene oxide); methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and Vinyl polymers such as methacrylic acid and poly(vinyl alcohol), poly(vinylpyrrolidone) and poly(vinyl acetate)--polyacrylates and polymethacrylates; poly(urethanes); cellulose, alkyl, hydroxyalkyl, ether, ester, nitrocellulose and various derivatives thereof, such as cellulose acetate; polysiloxanes; any chemical derivative thereof (e.g., substitution, addition, hydroxylation, oxidation, and other modifications of chemical groups such as alkyl, alkylene, etc., and other modifications routinely made by those skilled in the art). ), copolymers and mixtures thereof.

Poly(lactide-co-glycolide) microspheres may also be used. Microspheres are typically composed of lactic and glycolic acid polymers that are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with the components described herein. In some embodiments, the genome editing system, system components and/or nucleic acids encoding system components are delivered with block copolymers such as poloxamers or poloxamines.

Multimodal or Differential Delivery of Components Those skilled in the art will appreciate, in view of the present disclosure, that the different components of the genome editing system disclosed herein can be delivered together or separately and simultaneously or non-simultaneously. will understand. Separate and/or asynchronous delivery of genome-editing system components may be particularly desirable to provide temporal or spatial control over genome-editing system function and to limit specific effects caused by their activity. .

Differential or differential mode, as used herein, refers to delivery modes that confer different pharmacodynamic or pharmacokinetic properties on the component molecule of interest, eg, an RNA-guided nuclease molecule, gRNA, template nucleic acid or payload. For example, delivery modalities may result in, for example, different tissue distributions, different half-lives, or different temporal distributions in selected compartments, tissues or organs.

Some delivery modes, such as delivery by nucleic acid vectors that remain within the cell or cell progeny, for example by autonomous replication or insertion into cellular nucleic acids, result in more persistent expression and presence of the components. Examples include viral delivery, eg, AAV or lentiviral.

By way of example, components of genome editing systems such as, for example, RNA-guided nucleases and gRNAs are delivered by different modalities with respect to the half-life or persistence of the resulting components delivered to the body, specific compartments, tissues or organs. can be In certain embodiments, gRNA can be delivered by such modalities. The RNA-guided nuclease molecule component can be delivered by modes that result in less persistence or less exposure to the body or specific compartments or tissues or organs.

More generally, in certain embodiments, a first delivery modality is used to deliver a first component and a second delivery modality is used to deliver a second component. A first delivery mode provides a first pharmacodynamic or pharmacokinetic profile. The first pharmacodynamic property can be, for example, the distribution, persistence or exposure of the component or nucleic acid encoding the component within the body, compartment, tissue or organ. A second delivery mode provides a second pharmacodynamic or pharmacokinetic profile. The second pharmacodynamic property can be, for example, the distribution, persistence or exposure of the component or nucleic acid encoding the component within the body, compartment, tissue or organ.

In certain embodiments, a first pharmacodynamic or pharmacokinetic property, eg, distribution, persistence, or exposure, is more restrictive than a second pharmacodynamic or pharmacokinetic property.

In certain embodiments, the first delivery mode is selected to optimize, eg, minimize, pharmacodynamic or pharmacokinetic properties such as distribution, persistence, or exposure.

In certain embodiments, the second delivery mode is selected to optimize, eg, maximize, pharmacodynamic or pharmacokinetic properties such as distribution, persistence, or exposure.

In certain embodiments, the first delivery mode involves the use of relatively persistent elements such as nucleic acids, eg, plasmids or viral vectors, eg, AAV or lentivirus. Because such vectors are relatively persistent, the products transcribed from them will be relatively persistent.

In certain embodiments, the second delivery modality includes relatively transient components such as, for example, RNA or protein.

In certain embodiments, the first component comprises gRNA and the delivery mode is relatively persistent, eg, the gRNA is transcribed from a plasmid or viral vector, eg, AAV or lentivirus. Transcription of these genes is physiologically irrelevant because they do not encode protein products and gRNAs cannot act alone. The second component, the RNA-guided nuclease molecule, is delivered in a transient fashion, e.g., as mRNA or as a protein, to ensure that only the complete RNA-guided nuclease molecule/gRNA complex is present and active for a short period of time. to

Additionally, the components may be delivered in different molecular forms or different delivery vectors that complement each other to enhance safety and tissue specificity.

The use of differential delivery modalities may enhance performance, safety and/or efficacy, eg, reduce the likelihood of eventual off-target modifications. Since peptides from bacterial-derived Cas enzymes are presented on the cell surface by MHC molecules, delivery of immunogenic components such as, for example, Cas9 molecules in a less persistent manner may reduce immunogenicity. A two-part delivery system may alleviate these drawbacks.

Differential delivery modalities can be used to deliver the components to different but overlapping target areas. Formation-active complexes are minimized outside the overlap of target regions. Thus, in certain embodiments, a first component, eg, gRNA, is delivered by a first delivery modality resulting in a first spatial distribution, eg, tissue distribution. For example, a second component, such as an RNA-guided nuclease molecule, is delivered by a second delivery modality resulting in a second spatial distribution, such as tissue distribution. In certain embodiments, the first modality comprises a first element selected from liposomes, nanoparticles such as polymeric nanoparticles, and nucleic acids such as viral vectors. A second modality includes a second element selected from the group. In certain embodiments, the first delivery modality includes a first targeting element, eg, a cell-specific receptor or antibody, and the second delivery modality does not. In certain embodiments, the second delivery modality includes a second targeting element such as, for example, a second cell-specific receptor or a second antibody.

When RNA-guided nuclease molecules are delivered in viral delivery vectors, liposomes or polymeric nanoparticles, delivery to multiple tissues and therapeutic activity therein, where it may be desirable to target only a single tissue There is a possibility of A two-part delivery system may solve this problem and increase tissue specificity. If the gRNA and RNA-guided nuclease molecules are packaged in separate delivery vehicles with distinct but overlapping tissue tropisms, the fully functional complex will be restricted to tissues targeted by both vectors. formed in

The above principles and embodiments are further illustrated by the following non-limiting examples:
Example 1: Cpf1 RNPs Containing gRNAs Targeting the CCAAT Box Support Gene Editing in Human Hematopoietic Stem/Progenitor Cells Designed to target the CCAAT box. To determine the optimal nuclear localization signal configuration for Acidaminococcus sp. Cpf1 (“AsCpf1”) delivery in CD34+ cells, HBG1-1 gRNA was combined with the two nuclear localization signals of AsCpf1 ( NLS), namely His-AsCpf1-nNLS (SEQ ID NO: 1000) and His-AsCpf1-sNLS-sNLS-sNLS (SEQ ID NO: 1001). "His" refers to the 6-histidine purified sequence, "AsCpf1" refers to the Acidaminococcus sp. Cpf1 protein sequence, "nNLS" refers to the nucleoplasmin NLS, "sNLS" refers to the SV40 NLS point to

Briefly, mPB CD34+ cells were prestimulated with human cytokines in X-Vivo-10 for 2 days and then electroporated with 5 μM or 20 μM RNPs. Genomic DNA was extracted 3 days after electroporation and next generation sequencing was performed on the HBG PCR products. HBG1-1 gRNA complexed to any of the tested Cpf1 NLS variants (“His-AsCpf1-sNLS-sNLS_HBG1-1RNP” or “His-AsCpf1-nNLS_HBG1-1RNP”) stimulated CD34+ cells at 13nt target sites supported editing. At the highest dose tested, the His-AsCpf1-sNLS-sNLS_HBG1-1 RNP produced 60.6% edited alleles and the His-AsCpf1-nNLS_HBG1-1 RNP produced 51.1% edited alleles. (Fig. 3A).

Example 2: Co-delivery of Cpf1 RNPs targeting the CCAAT box and ssODN donors supports gene editing in human hematopoietic stem/progenitor cells with single-stranded oligodeoxynucleotide donor repair templates (ssODNs) by electroporation , RNPs containing HBG1-1 gRNA complexed to His-AsCpf1-sNLS-sNLS variants (“His-AsCpf1-sNLS-sNLS_HBG1-1 RNPs”) were co-delivered to mPB CD34+ cells. OLI16430 and OLI16424 ssODNs were designed to "encode" a 4-nucleotide deletion, and OLI16409 and OLI16410 ssODNs were designed to "encode" an 18-nucleotide deletion (Table 7). Both the 4nt deletion and the 18nt deletion disrupt the HBG distal CCAAT box and are associated with the induction of HBG expression. The ssODN contains 90 nucleotide long homology arms that flank the encoded deletion sequence, creating a complete deletion. The ssODNs were modified to contain phosphorothioate (PhTx) at the 5' and 3' ends (OLI16430, OLI16424, OLI16409 and OLI16410, Table 7). Briefly, human adult mPB CD34+ cells were pre-stimulated for 2 days in media supplemented with human cytokines and His-AsCpf1-sNLS-sNLS protein complexed to HBG1-1 gRNA (“His-AsCpf1-sNLS -sNLS_HBG1-1 RNP") at 5 μM alone or in combination with 2.5 μM of one of the ssODN donors (OLI16430, OLI164324, OLI16409 or OLI16410). Co-delivery of RNPs with an ssODN donor encoding an 18-nt deletion with a positive strand homology arm (OLI16409) was determined by sequencing of HBG PCR products from genomic DNA extracted 72 hours after electroporation. As such, the editing frequency increased from 39.5% without donor to 73.3% (Fig. 4A).

Further analysis of the specific type and size of the deletion at the target site revealed that the 18 nt plus strand was deleted compared to 7.5% of alleles when the His-AsCpf1-sNLS-sNLS_HBG1-1 RNP was delivered alone. In the presence of the donor (OLI16409), we revealed that 57.3% of alleles carried the 18nt deletion (Fig. 4B). The 18nt deletion represented 71.2% of the total indels generated by co-delivery of ssODN OLI16409 with His-AsCpf1-sNLS-sNLS_HBG1-1 RNPs, whereas the 18nt deletion represented 71.2% of the total indels generated by co-delivery of ssODN OLI16409 and His-AsCpf1-sNLS-sNLS_HBG1 Co-delivery of ssODNs mediated precise repair of DNA DSBs, as delivery of −1 RNP alone represented only 19.0% of the total indels generated (FIG. 4C).

Although the data in FIGS. 4A-4C originally suggested that co-delivery of Cpf1 RNPs by the ssODN donor increased precise gene editing of genomic DNA resulting in an 18-nt deletion, the experiment of Example 2 was repeated subsequently. studies confirmed that these data were experimental artifacts. Nonetheless, data from subsequent iterations testing the AsCpf1 RNP using the guide RNA HBG1-1 and the ssODN OLI16409 show that co-delivery of the ssODN donor directs the '−18nt deletion' of DNA Although not associated with precise repair of DSBs, it was shown to support increased total editing in human mPB-CD34+ cells (see Example 10).

Example 3: Cpf1 RNP containing gRNAs targeting the distal CCAAT box region of the HBG promoter support gene editing in human hematopoietic stem/progenitor cells promoting induction of HbF protein expression in erythroid progeny SEQ ID NO: Guide RNA HBG1-1 (Table 8) with a targeting domain containing 1002 (Table 9) targets sites within the HBG promoter (Fig. 5A). HBG1-1 gRNA (SEQ ID NO: 1022) was complexed to wild-type AsCpf1 (AsCpf1-sNLS-sNLS, SEQ ID NO: 1001) to form RNP (“AsCpf1-HBG1-1-RNP”, Table 8). This complex (5 μM or 20 μM) was electroporated into CD34+ cells from mobilized peripheral blood (mPB) using either an Amaxa nucleofector (Lonza) or MaxCyte GT (MaxCyte, Inc.) electroporation apparatus. Three days after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. AsCpf1-HBG1-1-RNP supported 43% and 17% on-target editing on Amaxa and MaxCyte electroporation systems, respectively (FIGS. 6A (Amaxa) and 6B (MaxCyte)). Following editing of mPB CD34+ cells, in vitro differentiation to the erythroid lineage was performed for 18 days (Giarratana 2011). Relative expression levels of γ-globin chains (relative to total β-like globin chains) were then measured by UPLC (FIGS. 6A (Amaxa) and 6B (MaxCyte)). AsCpf1-HBG1-1-RNP resulted in an increase in γ-globin expression up to 21% above levels detected in cells derived from mock-electroporated mPB CD34+ cells (FIG. 6A).

Example 4: Cpf1 RNP editing efficiency in the CCAAT box region of the HBG promoter Co-delivery with S. pyogenes Cas9 RNP enhances but does not affect viability Cpf1-RNP and closely targeting S. pyogenes It was hypothesized that co-delivery of Cpf1-RNP with S. pyogenes Cas9 RNP could enhance editing levels at the target site by introducing a catalytic incompetence or a single nick. In an attempt to enhance Cpf1-RNP-mediated editing in the distal CCAAT box region of the HBG promoter, HBG1-1-AsCpf1H800A-RNP (HBG1-1 gRNA with 3′ modifications as shown in Table 8 (complexed to SEQ ID NO: 1041 (1)(a) SpA gRNA (Tables 8, 15; FIG. 5B) ("SpA-D10A-RNP", Table 8) or (b) Cas9 D10A protein ( His-NLS-SpCas9D10A, SEQ ID NO: 1034). (2) (a) tSpA dgRNA (Tables 8, 9; FIG. 5E) (“tSpA-Cas9-RNP”, Table 8); (b) Sp182 dgRNA (Tables 8, 9) FIG. 5F) (“Sp182-Cas9-RNP”, Table 8); or (c) a truncated killing guide RNA selected from tSpA-Cas9-RNP and Sp182-Cas9-RNP (Tables 8, 9, FIG. 5D). (95-mer with shortened protospacer region) containing WT Cas9 protein (SpCas9WT, SEQ ID NO: 1033). mPB CD34+ cells were co-delivered with S. Pyogenes WT Cas9 RNP. RNP complexes were electroporated using a MaxCyte GT instrument (MaxCyte, Inc). Target site insertion/deletion levels were then analyzed by Illumina sequencing (NGS) of PCR amplified target sites 3 days after electroporation. In all combinations tested, the S. Regardless of S. Pyogenes Cas9 enzyme (D10A or WT) and PAM orientation, total editing was increased over levels observed following Maxcyte delivery of HBG1-1-AsCpf1H800A-RNP alone (FIGS. 7 and 7). Table 10). Furthermore, S. There was no adverse effect on viability of mPB CD34+ cells when co-delivered with S. Pyogenes Cas9 RNP (Fig. 8).

Example 5: The Cpf1 Editing Profile is S. HBG1-1-AsCpf1H800A-RNP can be engineered by co-delivery of S. pyogenes Cas9-D10A-RNP In addition to the increase in total editing observed when co-delivered to mPB CD34+ cells with S. Pyogenes Cas9-D10A-RNP (Fig. 7), changes in the indel profile were also observed. Introduction of a single-strand break proximal to the Cpf1-RNP target site by the D10A enzyme (FIG. 9A) altered the orientation, length and/or position of the indel (FIG. 9B). For example, Sp182-D10A-RNP strongly shifted the profile towards the nicking site introduced by the D10A RNP, with deletions of varying length extending from the Cpf1 cleavage site towards the nicking site upstream (Sp182- D10A-RNP, Figure 9B). SpA-D10A-RNP also promoted the generation of deletions extending from the HBG1-1-AsCpf1H800A-RNP cleavage site to the nicking site (here located downstream) introduced by the D10A enzyme, whereas in this case, clearly An additional deletion originating from the nicking site but not extending all the way to the HBG1-1-AsCpf1H800A-RNP cleavage site was also generated (SpA-D10A-RNP, FIG. 9B).

S. The orientation of the S. pyogenes Cas9-D10A-RNP target site, the distance to the target strand and the Cpf1-RNP target site lead to differences in the position and length of mutations driven by additional DNA nicks. , is almost certain (FIG. 9A). Of particular application, this directional manipulation of indel profiles, namely Cpf1-RNP and S. An increase in the frequency of indels occurring with the S. Pyogenes Cas9 D10A-RNP binding site facilitates the desired editing outcome and increases the rate of productive indels (e.g., indels that interfere with the target site). Note that it can be used for In a setting that interferes with the dCCAAT target region and induces expression of HbF protein, HBG1-1-AsCpf1H800A-RNP and S. Co-delivery of S. pyogenes D10A-RNP increased gamma globin by 16.7% (SpG-D10A-RNP) or 19.0% (SpA-D10A-RNP) above levels detected in mock-electroporated cells resulted in increased levels (Figure 7 and Table 10). Lower editing levels of HbF levels when HBG1-1-AsCpf1H800A-RNP and SpA-D10A-RNP are paired (42.6% by SpA-D10A-RNP vs. 88.6% by SpG-D10A-RNP) , a higher elevation (19% with Cas9D10A-SpA-RNP vs. 16.7% with SpG-D10A-RNP) was achieved, indicating that the frequency of productive indels (vs SpG-D10A-RNP) was higher (Figure 7 and Table 10).

Example 6: S. cerevisiae Containing Killing Guide RNA Increasing Cpf1 Editing Without Altering the Editing Profile in the Presence of S. Pyogenes Cas9 WT RNP Increasing HBG1-1-AsCpf1H800A-RNP (fixed dose of 6 μM) using a MaxCyte electroporation device When co-delivered to mPB CD34+ cells with a dose of Sp182-Cas9-RNP (complexed to Sp182, a truncated 95-mer dead gRNA), an editing enhancement of up to more than 10-fold was observed (the highest dose of Sp182 -8.0% for HBG1-1-AsCpf1H800A-RNP delivered alone, versus 85.4% editing for Cas9-RNP (FIG. 7). The level of editing achieved here by co-delivery of Sp182-Cas9-RNP is comparable to that obtained using the MaxCyte electroporation device when HBG1-1-AsCpf1H800A-RNP was delivered alone at a dose as high as 20 uM. was also significantly higher (Fig. 6B). In contrast to the results observed with co-delivery of D10A-RNP (Example 5), HBG1-1-AsCpf1H800A-RNP and Sp182-Cas9-RNP (Cas9-WT complexed with truncated gRNA) The increased editing achieved by co-delivery was not associated with a clear effect on the indel profile. Indels detected in electroporated cells were clustered around the Cpf1 cleavage site in a contiguous symmetrical fashion, with no indels detected at the Sp182-Cas9-RNP target site (FIG. 10A). Remarkably, 96% of all indels interfere with the distal-CCAAT box and 79.5% of all indels interfere with 3 or more nucleotides of the CCAAT box motif (Fig. 10B). Further administration using HBG1-1-AsCpf1H800A-RNP (consisting of His-AsCpf1-sNLS-sNLS-H800A (SEQ ID NO:1032) conjugated to HBG1-1 gRNA (SEQ ID NO:1022)) and Sp182-Cas9-RNP Optimization allowed editing levels of mPB-CD34+ cells up to 92% 120 hours after electroporation using the MaxCyte device, resulting in γ chain expression levels 32% above background in erythroid-derived cells. (41% gamma chain in treated cells and 8% in mock treated cells) (FIG. 11). These results demonstrate that WT S. cerevisiae complexed to truncated gRNAs RNPs Consisting of S. Pyogenes Cas9 Proteins Introduce Detectable Levels of Editing to Their Own Binding Sites or Significantly Affect the Length or Orientation of Indels Generated by Cpf1-RNPs We demonstrate that editing from proximally bound Cpf1-RNPs can be increased without the Here, the editing enhancement provided by Sp182-Cas9-RNP allows hyperediting of HBG1-1-AsCpf1H800A-RNP in the HBG promoter, with a high frequency of indels interfering with the distal CCAAT box target motif, leading to electroporation. resulting in therapeutically relevant γ-chain expression levels in the bulk erythroid progeny of the transfected cells.

Example 7: Clonal HbF Distribution in Cell Populations Edited with Cpf1 gRNA Targeting the Distal CCAAT Box mPB CD34+ cells from mPB CD34+ cells electroporated with a combination of His-AsCpf1-sNLS-sNLS-H800A (SEQ ID NO: 1032) complexed to HBG1-1 gRNA with modifications (SEQ ID NO: 1041) + Sp182-Cas9-RNP was assessed for the distribution of γ-chain expression levels in erythroid cells (Fig. 5F, Table 8). After a recovery period of 48 hours after electroporation, cells were sorted by fluorescence-activated cell sorting (FACS) at 1 cell/well in non-tissue culture treated 384-well plates. Cells were then differentiated, clonally expanded for 18 days and differentiated into the erythroid lineage (adapted from Giarratana 2011). UPLC analysis was performed to determine the distribution of γ-chain expression levels (percentage of γ-chains/[total β-like chains]) in clonal erythroid progeny of cells derived from the total population of mPB-CD34+ cells originally edited. bottom. It is believed that approximately 30% of red blood cells should have fetal hemoglobin levels greater than 30% to achieve functional benefit and alleviate symptoms associated with sickle cell disease. Of the clones analyzed, 83.1% had γ chain levels above the median level detected in erythroid clones derived from mock-electroporated mPB CD34+ cells (median = 18.8%); 64.2% had γ-chain levels greater than 30% of total β-like, and 35.8% had γ-chain levels greater than 48.8% of total β-like (30%+ in control cells). median level of 18.8%) (Fig. 12).

Example 8 Screening for Cpf1 gRNAs Targeting the HBG Promoter Region As single RNP components or in combination with "booster elements", increased editing of the HBG promoter region in CD34+ cells and fetal growth in erythroid progeny of modified cells To identify other AsCpf1 gRNAs that could be used to induce type globin expression, His-AsCpf1-NLS-NLS (“AsCpf1”, SEQ ID NO: 1000) targeting several domains of the HBG promoter; S542R/K607R (“AsCpf1 RR”, SEQ ID NO: 1036); or AsCpf1 S542R/K548V/N552R (“AsCpf1 RVR”, SEQ ID NO: 1037) gRNA sequences (Table 11) were designed (listed in Table 12). AsCpf1 RR and AsCpf1 RVR are engineered AsCpf1 variants that recognize TYCV/ACCC/CCCC and TATV/RATR PAMs, respectively (Gao 2017).

Complexed with RNP (5 μM) containing AsCpf1 protein (SEQ ID NO: 1000), AsCpf1 RR protein (SEQ ID NO: 1036) or single gRNA containing gRNA targeting domains of Table 12 using Amaxa electroporation apparatus (Lonza) AsCpf1 RVR (SEQ ID NO: 1037) (see gRNA ID name for specific Cpf1 molecule used) was delivered to mobilized peripheral blood (mPB) CD34+ cells. After 72 hours, genomic DNA was extracted from the cells and then analyzed for insertion/deletion levels by Illumina sequencing (NGS) of PCR amplified target sites. The percentage of editing (indels = deletions and insertions) for each gRNA is shown in Table 12 above. In certain embodiments, Cpf1 RNPs comprising one or more of the gRNAs listed in Table 12 can be used to target the regions listed in Table 11 to induce HbF expression and simultaneously can be delivered and achieve higher levels of editing compared to those of Cpf1 RNP alone.

Example 9: Co-delivery of HBG1-1-Cpf1 RNPs targeting the CCAAT box and ssODNs supports increased gene editing in human hematopoietic stem/progenitor cells

100 nt of ssODN (HBG delta-104:-121) (ie ssODN OLI16431 (SEQ ID NO: 1040), Table 7) was co-delivered with Cpf1 RNP to further investigate the editing results.

Briefly, human adult mPB CD34+ cells were pre-stimulated for 2 days in media supplemented with human cytokines and treated with modified HBG1-1 gRNA (SEQ ID NO: 1041, Table 8) (“His-AsCpf1-sNLS-sNLS H800A_HBG1- His-AsCpf1-sNLS-sNLS H800A protein (SEQ ID NO: 1032, Table 8) conjugated to 6 μM RNP alone or in combination with OLI16431 (0.5-6 μM) was electroporated. Co-delivery of RNPs with an ssODN donor encoding an 18-nt deletion with a positive strand homology arm (OLI16431) was determined by sequencing of HBG PCR products from genomic DNA extracted 72 hours after electroporation. As such, the editing frequency increased from 8.0% without donor to 75.6% (Fig. 13A). This level of editing allowed an increase in HbF levels approximately 24% above background (Fig. 13A). Furthermore, it should be noted that no reduction in cell viability (FIG. 13B) or colony forming ability (FIG. 13C) as measured by DAPI exclusion was observed at any dose tested.

Example 10 Optimization of HBG1-1-Cpf1 RNP and OLI16431 Dose to Maximize Editing in RNPs Targeting Distal CCAAT Box Sites Demonstrate increased editing when ssODNs and RNPs are co-delivered (See, eg, Example 9), the same method was used to optimize the dosage of each component in order to maximize the overall compilation. Briefly, His-AsCpf1 complexed to unmodified HBG1-1 gRNA (SEQ ID NO: 1022, Table 8) co-delivered at 0-12 μM with 0-12 μM OLI16431 (SEQ ID NO: 1040, Table 7). - A dosing matrix was prepared with an RNP ("His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP") consisting of the sNLS-sNLS H800A protein (SEQ ID NO: 1032, Table 8). Using a dose of 8 μM His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP co-delivered with 8 μM OLI16431, HBG PCR products from genomic DNA extracted from cells were isolated following 14 days of erythrocyte culture. A maximum of 89.4% indels were achieved as determined by sequence analysis (Fig. 14A). This was consistent with an increase in HbF levels >32% above background under these dosing conditions (Fig. 14A). The viability of this sample (His-AsCpf1-sNLS-sNLS H800A_HBG1-1 RNP [8 μM]+OLI16431 [8 μM]) was comparable to untreated control samples both 48 hours after electroporation and 14 days after erythrocyte culture. (Fig. 14B).

However, when >8 μM OLI16431 was electroporated into cells alone without RNP, artifacts were observed. When PCR amplification was performed 48 hours after electroporation under these conditions, there were false positive results likely due to excess ssODNs in the system (see Figure 14A, 8 μM and 12 μM at 48 hours). of OLI16431 alone (black bars)). At lower doses of ssODN and at later time points, this false positive was no longer evident (Fig. 14A, OLI16431 alone (black bars) at 4 and 6 μM at 48 hours and 4, 6, and 8 μM at day 14). and 12 μM OLI16431 alone (light gray bars)). Furthermore, the decrease in cell viability observed in the ssODN alone group (FIG. 14B, decrease to about 57% with 12 μM OLI16431 alone at 48 hours (black bars)) may be a compounding factor. The absence of false positive results at 4 μM and 6 μM ssODN doses suggests that this artifact may be due to excess ssODN. These factors were consistent with the baseline HbF levels observed in the 8 μM and 12 μM OLI16431 alone groups (FIG. 14A, gray dots compared to the negative control), along with a substantial and sustained enhancement of RNP editing by the addition of ssODNs. provide confidence that the is genuine and not an artifact.

Example 11: RNPs containing various Cpfl and gRNAs targeting the HBG promoter region support gene editing in human hematopoietic stem/progenitor cells, promoting induction of HbF protein expression in erythroid progeny.
Guide RNAs comprising SEQ ID NOs: 1022, 1023, 1041-1093, 1098-1106 (Table 13) were conjugated to various Cpf1 mutant enzymes (SEQ ID NOs: 1032, 1094-1097, 1107-1109, Table 14). , formed various RNP complexes (Table 15). RNPs contained gRNAs with modifications to the 5' end and/or modifications to the 3' end (Table 13). Guide RNAs containing SEQ ID NOs: 1022, 1023, 1041-1084, 1098-1106 (Table 13) have the same predicted cleavage site in the distal CCAAT box target region, and the relevant targeting domains are 20-mer, 21-mer SEQ ID NO: 1002 (HBG1-1), SEQ ID NO: 1254 (HBG1-1-21-mer), SEQ ID NO: 1256 for gRNAs containing 22-mer or 23-mer protospacer sequences, respectively (Tables 15 and 16) (HBG1-1-22mer), containing the sequence set forth in SEQ ID NO: 1258 (HBG1-1-23mer). optionally SEQ ID NO: 1260 (AsCpf1 HBG1 promoter-1 (21-mer)), SEQ ID NO: 1262 (AsCpf1 HBG1 promoter-2 (21-mer)) or SEQ ID NO: 1264 (AsCpf1 HBG1 promoter-6 (21-mer) gRNAs targeting other locations within the HBG promoter were also tested, including the guide RNAs of SEQ ID NOS: 1085-1096, which contain targeting domains containing the sequences described (Table 16, Table 17). provides a list of the SEQ ID NOs of the gRNAs and Cpf1 variants forming each RNP and each RNP complex tested in Examples 11-13.Additional information on each gRNA and Cpf1 variant can be found at , can be found in Tables 13 and 14, respectively.

The gRNAs used in Examples 11 and 12 were chemically synthesized. Chemicals for oligonucleotide synthesis were purchased from BioAutomation, Glen Research, Millipore Sigma, Sigma-Aldrich, ChemGenes and Thermo Fisher Scientific. The solid support used for synthesis was either Unilinker 2000 Å CPG resin, 2′-TBDMS rU 2000 Å CPG resin or 2′-O-methyladenosine (N-Bz) Icaa 2000 Å CPG resin from ChemGenes. rice field. RNA (TBDMS protected) and DNA phosphoramidites were obtained from Thermo Fisher Scientific. In certain embodiments, the phosphorothioate was introduced during the sulfurization step with a solution of DDTT (3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazol-3-thione) from Glen Research. . Oligonucleotides were synthesized using standard RNA and DNA phosphoramidite chemistry on either a BioAutomation MerMade 12 synthesizer or a GE Aekta OligoPilot 100 synthesizer. Following synthesis, oligonucleotides were cleaved from the solid support and deprotected in a two step process using ammonium hydroxide/methylamine and TEA-3HF. After desalting, oligonucleotides were purified using reverse-phase chromatography on preparative HPLC.

First, the effect of editing was tested using RNPs containing gRNAs modified at the 5' end of the gRNA. Briefly, RNP complexes (6.0 μM and 12 μM) were applied to 1×10 ⁶ mPB CD34+ cells following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. Delivery was via MaxCyte electroporation. Seventy-two hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. The results were 5 nt RNA (RNP37), 10 nt RNA (RNP38) against the 5′ end of the RNP (RNP33, also referred to as “HBG1-1-AsCpf1 RNP”, Table 8) containing gRNA without modification or gRNA. , 25 nt of RNA (RNP39), 60 nt of RNA (RNP40), 5 nt of DNA (RNP41), 10 nt of DNA (RNP42), 25 nt of DNA (RNP43) and 60 nt of DNA (RNP44). We demonstrate that certain RNPs (Table 15) support on-target editing (Figure 15).

Then Cpf1 RNP and Sp182 killing RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed with S. pyogenes Cas9 (SEQ ID NO: 1033) or ssODN OLI16431 (SEQ ID NO: 1040, Table 7) was tested for the effect of co-delivery. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, various concentrations (6 μM, 8 μM, 8 μM and 12 μM) of RNP33 (5′ or 3′ gRNA No modification, Table 15) The administered matrix with conjugates was injected with Sp182 RNP (8 μM, 8 μM, 6 μM and 4 μM) or ssODN OLI16431 (8 μM, 8 μM, 6 μM and 4 μM) into 5.25×10 ⁶ mPB CD34+ cells with MaxCyte Co-delivered via electroporation. Following electroporation, cells were returned to culture for an additional 48 hours. A portion of the CD34 cells were then split for gDNA extraction and indel quantification in the bulk cell population. In addition, to examine editing in phenotypic progenitor cells and phenotypic hematopoietic stem cells (HSCs), HSPC subpopulations were characterized (among the remaining CD34 cells) by immunophenotyping (Notta 2011) and fluorescence-activated cells Separated by sorting (FACS). Immunophenotyping 48 hours after electroporation was performed by staining cells with antibodies against hCD34, hCD38, hCD45RA, hCD90 and hCD123. Phenotypic HSCs are defined as hCD34bright hCD38 hCD90+ hCD45RA− and progenitor cells are defined as hCD34bright CD38+. These two populations were FACS sorted and DNA extracted to determine the level of editing of these subpopulations. Target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified targets. Results demonstrate that RNP33 co-delivered with the 'booster element' Sp182 RNP or ssODN OLI16431 supports on-target editing at levels higher than those observed when RNP33 is delivered alone (Fig. 15). (FIGS. 16A-16B).

then RNP33 (no 5′ or 3′ gRNA modifications, Table 15), RNP43 (+25 DNA 5′ gRNA modifications, Table 15) or RNP34 (1×PS2-OMe+1×OMe 3′ gRNA modifications, Table 15); The effect of co-delivery with Sp182 dgRNA (Table 9) or ssODN OLI16431 (Table 7) was tested. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNP33, RNP34 or RNP43 complexes (8 μM) were added to Sp182 RNP (8 μM) or ssODN OLI16431 (8 μM). ) to 5.25×10 ⁶ mPB CD34+ cells via MaxCyte electroporation. Following electroporation, cells were returned to culture for an additional 48 hours and sorted into phenotypic progenitor and phenotypic HSC fractions for indel quantification. Forty-eight hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. The results demonstrate that RNPs containing gRNAs with 5′ or 3′ modifications co-delivered with the “booster element” Sp182 RNP or ssODN OLI16431 support on-target editing (FIG. 17). Both booster elements tested provided comparable levels of editing to RNP34 and RNP33. RNP33 editing was enhanced by both booster elements, while RNP43 editing was enhanced only by the addition of Sp182 RNP, when compared to the level of editing obtained without booster elements (Figs. 15, 17).

Next, the effect of different Cpf1 proteins on RNP editing was tested. A stoichiometric comparison (gRNA:Cpf1) with RNPs containing various Cpf1 proteins was performed. Briefly, RNPs (RNP64, RNP63, RNP45, Table 15) were delivered at stoichiometric ratios (gRNA:Cpf1 complexation ratios) of either 2 or 4 with a molar excess of gRNA. All RNPs were delivered to 1×10 ⁶ CD34 + cells at 8 μM via MaxCyte electroporation following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. Following electroporation, cells were returned to culture for an additional 72 hours prior to indel quantification. The results demonstrated that at the concentrations tested, there was no difference in editability when the stoichiometry was changed (Figure 18). RNP45 (consisting of Cpf1 protein SEQ ID NO: 1094) outperforms RNP63 (consisting of Cpf1 protein SEQ ID NO: 1095) and RNP64 (consisting of Cpf1 SEQ ID NO: 1109).

Next, the effect of co-delivery of Sp182 RNP or ssODN OLI16431 with various RNPs containing different Cpf1 proteins was tested. Briefly, RNPs (RNP33, RNP64, RNP63, RNP45, Table 15) were delivered alone or in combination with Sp182 RNP or ssODN OLI16431. All reagents were delivered to 1×10 ⁶ CD34+ cells at 8 μM via MaxCyte electroporation following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. Following electroporation, cells were returned to culture for an additional 72 hours prior to indel quantification. The results show that the 'booster element' Sp182 RNP or ssODN OLI16431 enhanced editing for all RNPs tested (FIG. 19).

The effect of editing using RNPs containing gRNAs with various 5' DNA extensions or RNPs without 5' DNA extensions (RNP45) was tested with or without co-delivery of ssODN OLI16431. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNPs (RNP45 (no 5′ modifications), RNP46 , RNP47, RNP48, RNP49, RNP50, RNP51, RNP52, RNP53, RNP54, RNP55, RNP56 and RNP57, Table 15) delivered alone or co-delivered with 8 μM ssODN OLI16431 to 1×10 ⁶ CD34+ cells. bottom. Following electroporation, cells were returned to culture prior to indel quantification. Results demonstrate that all RNPs support on-target editing (Fig. 20).

The effect of editing using RNPs containing gRNAs with identical 5′ DNA extensions but different 3′ gRNA modifications was tested to assess the impact of 3′ gRNA modifications. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, the 5′-end matched but 3′-end matched at a concentration of 8 μM via MaxCyte electroporation. RNPs containing gRNAs with different gRNA modifications (RNP49 vs. RNP58 and RNP59 vs. RNP60) were delivered to 1×10 ⁶ CD34+ cells. Following electroporation, cells were returned to culture prior to indel quantification. In both comparisons, RNP containing gRNA with 3'PS-OMe outperformed the unmodified 3' version 24 hours after electroporation (Figure 21).

Next, different concentrations of Cpf1 and gRNA were tested for RNP58. Briefly, 48 hours of pre-stimulation at either a 2:1, 1:1 or 0.5:1 molar ratio stoichiometric ratio (gRNA:Cpf1 complex formation ratio) was followed by RNP58 (+25 DNA 5′gRNA modified and 1×PS-OMe 3′gRNA modified, Table 15) was delivered to 1×10 ⁶ CD34+ cells via MaxCyte electroporation. Following electroporation, cells were returned to culture prior to indel quantification. At all doses tested, editing was optimal when RNPs were complexed at a 2:1 ratio (FIGS. 22A-22B).

The effect of editing using RNPs containing gRNAs with identical 5′ DNA extensions but different 3′ gRNA modifications was further tested to assess the impact of 3′ gRNA modifications. Briefly, following 48 hours of pre-stimulation, RNPs containing gRNAs with identical 5′ ends but different 3′ gRNA modifications (RNP58, RNP2, RNP3, RNP4, RNP5, RNP6, RNP7, RNP8, RNP9 , RNP10) were delivered at various concentrations (1 μM, 2 μM, 4 μM) to 1×10 ⁶ CD34+ cells via MaxCyte electroporation. Following electroporation, cells were returned to culture prior to indel quantification. The results show that all RNPs support on-target editing (Figures 23A-23B).

Editing by RNPs containing gRNAs with 3′ gRNA modifications or with 5′ and 3′ modifications targeting different regions of the HBG promoter was next tested. These include targeting domains SEQ ID NOs: 1260 (AsCpf1 HBG1 promoter-1 (21-mer)), 1262 (AsCpf1 HBG1 promoter-2 (21-mer)), 1264 (AsCpf1 HBG1 promoter-6 (21-mer)) (Table 16, Table 17). Altering to the distal CCAAT box target region, these gRNAs are configured to provide editing events within the region selected from Table 11. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNPs containing unmodified 5′ gRNA and gRNA containing 1×PS-OMe 3′ gRNA modifications ( RNP11, RNP16, RNP19 and RNP22, Table 15), RNPs containing gRNAs containing +20 DNA + 2xPS 5'gRNA modifications and 1xPS-OMe 3'gRNA modifications (RNP12, RNP21 and RNP24, Table 15), +25 DNA 5'gRNA modifications and RNPs containing gRNAs containing 1xPS-OMe 3' gRNA modifications (RNP58 and RNP20, Table 15) were delivered at a concentration of 8 μM to 1x10 ⁶ CD34+ cells via MaxCyte electroporation. Editing of mPB CD34+ cells was followed by in vitro differentiation to the erythroid lineage for 18 days (Giarratana 2011) and gDNA was isolated on day 14 of culture for indel analysis. Briefly, RNPs were delivered to CD34+ cells as described above. Following 48 hours of recovery in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, treated cells were counted and transferred to erythroid differentiation medium for cell counting and feeding at 4, 7, 10 and 14. Red blood cells were collected on day 18, performed on day 1. These CD34+ derived red blood cells were counted, lysed in HPLC grade water, and filtered to remove cell debris. The relative expression levels of γ-globin chains (relative to total β-like globin chains) were then determined by UPLC for each sample, and the ratio of acetonitrile containing 0.1% trifluoroacetic acid to water containing 0.1% trifluoroacetic acid was calculated. A gradual increase achieved protein separation (FIGS. 24A-24C). Figures 24A-24C showed that all RNPs supported on-target editing. However, only editing at specific target sites results in increased HBF expression. Identical experiments were performed with varying concentrations of RNP58 (0, 1 μM, 2 μM, 4 μM) (FIG. 25).

Next, the effect of different Cpf1 proteins on RNP editing was tested. Briefly, SEQ ID NO: 1051 (+25 DNA 5'gRNA modifications and 1xPS RNP58, RNP26, RNP27 and RNP28 (Table 15) containing gRNAs containing -OMe 3'gRNA modifications, Table 13) were delivered at 8 μM to 1×10 ⁶ CD34+ cells via MaxCyte electroporation. Results demonstrated that all RNPs support on-target editing (Fig. 26).

Next, we tested the effect of different RNPs containing gRNAs with different 5′ gRNA modifications and gRNAs with the same 3′ modification. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNPs (RNP58 (+25 DNA 5′ gRNA modification and 1×PS-OMe 3′ gRNA modification), RNP29 (+25 DNA + 2xPS 5'gRNA modifications and 1xPS-OMe 3'gRNA modifications), RNP30 (polyA RNA + 2xPS 5'gRNA modifications and 1xPS-OMe 3'gRNA modifications) and RNP31 (PolyU RNA + 2xPS 5'gRNA modifications and 1xPS-OMe 3'gRNA modifications). gRNA modifications) (Table 15)) were delivered at 1 μM, 2 μM or 4 μM to 1×10 ⁶ CD34+ cells via MaxCyte electroporation (RNP30 was not tested at 1 μM due to cell availability). . Results demonstrated that all RNPs support on-target editing (Fig. 27).

Next, the effect of different Cpf1 proteins on RNP editing was tested. Briefly, SEQ ID NO: 1051 (+25 DNA 5'gRNA modifications and 1xPS - RNP58, RNP27 and RNP26 (Table 15) containing gRNAs containing OMe 3'gRNA modifications, Table 13) were delivered at 2 μM or 4 μM to 1×10 ⁶ CD34+ cells via MaxCyte electroporation. To examine editing in bulk CD34, phenotypic progenitor cells and phenotypic hematopoietic stem cells (HSCs), HSPC subpopulations were characterized. Thus, following electroporation, cells were returned to culture for an additional 48 hours and sorted into progenitor and HSC fractions for indel quantification. Forty-eight hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. Results demonstrated that all RNPs supported on-target editing in bulk CD34, phenotypic progenitor cells and phenotypic hematopoietic stem cells (Figure 28).

Then RNP and Sp182 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)) against RNPs containing different Cpf1 proteins or The effect of co-delivery with ssODN OLI16431 (SEQ ID NO: 1040, Table 7) was tested. Briefly, following 48 hours of prestimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNP61, RNP62, RNP34 (Table 15) were added at 8 μM to Sp182 RNP (8 μM) or ssODN OLI16431 ( 8 μM) to 25×10 ⁶ mPB CD34+ cells via MaxCyte electroporation. To examine editing in bulk CD34, phenotypic progenitor cells and phenotypic hematopoietic stem cells (HSCs), HSPC subpopulations were characterized. Thus, following electroporation, cells were returned to culture for an additional 48 hours and sorted into progenitor and HSC fractions for indel quantification. Forty-eight hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. The results demonstrate that RNPs co-delivered with the 'booster element' Sp182 RNP or ssODN OLI16431 support on-target editing (FIG. 29). Some of these cells were also cryopreserved 24 hours after electroporation for further characterization in an in vivo engraftment model (see Example 12).

Next, the effects of RNP58 and RNP32 editing were tested. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNP58 and RNP32 (Table 15) were added to 6×10 ⁶ mPB CD34+ cells at 2 μM MaxCyte Delivered via electroporation. To examine editing in bulk CD34, phenotypic progenitors and phenotypic HSCs, HSPC subpopulations were characterized. Thus, following electroporation, cells were returned to culture for an additional 48 hours and sorted into progenitor and HSC fractions for indel quantification. Forty-eight hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. The results demonstrate that RNP58 and RNP32 support on-target editing (Fig. 30).

Following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, RNP58 and RNP1 (Table 15) were applied to 25×10 ⁶ mPB CD34+ cells at concentrations of 2 μM to 8 μM by MaxCyte stimulation. Delivered via perforation. To examine editing in bulk CD34, phenotypic progenitor cells and phenotypic hematopoietic stem cells (HSCs), HSPC subpopulations were characterized. Thus, following electroporation, cells were returned to culture for an additional 48 hours and sorted into progenitor and HSC fractions for indel quantification. Forty-eight hours after electroporation, target site insertion/deletion levels were analyzed by Illumina sequencing (NGS) of PCR amplified target sites. The results demonstrate that the RNPs tested support on-target editing (Fig. 31). Some of these cells were also cryopreserved 24 hours after electroporation to further characterize the in vivo engraftment model (see Example 12).

Example 12: Edited mPB CD34+ cells NOD, B6. Infusion into SCID Il2rγ-/-Kit (W41/W41) mice results in long-term engraftment and HbF induction.
Mobilized peripheral blood (mPB ) by non-irradiated NOD, B6. SCID Il2rγ−/− Kit (W41/W41) (Jackson lab strain name: NOD.Cg-Kit<W-41J>Tyr<+>Prkdc<scid>Il2rg<tm1Wjl>/ThomJ) (“NBSGW”) infused into mice bottom. Briefly, following 48 hours of pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, 62.5×10 ⁶ /mL of RNP at a dose of 8 μM and ssODN OLI16431 at 6 μM. mPB CD34+ cells were electroporated via MaxCyte electroporation. After 24 hours, mCD34+ cells were cryopreserved. After 5 days, mPB CD34+ cells were thawed and infused into NBSGW mice at 1 million cells per mouse via tail vein injection. After 8 weeks, mice were euthanized and bone marrow (BM) was harvested from femur, tibia and pelvic bones. Bone marrow subpopulations of cells identified as CD15+, CD19+, glycophorin A (GlyA, CD235a+) and lineage negative CD34+, respectively, were isolated by FACS and DNA extracted to determine the level of editing in each of these fractions. . FIG. 32 shows the frequency of indels in unfractionated BM or individual flow sorted BM subpopulations as determined by next generation sequencing.

Next, RNP34 or RNP33 (Table 15) co-delivered with Sp182 RNP (killing gRNA comprising SEQ ID NO: 1027 (Table 8) complexed to S. pyogenes Cas9 (SEQ ID NO: 1033)). To determine whether delivery achieves editing in long-term repopulating hematopoietic stem cells, human adult CD34+ cells from mobilized peripheral blood (mPB) were infused into non-irradiated NBSGW mice. Briefly, following 48 hours of prestimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3, various doses of RNP and various doses of Sp182 RNP were administered to 62.5×10 ⁶ /mL. mPB CD34+ cells were electroporated via MaxCyte electroporation. After 24 hours, mCD34+ cells were cryopreserved. After 5 days, mPB CD34+ cells were thawed and infused into NBSGW mice at 1 million cells per mouse via tail vein injection. After 8 weeks, mice were euthanized and bone marrow (BM) was harvested from femur, tibia and pelvic bones. Bone marrow subpopulations of cells identified as CD15+, CD19+, glycophorin A (GlyA, CD235a+) and lineage negative CD34+, respectively, were isolated by FACS and DNA extracted to determine the level of editing in each of these fractions. . FIG. 33A shows the frequency of indels in unfractionated BM or individual flow sorted BM subpopulations as determined by next generation sequencing.

Finally, long-term HbF induction by BM-derived CD34+ cells was analyzed. Aliquots of BM cells were cultured under erythroid differentiation conditions for 18 days (Giarratana 2011) and assessed for HbF expression by UPLC. Briefly, unfractionated BM cells extracted from mice 8 weeks post-infusion were placed in red blood cell culture conditions for 18 days. Cell counts and feeding were performed on days 7, 10 and 14, and red blood cells were collected on day 18. These bone marrow-derived red blood cells were counted, lysed in HPLC grade water, and filtered to remove cell debris. The relative expression levels of γ-globin chains (relative to total β-like globin chains) were then determined by UPLC for each sample, and the ratio of acetonitrile containing 0.1% trifluoroacetic acid to water containing 0.1% trifluoroacetic acid was calculated. A gradual increase achieved protein separation (Fig. 33B). These data demonstrate that robust long-term HbF induction is achieved by RNP34 and RNP33 co-delivered with Sp182 RNP editing of human CD34+ cells.

Next, to determine whether delivery of RNP61 or RNP62 (Table 15) co-delivered with ssODN OLI16431 (SEQ ID NO: 1040, Table 7) would achieve editing in long-term repopulating hematopoietic stem cells, of human adult CD34+ cells were infused into non-irradiated NBSGW mice. Briefly, 8 μM RNP and 8 μM ssODN OLI16431 were applied to 62.5×10 ⁶ /mL mPB CD34+ cells following 48 h pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. Cells were electroporated via MaxCyte electroporation. After 24 hours, mCD34+ cells were cryopreserved. After 4 days, mPB CD34+ cells were thawed and infused into NBSGW mice at 1 million cells per mouse via tail vein injection. After 8 weeks, mice were euthanized and bone marrow (BM) was harvested from femur, tibia and pelvic bones. Bone marrow subpopulations of cells identified as CD15+, CD19+, glycophorin A (GlyA, CD235a+) and lineage negative CD34+, respectively, were isolated by FACS and DNA extracted to determine the level of editing in each of these fractions. . FIG. 34A shows the frequency of indels in unfractionated BM or individual flow sorted BM subpopulations as determined by next generation sequencing.

Finally, long-term HbF induction by BM-derived CD235a+ (GlyA+) erythroid cells was analyzed. Aliquots of BM cells were cultured under erythroid differentiation conditions for 18 days and assessed for HbF expression by UPLC. Briefly, unfractionated BM cells extracted from mice 8 weeks post-infusion were placed in red blood cell culture conditions for 18 days. FIG. 34B shows HbF expression calculated as γ/β-like chains (%) by red blood cells. These data demonstrate that robust and long-term HbF induction is achieved by RNP61 and RNP62 co-delivered with ssODN OLI16431 editing of human CD34+ cells.

Human adult CD34+ cells from mPB were then infused into non-irradiated NBSGW mice to determine whether delivery of RNP1 or RNP58 (Table 15) would achieve editing in long-term repopulating hematopoietic stem cells. Briefly, 48 h of prestimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3 was followed by 4 μM or 8 μM RNP1 or 2 μM, 4 μM or 8 μM RNP58 at 62.5×10 ⁶ /. mL of mPB CD34+ cells were electroporated via MaxCyte electroporation. After 24 hours, mCD34+ cells were cryopreserved. After 4 days, mPB CD34+ cells were thawed and infused into NBSGW mice at 1 million cells per mouse via tail vein injection. After 8 weeks, mice were euthanized and bone marrow (BM) was harvested from femur, tibia and pelvic bones. Human chimerism in BM and lineage rearrangement (CD45+, CD14+, CD19+, glycophorin A (GlyA, CD235a+), lineage and CD34+ and mouse CD45+ marker expression) in BM were determined and analyzed by flow cytometry (Figure 35). The frequency of GlyA+ cells was calculated as GlyA+ cells/total cells in BM. Human chimerism was defined as human CD45/(human CD45+mCD45).

Similar chimerism and lineage distribution was achieved by RNP-transfected mPB CD34 cells 8 weeks after transplantation compared to mock-transfected mPB CD34 cells, indicating that editing reduced the engraftment potential of hematopoietic stem cells. Demonstrate that it is equivalent to maintaining. Figure 36 shows indels determined by next generation sequencing of unfractionated BM 8 weeks after infusion. FIG. 37 shows the frequency of indel rates of BM, CD15+, CD19+, glycophorin A (GlyA, CD235a+) and lin-CD34+ cells with 2 μM, 4 μM or 8 μM RNP58.

Long-term HbF induction by CD235a+ (GlyA+) erythroid cells derived from edited CD34+ cells was also analyzed. GlyA+ cells obtained from bone marrow were isolated and collected by fluorescence activated cell sorting (FACS) and lysed in HPLC grade water. Lysates were then assessed for HbF expression by UPLC. Figure 38 shows HbF expression calculated as γ/β-like chains (%) by GlyA+ cells. These data demonstrate that RNP58 editing of human CD34+ cells with varying concentrations of RNP58 achieves robust and long-term HbF induction.

Importantly, CD34 ⁺ cells electroporated with different concentrations of RNP1 or RNP58 maintained their ex vivo hematopoietic activity as determined by the hematopoietic colony-forming cell (CFC) assay (i.e., untreated donors). There was no difference in the amount or diversity of erythroid and myeloid colonies compared to matched CD34 ⁺ cell negative controls) (FIG. 39).

Example 13: NHEJ-mediated deletion of 4 or more nucleotides in the distal CCAAT box region of the HBG promoter is long-lasting and promotes elevated HbF expression.
Delivery of RNPs containing Cas9 or Cpf1 enzymes targeting the HBG promoter region (Fig. 41A) results in the generation of multiple insertions and deletions (indels). These include indels derived from microhomology-mediated end joining (MMEJ) and non-homologous end joining (NHEJ) repair mechanisms. As shown below, the MMEJ repair machinery is underutilized during editing of long-term stem cell populations (HSCs) and this type of editing is lost over time.

Briefly, 62.5×10 ⁶ /mL mPB CD34+ cells were stimulated by MaxCyte electrolysis with Sp35 RNP after 48 h pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. Electroporation was performed through the perforation. The Sp35 RNP is the S. Sp35 gRNA, complexed with S. pyogenes wild-type (Wt) Cas9 protein, SEQ ID NO: 339 (i.e., containing the targeting domain of CUUGUCAAGGCUAUUGGUCA (RNA)); SEQ ID NO: 917 (i.e., CTTGTCAAGGCTATTGGTCA (DNA) ))including. Twenty-four hours later, an aliquot of mPB CD34+ cells was taken for pre-injection (CD34) indel analysis and another aliquot was placed into erythroid differentiation for indel analysis in erythroid progenitor cell progeny. The remaining cells were cryopreserved and stored in liquid nitrogen until engraftment studies were initiated. mPB CD34+ cells were thawed at the time of infusion, non-irradiated NOD, B6. SCIDIl2rγ−/−Kit (W41/W41) (Jacksonlab strain name: NOD.Cg-Kit<W-41J>Tyr<+>Prkdc<scid>Il2rg<tm1Wjl>/ThomJ) (“NBSGW”) mice were injected with tail vein One million cells per mouse were injected via intraperitoneal injection. After 16 weeks, mice were euthanized, bone marrow (BM) was harvested from femurs, tibiae and pelvic bones, lineage-negative CD34+ was isolated by FACS, cells harvested prior to injection, their erythroid progeny (ex vivo) were isolated. ), and cells harvested at week 16 in vivo to determine indels (insertions/deletions) using the following primers: forward = CATGGCGTCTGGACTAGGAG (SEQ ID NO: 1266) and reverse = AAACACATTTCACAATCCCTGAAC (SEQ ID NO: 1267). Read sequences surrounding all detected deletions were analyzed to determine if they were likely the result of DSB repair by the MMEJ or NHEJ pathways. MMEJ is distinguished from NHEJ by its use of microhomology sequences. MMEJ repair depends on strand excision and annealing of proximally located repeated stretches of nucleotides (microhomology) around the DSB. The resulting deletion deletes one of the microhomology sequences along with the entire intervening sequence between the two microhomologies. Thus, likely MMEJ-mediated deletions can be identified by searching either end of the deletion sequence for the presence of repeated base sequences in the regions flanking the other end of the deletion. On this basis, deletions were classified as 'MMEJ' if a stretch of 2 base pairs (bp) or more was detected at the deletion boundary and repeated in the region flanking the other end of the deletion. All other deletions were classified as NHEJ.

Prior to injection, approximately 30% of indels were derived from MMEJ repair (Fig. 41B, CD34, black striped bars = MMEJ, white bars = NHEJ). In erythroid progenitor cells (based on indels detected after erythroid differentiation of CD34 cells), MMEJ indels accounted for approximately 36% of indels (Fig. 41B, Prog, black striped bars = MMEJ, white bars = NHEJ). After 16 weeks of engraftment, most of the MMEJ indels were lost (Fig. 41B, HSC, black-striped bars = MMEJ, white bars = NHEJ) and NHEJ-derived editing was maintained. This observation has been observed by others (Bauer 2019, Wu 2019, Weber 2020) and does not appear to be site-specific. This shifts from a predominant population of MMEJ-prone short-lived progenitors that are actively cycling and make up most of the pre-infusion CD34+ cells, to a quiescent (and MMEJ-refractory) autologous population at later time points in vivo. It was thought to be the result of the migration of regenerated hematopoietic stem cells (HSCs) into production. While HSCs lead to hematopoietic reconstitution long after engraftment, they represent a very rare population among the pre-infusion CD34+ bulk population. This finding highlights the need to achieve sustained HbF expression using editing tools that generate HbF-induced indels via NHEJ-mediated repair rather than MMEJ repair.

In addition, genotypic to phenotypic analysis in the distal CCAAT box region of the HBG1/2 promoter identified mutations leading to the highest elevation of HbF expression. Single-cell experiments were performed to assess the distribution of γ-chain expression levels in erythroid cells derived from mPB CD34+ cells edited in the distal CCAAT box region. Indels were generated at this site using Sp35 RNP or RNP34 (Table 15) plus Sp182-Cas9-RNP (Fig. 5F, Table 8). Briefly, 6.25×10 ⁶ /mL mPB CD34+ cells (100 μl) were subjected to MaxCyte electroporation after 48 h pre-stimulation in X-Vivo 10 medium supplemented with SCF, TPO and FLT3. 8 μM HBG1-1-AsCpf1H800A-RNP co-delivered with 4 μM Sp35 RNP via Via or with 8 μM Sp182-Cas9-RNP (all RNPs at a 4:1 molar ratio of gRNA:RNA-induced nuclease) was electroporated. The Sp35 RNP is the S. Sp35 gRNA, complexed with S. pyogenes wild-type (Wt) Cas9 protein, SEQ ID NO: 339 (i.e., containing the targeting domain of CUUGUCAAGGCUAUUGGUCA (RNA)); SEQ ID NO: 917 (i.e., CTTGTCAAGGCTATTGGTCA (DNA) ))including. RNP34 is HBG1-1-AsCpf1H800A-RNP (His-AsCpf1-sNLS-sNLS-H800A (SEQ ID NO: 1032 ) consisting of ). After a recovery period of 48 hours after electroporation, cells were sorted by fluorescence-activated cell sorting (FACS) at 1 cell/well in non-tissue culture treated 384-well plates. Cells were then differentiated, clonally expanded for 18 days and differentiated into the erythroid lineage (adapted from Giarratana 2011). UPLC analysis was performed to determine the distribution of Gγ chain expression levels (percentage of Gγ/[total β-like chains]) in clonal erythroid progeny of cells derived from the total population of mPB-CD34+ cells originally edited. bottom. Sequence analysis (identification of alleles and detection of indels using the following primers: forward = CATGGCGTCTGGACTAGGAG (SEQ ID NO: 1266) and reverse = AAAACATTTCACAATCCCTGAAC (SEQ ID NO: 1267), and ddPCR (deleting the g-γ coding sequence Deletion of a 4.9 kb fragment) was performed on gDNA from an aliquot of each clonal culture taken 14 days prior to nuclear depletion to associate with a specific indel, encoding G-γ. Indels disrupting more than 3 nucleotides were generally associated with higher HbF expression compared to smaller indels (Fig. 41C). ).This finding suggests that using editing tools that generate larger deletions in the distal CCAAT box region is more effective at disrupting binding at this site of regulators that repress HBG expression. In particular, most of the larger deletions evaluated were detected in clones derived from CD34 cells edited with Cpf1 RNP, whereas the evaluated Most of the smaller deletions detected were detected from clones derived from CD34 cells edited with Cas9 RNP (Fig. 41D), with larger indels occurring more frequently with Cpf1, compared to Cas9. This suggests that editing with Cpf1 leads to a potentially high proportion of HbF-elevated cells in the erythroid progeny of CD34 cells, consistent with this result that editing primarily by either Cpf1 or Cas9 RNP The distribution of γ-chain expression levels (percentage of γ-chains/[total β-like chains]) in clonal erythroid progeny of cells from the total population of mPB-CD34+ cells treated with Cpf1 samples compared to spCas9 samples: A higher frequency of cells with high HbF was shown (Fig. 41E).In analyzed clones derived from CD34+ cells edited with Cpf1 RNP, 83.1% were mock-electroporated mPB CD34+ had γ-chain levels greater than the median detected in cell-derived erythroid clones (median = 18.8%), 64.2% had γ-chain levels greater than 30% overall β-like, 35.8% had γ-chain levels greater than 48.8% of total β-like (median level in control cells was 30% + 18.8%) Analysis from CD34+ cells edited with Cas9 RNP Of clones detected, 71.4% had γ-chain levels above the median detected in erythroid clones from mock-electroporated mPB CD34+ cells (median = 18.8%), 49.5 % had γ-chain levels greater than 30% of total β-like and 23.1% had γ-chain levels greater than 48.8% of total β-like (median level in subject cells was 30% + 18 .8%).

Example 14: Cpf1 RNPs Targeting the Distal CCAAT Box Region of the HBG Promoter Efficiently Generate NHEJ-Mediated Persistent Deletions of ≥4 Nucleotides Complexed with Either Cpf1 or Cas9 Enzymes RNPs consisting of guide RNAs targeting the distal CCAAT box region of the HBG promoter were electroporated into CD34+ cells from mobilized peripheral blood (mPB) using a MaxCyteGT (MaxCyte, Inc.) electroporation device. Briefly, 62.5×10 ⁶ /mL mPB CD34+ cells were treated with 4 μM Sp35 RNP (gRNA:RNA Electroporation was performed with 2:1 molar ratio of inducing nuclease) or 8 μM RNP58 (Table 15) (4:1 molar ratio of gRNA:RNA-inducing nuclease). The Sp35 RNP is the S. Sp35 gRNA (comprising the targeting domain of SEQ ID NO: 339 (i.e., CUUGUCAAGGCUAUUGGUCA) (RNA)), complexed with S. pyogenes wild-type (Wt) Cas9 protein; DNA))).

Twenty-four hours after electroporation, the levels and profiles of insertions/deletions at target sites were analyzed. The proportion of small indels present in the samples was assessed by Illumina amplicon sequencing (ILL-seq) following library construction methods and analysis. A 15 bp window around the expected cleavage site was used in the analysis to calculate the edit rate. Oligonucleotides and amplicons used to generate targeted amplicon sequencing products are provided in Figures 55A and 55B.

Indels generated in each sample were analyzed and processed to characterize editing by RNP32. The results were summarized by looking at 1) the deletion percentage of each base within the target region, 2) the distribution of insertion and deletion center positions, and 3) the distribution of insertion and deletion lengths. To perform these analyses, the CIGAR strings of reads in the alignment bam files were processed using the following procedure:
1) Group indels that occur in the same read.
2) Create an indel_id for the indels from each read. Indel_id is a string that identifies an indel and is indicated as indel_start_position+_+indel_length+_+ID. ID is NA for deletions and inserted sequence for insertions. See example in Figure 55D.
3) Group reads with the same indel_id.
4) Count the number of reads with the same indels and calculate their total percentage (including wt) and percentage of indels (excluding wt). Percentages in indels allow comparisons between samples when the samples have different amounts of total editing.

Deletions were classified as 'MMEJ' if a stretch of 2 base pairs or more bounded the deletion and was repeated in the region flanking the other end of the deletion. All other deletions were classified as HEJ. Table 24 shows indels detected in samples edited with RNP58 or Sp35 RNP.

Delivery of RNPs containing Cas9 or Cpf1 enzymes targeting the HBG promoter region (Fig. 41A) results in the generation of multiple insertions and deletions (indels). Different enzymes have different mechanical properties, including their engagement with DNA target sites and their relationship to their cleavage activity, leaving different DNA ends that influence repair outcomes (Sternberg 2014, S, trohkendl 2018, Swarts 2018). Notably, Cpf1 editing results in a 4-nucleotide 5′ overhang, which is quite different from the blunt end of SpCas9 editing (see FIG. 41A).
Evaluation of the editing profile mediated by Cpf1 and Cas9 RNPs (RNP58 and Sp35 RNP, respectively) targeting the distal box revealed that indels generated by both enzymes were generally centered around the distal CCAAT box region (Fig. 42H). , indicated that the most frequently deleted base was TSS:-113 of both RNPs (FIGS. 42E and 42F). However, evaluation of individual indels defined by position, length and, in the case of insertions, sequence relative to HBG-TSS showed differences in editing results after editing this site with SpCas9 or Cpf1 RNPs. Demonstrate. Figures 42C and 42D show the top 20 indels generated by Cpf1 and Cas9 RNPs, respectively. The most abundant indel generated by Cas9 is the MMEJ-mediated 13bp deletion 157_-13_NA (HBG1/2c. -102 to -114, Table 24), more commonly known as the 13bp HPFH deletion. , was detected in 31.88% of Cas9 samples compared to 3.36% in Cpf1 samples (Figures 42D and 42C, respectively). The most abundant indel generated by Cpf1 was the MMEJ-mediated 18nt deletion 159_-18_NA (HBG1/2c. -104 to -121, Table 24), with 18.53% of Cas9 samples compared to 18.53% of Cpf1 samples. was detected at 1.35% (FIGS. 42C and 42D, respectively). The five most common NHEJ indels detected in each sample were 3-7 bp deletions in length in Cpf1 samples and 1-4 bp deletions and 1 bp insertions in Cas9 (Fig. 42C, respectively). and FIG. 42D). A complete list of indels detected >0.1% in either of the two samples is shown in Table 24.

Most indels detected at >0.1% in either of the two samples were also detected in the other samples (greater than or less than 0.1%), while the relative frequencies among all indels were enzymatically different (Table 24 and Figure 42I). Notably, insertions were detected in 14.09% of Cas9 (Sp35 RNP) samples, while almost none were detected in Cpf1 (RNP58) samples (0.47%). Larger indels were also enriched in CD34 cells edited with Cpf1 RNP (RNP58) compared to Cas9 RNP (Sp35 RNP) (FIG. 42J). FIG. 42G shows the distribution of indel lengths generated by each RNP, and FIG. 42A shows the distribution of indel lengths generated by each RNP for indels classified as mediating NHEJ. Most indels produced by Cpf1 were larger than 3 bp (Fig. 42G), which were shown to be associated with elevated HbF in erythroid cells (Fig. 41C). Cas9 also generated deletions greater than 3 bp, but these are primarily generated via the MMEJ repair pathway, which is underutilized by the rare population of HSCs within CD34+ cells, thus injecting The frequency of later in vivo observations is expected to be low. NHEJ is the major repair pathway utilized by HSCs (Fig. 41B), thus evaluation of NHEJ-mediated indel profiles revealed the types of indels expected to be maintained in vivo long-term after infusion of CD34+ cells. provide information about

When compared to the Cas9 RNP (Sp35 RNP), the Cpf1 RNP (RNP58) showed a greater shift to NHEJ-mediated indels (Fig. 42A), with the most commonly observed NHEJ indel lengths compared to 5 bp for Cpf1. 1 bp for Cas9, suggesting a more favorable CD34+ cell editing profile by Cpf1 for robust and sustained induction of HbF.

The percentage of NHEJ-mediated indels greater than 3 bp, MMEJ-mediated indels greater than 3 bp, and indels less than or equal to 3 bp at the distal CCAAT box resulting from editing by RNP58 (Cpf1) or Sp35 RNP (Cas9) are shown in FIG. 42B ( Indels generated by SpCas9 (left pie chart): NHEJ > 3 bp = 21.42%; ≤ 3 bp = 48.84%; MMEJ > 3 bp = 29.74%; ): NHEJ > 3 bp = 64.86%; ≤ 3 bp = 11.11%; MMEJ > 3 bp = 24.02%). Cas9 (Sp35 RNP)-edited cells constituted the majority of small indels less than 3 bp in length, while indels greater than 3 bp in length (associated with the highest levels of HbF) were mainly mediated by the MMEJ pathway ( Figure 42B). Instead, nearly 90% of Cpf1 (RNP58)-mediated indels were greater than 3 bp in length, approximately two-thirds of NHEJ-mediated indels (FIG. 42B), and were greater than 3 bp. A high proportion of indels is expected to be maintained in vivo for an extended period of time, suggesting that the proportion of MMEJ-mediated indels is reduced in favor of NHEJ-mediated indels (Fig. 41B). Taken together, this suggests that the most persistent NHEJ-mediated indels are productive (greater than 3 bp) in RNP58, whereas Sp35 RNPs have a much lower proportion of highly productive indels, many of which are non-persistent. (MMEJ meditated). This indicates that RNP58 is an advantageous editing tool that generates HbF-induced indels not only by MMEJ repair, but also by NHEJ-mediated repair, resulting in sustained HbF expression.

Example 15: NOD of RNP32-edited mPB CD34+ cells, B6. Injection into SCIDIl2rγ−/−Kit(W41/W41) mice results in long-term engraftment, maintenance of indels, and high HbF induction. To determine human adult CD34+ cells from mobilized peripheral blood (mPB), non-irradiated NOD, B6. SCIDIl2rγ−/− Kit (W41/W41) (Jackson Lab strain name: NOD.Cg-Kit<W-41J>Tyr<+>Prkdc<scid>Il2rg<tm1Wjl>/ThomJ) (“NBSGW”) mice were injected . Briefly, mPB CD34+ cells at 62.5×10 ⁶ /mL and 6 μM RNP32 (Table 15) after 48 h pre-stimulation in X-Vivo10 medium supplemented with SCF, TPO and FLT3 ( Electroporation was via MaxCyte electroporation using gRNA:RNA-guided nuclease molar ratio 2:1) or buffer alone (“mock”). After an additional 24 hours, mCD34+ cells were cryopreserved. A portion of the cells was placed in ex vivo culture for up to 72 hours after electroporation and gDNA was harvested daily for indel analysis. On the day of injection, mPB CD34+ cells were thawed and NBSGW mice were injected via tail vein injection at 1 million cells per mouse. After 16 weeks, mice were euthanized and bone marrow (BM) was harvested from femur, tibia, and pelvic bones.

Genomic DNA was isolated from both pre-injection CD34+ cells (24-72 hours after electroporation) and bone marrow after 16 weeks engraftment. Twenty-four hours after electroporation, approximately 92% indels were achieved as determined by Illumina sequencing of the on-target PCR product using the following primers: forward = CATGGCGTCTGGACTAGGAG (SEQ ID NO: 1266) and reverse = AAACACATTTCACAATCCCTGAAC ( SEQ ID NO: 1267).

Distribution of indel lengths and indels up to 3 bp in length classified as either MMEJ- or NHEJ-mediated indels and lengths for pre-injected cell populations after in vitro culture 24 h to 72 h post-electrophoresis. An analysis of the distribution of indels with >3 bp was performed. Deletions were classified as 'MMEJ' if a stretch of 2 base pairs (bp) or more was detected at the deletion boundary and repeated in the region flanking the other end of the deletion. All other deletions were classified as NHEJ.

The length distributions of indels shown in Figures 43H-K (only NHEJ indels in Figures 43D-G) were very similar at all time points (Figures 43D and 43K). Few insertions were detected and the most commonly observed deletion size was −18, which is particularly noteworthy for the 18 bp MMEJ deletion HBG1/2c. It corresponds to a size of -104 to -121 and is the most frequent repair result after editing this site with Cpf1 (Fig. 42C, 57, Table 25). The most commonly observed deletion size among NHEJ indels was 5 bp, as shown in Figures 43D-G.

At all time points, indels greater than 3 bp in size that were observed to be associated with the highest levels of HbF induction (Fig. 41C) represented nearly 90% of all indels (Fig. 43A-C). Approximately two-thirds of them are mediated by NHEJ, and a high proportion of indels greater than 3 bp in length is expected to be maintained in vivo for long periods of time, reducing the proportion of MMEJ-mediated indels and reducing the proportion of NHEJ-mediated indels. (Fig. 41B).

After 16 weeks of engraftment, approximately 96% indels were measured, demonstrating the maintenance of indels within the long-term repopulation of CD34+ cells (Fig. 43A).

Subpopulations of cells within the bone marrow were then identified by FACS analysis to determine the level of multilineage engraftment. Human chimerism and lineage rearrangement (CD45+, CD15+, CD19+, glycophorin A (GlyA, CD235a+), lineage, and CD34+, and mouse CD45+ marker expression) in BM were determined and analyzed by flow cytometry. The frequency of GlyA+ cells was calculated as GlyA+ cells/total cells in BM. Human chimerism was defined as human CD45/(human CD45+mCD45). Analysis revealed high levels of human chimerism and no difference compared to mock-electroporated cells. Greater than 90% human chimerism was achieved in both mock and RNP32 edited mouse groups (Fig. 43M). Furthermore, comparable reconstitution of B-cell, neutrophil, erythroid, and CD34+ cell lineages was observed in mice injected with RNP32 or mock-electroporated cells (Fig. 43M).

Long-term HbF induction by CD235a+ (GlyA+) erythroid cells derived from RNP32-edited CD34+ cells was also analyzed. GlyA+ cells obtained from bone marrow were stained for γ-globin expression and analyzed via flow cytometry. Edited cells exhibited a pancellular distribution, with approximately 90% of cells being F-positive (Fig. 43N). Additionally, the GlyA+ cell population was isolated and harvested by fluorescence activated cell sorting (FACS) and dissolved in HPLC grade water. Lysates were then assessed for HbF expression by UPLC. FIG. 43O depicts HbF expression within the GlyA+ population over 50%, calculated as γ/β-like chains (%) by GlyA+ cells.

These data demonstrate that robust long-term HbF induction is achieved with RNP32 editing of human CD34+ cells. Furthermore, RNP32 editing does not adversely affect the engraftment and lineage reconstitution capacity of CD34+ cells.

Example 16: Injection of RNP32-edited mPB CD34+ cells into NSG (NOD-SCIDIl2rγ ^Null ) mice demonstrates highly polyclonal and stable engraftment of edited cells for 20 weeks without clonal expansion RNP32 (Table 15) )-derived indels longitudinally maintain a polyclonal profile, human adult CD34+ cells from mobilized peripheral blood (mPB) were subjected to non-irradiated NSG (NOD-SCIDIl2rγ ^Null ) (Jacksonlab strain name: NOD .Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ (“NSG”) mice were injected: Briefly, after 48 hours of pre-stimulation in X-Vivo10 medium supplemented with SCF, TPO and FLT3, 62.5× 10 ⁶ /mL of mPBCD34+ cells were electroporated via MaxCyte electroporation with 6 μM RNP32 or buffer alone (“mock”) After an additional 24 hours, mCD34+ cells were cryopreserved on the day of injection. mPB CD34+ cells were thawed and injected into busulfan-treated NSG mice at 5 million cells per mouse via tail vein injection Tail incision was performed from mice at 8, 12, 16 and 20 weeks post-injection. Indel profile analysis was performed on these samples, along with pre-injection CD34+ cell products (24 hours after electroporation), to demonstrate the maintenance of polyclonality. The following primers were used for the detection of indels: forward = CATGGCGTCTGGACTAGGAG (SEQ ID NO: 1266) and reverse = AAACACATTTCACAATCCCTGAAC (SEQ ID NO: 1267) No significant clonal expansion was observed in any of the mice analyzed. did not occur, demonstrating maintenance of a diverse indel profile over 20 weeks (Fig. 44A).In addition, 20 weeks after engraftment, mice were euthanized and bone marrow (BM) was harvested from femurs and tibiae. Genomic DNA was isolated and indel profile analysis was performed using the above primers A wide variety of indel sequences were observed as found in peripheral blood (Fig. 44B). demonstrated reconstitution by large numbers of edited hematopoietic stem cells, suggesting that RNP-mediated genetic modification did not impair the in vivo engraftment and self-renewal capacity of HSCs within the edited CD34 cell population be done. Furthermore, no clonal expansion was observed, suggesting that cells with uncontrolled growth of tumorigenic potential were not generated after modification of CD34 cells with RNP. The emergence of dominant clones is a potential sign of uncontrolled growth or tumorigenesis, which is not seen with RNP32 editing.

Example 17 Use of RNP32 for Treatment of Sickle Cell Disease As described herein, AsCasl2a (Cpf1) in the HBG1 and HBG2 promoters to induce expression of anti-sickling fetal hemoglobin. ) An autologous cell therapy for sickle cell disease (SCD) can be developed comprising CD34+ cells from patients with SCD edited with RNP. This autologous cell therapy is a therapeutic approach to SCD that promotes the expression of anti-sickling fetal hemoglobin by directly targeting the promoters of the HBG1 and HBG2 genes, which encode fetal gamma globin chains. There is (Fig. 45).

The efficiency of RNP32 editing for such cell therapy was determined and compared using CD34+ cells obtained from 3 independent normal adult donors and 4 SCD patient donors. Seven batches of mobilized peripheral blood CD34+ cells were used in two independent experiments (Table).

Briefly, CD34+ cells from normal or SCD donors were treated with 1×Glutamax, 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin in a 37° C., 5% carbon dioxide (CO2) humidified incubator. (TPO), pre-stimulated for 2 days in medium consisting of X-Vivo 10 supplemented with 100 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L). After 2 days of culture, cells were harvested and resuspended in MaxCyte electroporation buffer. RNP32 (6 μM, gRNA/protein molar ratio of 2) was delivered to CD34+ cells via the MaxCyte GT electroporation device. Between 1×10 ⁶ and 6.25×10 ⁶ cells can be used per OC-100 cartridge for electroporation. However, due to the limited number of cells available from SCD donors, 0.4×10 ⁶ to 1×10 ⁶ cells of each batch were used for electroporation (FIG. 46B). Prewarmed complete medium was then added to the cells to give a final cell density of approximately 1×10 ⁶ cells/mL (assuming approximately 10% cell loss during processing). Electroporated cells were then placed in a 37° C., 5% CO ₂ humidified incubator along with untreated control cells (cells that did not undergo electroporation). On days 1, 2, and 3 after electroporation, aliquots of cells were counted to determine viability and cells were harvested for further analysis.

Comparable viability was obtained between normal and SCD CD34+ cells treated with RNP32 1-3 days after electroporation (FIGS. 46A, 46B). The two samples with the lowest viability (1 SCD and 1 normal donor CD34+ cells) were consistent with the low cell numbers used for electroporation (Fig. 46B).

Electroporated CD34+ cells were resuspended in Quick Extract at a concentration of 2,000-4,000 cells/μL. Crude genomic deoxyribonucleic acid (gDNA) extraction was performed by exposing the lysate to the following conditions in a thermocycler: 65°C for 15 minutes followed by 95°C for 10 minutes. Crude gDNA was then analyzed for indels by next-generation sequencing using the following primers: forward = CATGGCGTCTGGACTAGGAG (SEQ ID NO: 1266) and reverse = AAACACATTTTCACAATCCCTGAAC (SEQ ID NO: 1267). RNP32 edited normal donor CD34+ cells as efficiently as SCD CD34+ cells (FIGS. 47A, 47C). Indel levels varied on day 1 post-electroporation (normal, 40.07%-84.39%; SCD, 46.78%-79.50%) but were normal by day 3 post-electroporation (83.13%-97.17%) and SCD CD34+ cells (83.04%-95.78%) increased to about 90%. (Fig. 47C, Fig. 47B (day 3)). The variable level of editing observed on day 1 was likely related to the low number of cells used in this experiment, which as described above was due to the availability of cells from SCD donors. Limited.

RNP32 recognizes both the HBG1 and HBG2 promoters. Truncation at both sites can lead to deletion of the 4.9 kb intervening sequence. Two ddPCR assays, an on-target amplicon and a reference amplicon, were designed to assess the frequency of the 4.9 kb deletion (Figure 48A). The relative positions of primers and probes are shown in Figure 48A and the sequences are listed in Figure 48B. Approximately 1.1 ng of gRNA for each sample was placed in a PCR plate droplet generator (BioRad) to generate droplets according to the manufacturer's instructions. The plate was then transferred to a thermocycler and the following protocol was run: 1 cycle of 98°C for 10 min, 40 cycles of 94°C for 30 sec, and 94°C for 2 min followed by 98°C for 10 min. 1 cycle and hold at 4°C. Plates were then plated for quantification and counting of droplets that were a) negative, b) positive for both on-target and reference amplicons, c) positive only for on-target, d) positive only for reference. Transferred to a droplet reader (BioRad). To determine the percentage of 4.9 kb deletion, the following formula was applied to each sample.

^* Reference correction factor was calculated as the mean of [(on-target concentration)/(reference concentration)] of control samples.

On-target editing by RNP32 also resulted in the deletion of a 4.9 kb fragment between the HBG1 and HBG2 promoters, which resulted in normal (16.4%-38.5%) and SCD at day 1 post-electroporation. It occurred at approximately 27% frequency of the β-globin locus in both CD34+ cells (20.7%-32.4%) (Figure 49A, Figure 49B).

Erythroid progeny of normal and SCD CD34+ cells were also characterized to determine the ability of RNP32-edited CD34+ cells to differentiate into erythroid cells. Briefly, on day 1 after electroporation (described above), 120,000 cells were cultured in erythrocyte induction medium and erythrocytes were induced using a modified three-step differentiation protocol developed by Giarratana et al. (Giarratana, 2005). generated. CD34+ cells were treated with 1×GlutaMAX (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin, 5% human AB+ plasma, 330 μg/mL human holo transferrin, 20 mg/mL human insulin, 2 U/mL heparin, 1 μM hydrocortisone, 3 U/mL Cultured for 7 days in Step 1 medium consisting of Iscove's Modified Dulbecco's Medium (IMDM) supplemented with mL recombinant human erythropoietin (EPO), 100 ng/mL SCF, and 5 ng/mL interleukin (IL)-3. On day 7, cells were transferred to step 2 medium, which was identical to step 1 medium except that hydrocortisone and IL-3 were absent, and cultured for 4 days. Cells were then cultured for 7 days in Step 3 medium, which was similar to Step 2 medium but without the addition of SCF and in which 5% human AB plus plasma was replaced with 5% knockout serum replacement (Gibco). At the end of 18 days of culture, 10 μL of cells were stained with 90 μL of erythrocyte fluorescence-activated cell sorting (FACS) master mix for 15 min at 4° C. and acquired on a Guava easyCyte 12HT flow cytometer to determine nuclear elimination frequency. Judged. A maximum of 120 mL of cell suspension per sample was spun down at 500 xg for 5 minutes, reducing the total volume of each sample to 20 mL. The cell suspension was then passed through an Acrodisc WBC syringe filter to remove nucleated cells and the resulting RBCs were used for further analysis in the experiments described in this example.

When placed under erythroid-inducing conditions, normal and SCD CD34+ cells (RNP32-edited and unedited cells) underwent a robust proliferation that averaged 23,000-fold across all experimental groups in 18 days (Fig. 50A, Table 20). At day 18, cultures consisted of erythroblasts, RBCs, and extruded nuclei (pirenocytes) (data not shown). Approximately 80% of erythroid cells in all groups reached terminal maturation and lost their nuclei (Fig. 50B). In summary, comparable fold expansion and terminal maturation were achieved by normal and SCD CD34+ cells with and without RNP32 electroporation.

HbF induction in erythroid progeny of normal and SCD CD34+ cells after editing with RNP32 was also characterized and globin chain analysis was performed. Expression of various hemoglobin subunits was analyzed using reverse-phase ultra-performance liquid chromatography (RP-UPLC) modified from the method described by Masala and Manca (Masala 1994). 1×10 ⁶ cells after filtration from the above erythroid differentiation were washed once with phosphate-buffered saline (PBS)-0.5% bovine serum albumin (BSA) and 50 μL of liquid chromatography-mass Dissolved in analytical (LC-MS) grade water. Samples were loaded onto an Agilent 1290 UPLC system. Elution was followed at 220 nm without reference wavelength. The globin chains were eluted in this order: β, α, AγT (common Aγ variant, gene product of HBG1), Gγ (gene product of HBG2), and Aγ (gene product of HBG1). The area under the curve under each peak approximated the relative abundance of each globin chain and was used to calculate levels of HbF. Since HbA is composed of α2β2 and HbF is composed of α2γ2, the level of HbF expression is calculated as (Aγ+Gγ)/(Aγ+Gγ+β) (%) or (AγT+Aγ+Gγ)/(AγT+Aγ+Gγ+β) (%), where γ/ Labeled as β-like (%).

The frequency of HbF+ cells (HbF-expressing cells) in RBCs after filtration was determined using the method described by Thorpe (Thorpe 1994). Briefly, RBCs from the erythroid differentiation described above were fixed with 4% formaldehyde, permeabilized with ice-cold acetone, washed with PBS-0.5% BSA, and stained with HbF+ cell FACS master mix. bottom. Cells were washed with PBS-0.5% BSA, resuspended in PBS 0.5% containing NucRed (2 drops/mL) and acquired on a Guava flow cytometer.

Erythroid cells from normal and SCD CD34+ cells electroporated with RNP32 demonstrated elevated pancellular HbF expression. An average of 43.26% HbF (38.38%-51.60%) in the treated normal donor group and 54.21% HbF (48.63%-58.99%) in the treated SCD donor group ) was measured, with 13.98% (10.49%-17.97%) and 21.16% (16.54%-27.92%) backs in the untreated normal and SCD donor groups, respectively. Significantly increased from ground (Fig. 51A, Table 20). Furthermore, RBCs derived from RNP32-electroporated CD34+ cells of either donor type (normal or SCD donor) averaged over 92% HbF+ (normal, 90.61%-93.85%; SCD, 93 .29%-94.50%) (FIG. 51B, Table 20), which is higher than that of RBCs from each untreated CD34+ cell (normal, 37.76%-58.23%; SCD, 50.09%-71%). .27%). Thus, high RNP32 editing levels resulted in elevated HbF levels after in vivo erythroid differentiation, with more than 50% of HbF measured in RNP32-derived erythrocytes (FIG. 51C).

As shown in Figure 51B, several assays were performed to demonstrate the clinical relevance of elevated fetal hemoglobin in untreated sickle cell patient cells and, to a lesser extent, in untreated cells from normal donors. Proven. Untreated cells from SCD patients have a high background of HbF due to the ex vivo culture used to differentiate CD34+ cells into RBCs to perform these assays. This is expected under these culture conditions and has been reported by Metais et al. (Metais 2019) previously reported. This likely partially obscures the full benefits obtained with modified cells over unmodified cells.

To assess the effect of HbF induction on RBC sickling as a result of hypoxia-induced HbS polymerization, untreated RBCs from unedited SCD CD34+ cells (unedited control cells) and RBCs from edited SCD CD34+ cells were , oxygen was removed from the cell suspension by incubating with the oxygen scavenger sodium metabisulfite solution, thereby placing the cells under a very low partial pressure of oxygen. The percentage of sickle RBCs from unedited SCD CD34+ cells and sickle red blood cells from RNP-32 edited SCD CD34+ cells was then determined and compared.

Briefly, cells were initially induced to generate red blood cells. One million RNP32-edited RBCs were harvested from the erythroid differentiation assay as described above and washed with PBS-0.5% BSA. The cell pellet was then resuspended in 20 μL of PBS and mixed with 20 μL of 2% (weight/volume) sodium metabisulfite in water. A drop (approximately 20 μL) of this cell mixture was then placed on a microscope slide, covered with a cover slip, and the edges sealed. Slides were stored at room temperature for 1-4 hours prior to imaging and analysis of morphological changes. The mean sickling frequency went from 38.3% in untreated SCD RBCs (with a mean HbF of 19.9%) to 10.6% in RNP32-edited SCD RBCs (with a mean HbF of 53.8%). decreased, corresponding to an approximately four-fold reduction in the sickling morphology of RNP32-edited RBCs from SCD patients compared to unedited RBCs from SCD patients (FIG. 52). Sodium metabisulfite leads to extreme hypoxia below levels commonly observed in physiology, and thus is expected to result in a lower proportion of sickle RBCs from edited cells in vivo.

The deformability of SCD RBCs was also evaluated. To assess whether HbF induction reduces the stiffness of SCD RBCs and improves deformability when deoxygenated, untreated RBCs from patients with SCD and RNP32-edited RBCs from SCD patients were analyzed on a Lorrca ektacytometer in which shear stress was applied with decreasing oxygen partial pressure (Fig. 53A). Fifteen million RBCs after filtration from erythroid differentiation (as above) were harvested and washed with PBS-0.5% BSA. Cells were centrifuged at 500×g for 5 minutes, resuspended in 1.5 ml of OXY ISO solution (Lorrca) and gently mixed by inversion. Cell suspensions were then loaded onto a Lorrca ektacytometer and oxygen scans were performed according to the manufacturer's instructions to measure the deformability of RBCs under shear stress during deoxygenation. RBC deformability was expressed as the elongation index (EI) (Fig. 53A), and the "sickling point" is the relative point when SCD RBCs begin to sickle and lose more than 5% EI during deoxygenation. Corresponds to oxygen pressure. Normal RBCs containing healthy adult hemoglobin (HbA and HbF) from three different donors were unaffected by the reduction in oxygen tension (Fig. 53B). In contrast, SCD RBCs from four donors stiffened upon deoxygenation, as indicated by a gradual decrease in elongation index corresponding to oxygen depletion (Fig. 53C (black lines)). RNP32-edited CD34+ cell-derived SCD RBCs began to sickle at lower oxygen tensions (represented as sickling points) compared to untreated CD34+ cell-derived RBCs (Fig. 53C (gray line); 53D, Table 21). Furthermore, deoxygenated RNP32-treated SCD RBCs remained more flexible than untreated SCD RBCs, as indicated by the higher minimum elongation index observed in RNP32-treated SCD RBCs compared to untreated SCD RBCs ( Fig. 53E, Table 21).

Next, to assess whether the reduced sickling and increased flexibility of RNP32-edited CD34+ cell-derived SCD RBCs lead to improved rheological behavior, RBCs were evaluated using a microfluidic platform. A microfluidic platform replicated microvasculature blood flow to directly observe the bulk flow of SCD RBCs cultured under various oxygen conditions. Upon deoxygenation, intracellular polymerization of HbS causes RBCs to become inflexible and rigid, which leads to reduced velocity through microchannels. Briefly, following filtration after red blood cell induction as described above, 150 million RBCs were spun down and resuspended in fresh Step 3 medium to a final concentration of 10×10 ⁶ cells/mL. made it Cultured RBCs were washed with PBS and resuspended in PBS to achieve a target hematocrit of 20%. A 15 μL volume of cultured RBCs was loaded onto the device under constant pressure and exposed to varying levels of oxygen controlled via a gas mixer. The flow was imaged with a high speed camera and Kanade-Lucus-Tomasi feature tracking in MATLAB® was used to determine blood flow velocity through the channel. Cell velocities were then measured under a range of oxygen levels. Typical oxygen levels observed in the venous circulation are approximately 4% to 6% oxygen.

RBCs from normal donors did not show oxygen-dependent rheological disturbances (Figs. 54A, 54B). Although the rheological behavior of RBCs derived from RNP32-edited SCD CD34+ cells was not completely normalized, they exhibited a lower degree of injury and decreased velocity during hypoxia compared to RBCs derived from untreated SCD CD34+ cells. (10% oxygen for RBCs from untreated SCD CD34+ cells, 6% oxygen for RBCs from RNP32-edited SCD CD34+ cells) was required before the development of . As shown in FIG. 54C, RBCs derived from RNP32-edited SCD CD34+ cells (circles) exhibited dramatically improved rheological changes at physiological oxygen levels compared to RBCs derived from untreated SCD CD34+ cells (triangles). demonstrated behavior. Unedited RBCs (diamonds) from normal donors showed no oxygen-dependent rheological impairment. Although the rheological behavior of RBCs derived from RNP32-edited SCD CD34+ cells did not completely normalize, they exhibited a lower degree of injury during hypoxia and increased kinetics compared to RBCs derived from unedited SCD patient CD34+ cells. Extreme hypoxia was required due to the onset of depression. Notably, at oxygen levels typically observed in venous circulation, the velocity of red blood cells from RNP32-edited CD34 cells was almost completely normalized compared to RBCs from normal donor cells. Decreased rheological disturbances under hypoxia were also strongly correlated with increased HbF levels in RBCs (Figs. 54D, 54E). For example, FIG. 54E shows that RBCs with the highest HbF levels exhibited the highest velocities at a physiological oxygen level of 4%. This indicates that phenotypic correction of RBCs from sickle cell patients is maximal when HbF expression is very high, as observed in RBCs derived from RNP32-edited SCD CD34+ cells ( 54D, 54E).

In summary, two studies were performed to investigate editing of SCD and normal CD34+ cells with RNP32 to determine functional consequences in erythroid progeny. RNP32 efficiently edited normal and SCD CD34+ cells, achieving approximately 90% insertions and/or deletions (indels) 3 days after electroporation. Editing of CD34+ cells with RNP32 did not adversely affect erythroid differentiation or maturation. Robust HbF expression was obtained and an average of 43.26% and 54.21% of total hemoglobin in RBCs from RNP32-edited normal and SCD CD34+ cells, respectively, was distributed in a pan-cellular manner (92% on average). RBC exceeding). High levels of HbF expression in RBCs derived from RNP32-edited SCD CD34+ cells were associated with reduced sickling upon deoxygenation, improved deformability under shear stress, and improved deformability under shear stress compared to RBCs from untreated SCD CD34+ cells. Consistent with improved flow through microfluidic channels. Potentially therapeutically relevant levels of HbF were achieved by highly efficient RNP32 editing of CD34+ cells at the HBG1 and HBG2 promoter regions. This leads to reduced sickling and improved rheological properties of red blood cells, which is advantageous for autologous cell therapy using RNP32 for the treatment of sickle cell disease.

Example 18: Meta-analysis of on-target indels generated by RNP32 in CD34+ cells , yielding highly heterogeneous repair outcomes involving hundreds of different genotypes. The editing results that occur when using Cpf1 (also called AsCasl2a), the enzyme used for RNP32, have not been characterized in detail. Cpf1 editing results in a 4-nucleotide 5′ overhang (see FIG. 55A), which is quite different from the blunt end of SpCas9 editing (see FIG. 41A). "Cleavage site" as used in this example is used to refer to the central position of the expected overhang resulting from editing with RNP32 (Fig. 55A, gray dashed line).

DNA repair mechanisms often produce indels of size from 1 to approximately 50 base pairs (bp), which are commonly detected by PCR-based targeted sequencing assays. The consequences of more complex genome repair have also been described (2018; 2018; 2020) and include deletions at on-target loci of greater than about 50 bp, termed excisions, and translocations. These more complex rearrangements require other methods for accurate quantification (see, eg, PCT/US2018/012652).

To determine the indel profile generated by RNP32, a PCR-based targeted sequencing assay was used to characterize indels ranging in size from 1 bp to approximately 50 bp in the distal CCAAT box generated by RNP32. Indel patterns were examined in different samples across different genotypes (normal vs. sickle cell disease) and mobilization regimens (plelixafor alone vs. G-CSF alone vs. G-CSF and plelixafor) (Table 12). . This meta-analysis demonstrates that RNP editing in the CCAAT box results in distinct indel profiles that are reproducible across different samples and editing levels.

This meta-analysis used mobilized peripheral blood CD34+ cells from 12 different donors (normal and SCD donors) (Table 22). Five samples from normal donors were generated using the large scale process (Table 22, experiment SCD1). Briefly, leukopacs (HemaCare or KeyBiologics) were obtained from normal donors mobilized with granulocyte colony-stimulating factor (G-CSF) and plelixafor. CD34+ cells were enriched using the CliniMACS Plus system, aliquoted and cryopreserved in a Cryostor CS10 and stored in liquid nitrogen vapor phase. Thaw CD34+ cells and complete medium consisting of X-Vivo 10 supplemented with 1×Glutamax, 100 ng/mL Stem Cell Factor (SCF), 100 ng/mL Thrombopoietin (TPO), 100 ng/mL FMS-like Tyrosine Kinase 3 Ligand (Flt3L) for 2 days, mixed with RNP32 (gRNA/protein molar ratio of 2) to a final concentration of 8 μM, and electroporated using a Maxcyte GT electroporator according to the manufacturer's instructions. Cellular material was cultured overnight following electroporation, aliquoted and cryopreserved in a Cryostor CS10 and stored in liquid nitrogen gas phase until ready for experiments.

A research-scale process was used to generate nine samples (Table 22, SCD011 and SCD014). Cells were thawed, washed and added at 1 x ¹⁰⁶ cells/mL with 1 x Glutamax, 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO), 100 ng/mL FMS-like tyrosine kinase 3 ligand ( The cells were cultured in a complete medium consisting of X-Vivo 10 supplemented with Flt3L) at 37° C. and 5% carbon dioxide (CO 2 ) in a wet incubator for 2 days. After 2 days of culture, cells were harvested and resuspended in Maxcyte electroporation buffer. RNP32 (gRNA/protein molar ratio of 2) was added to the cell suspension to a final concentration of 6 μM. The mixture was transferred to a Maxcyte OC-100 cartridge and electroporated using a Maxcyte GT electroporator according to the manufacturer's instructions. After electroporation, cells were cultured in complete medium for 1 day before being harvested for analysis.

The proportion of small indels present in the samples was assessed by Illumina amplicon sequencing (ILL-seq) following library construction methods and analysis. A 15 bp window around the expected cleavage site was used in the analysis to calculate the edit rate. Oligonucleotides and amplicons used to generate targeted amplicon sequencing products are provided in Figures 55A and 55B.

Indels generated in each sample were analyzed and processed to characterize editing by RNP32. The results were summarized by looking at 1) the deletion percentage of each base within the target region, 2) the distribution of insertion and deletion center positions, and 3) the distribution of insertion and deletion lengths. To perform these analyses, the CIGAR strings of reads in the alignment bam files were processed using the following procedure:
5) Group indels that occur in the same read.
6) Create an indel_id for the indels from each read. Indel_id is a string that identifies an indel and is indicated as indel_start_position+_+indel_length+_+ID. ID is NA for deletions and inserted sequence for insertions. See example in Figure 55D.
7) Group reads with the same indel_id.
8) Count the number of reads with the same indels and calculate their total percentage (including wt) and percentage of indels (excluding wt). Percentages in indels allow comparisons between samples when the samples have different amounts of total editing.

To analyze the reproducibility of individual indels detected across samples, we estimated the number of times they were detected across samples and the average percentage among indels. If no indels were detected in a particular sample, the percentage was assumed to be 0%.

The total percentage of indels for each RNP32 edited sample in this analysis is shown in Table 23. All samples (including normal and SCD donors) showed an overall compilation greater than 72.6% (72. 6% to 89.2%). Samples that showed lower editing had lower viability, potentially related to the lower number of cells used for electroporation.

A total of 385 unique indels were detected, and only indels detected at a percentage greater than 0.1% (of total indels) in at least one of the samples in Table 25 were counted.

The wt alleles and the top 20 indels are shown in Figure 57 along with their relative average frequencies (among all samples). The most abundant indel was 159_-18_NA, present in an average of 15.15% of all samples. In particular, the indel 157_-13_NA, more commonly known as the 13bpHPFH deletion, was detected in an average of 2.63%. A 13 bp deletion is a spontaneous mutation associated with elevated HbF.

Indel analysis of the bulk CD34+ population (progenitor cells) 24-72 hours after electroporation can determine whether editing at this site is derived from Cas12a or Cas9. For Cas12a, the most prevalent indel detected at this time is the 159_-18 deletion, whereas for Cas9 the most frequent indel is the 157_-13 deletion. Furthermore, the most common NHEJ indel generated by Cas9 at this site was the -1bp deletion (169_-1_NA, Table 24), which occurred in about 9.6% here. In contrast, the most common NHEJ indels generated after editing with Casl2a are 6bp and 4bp deletions (165_-6_NA and 167_-4_NA; Table 24, RNP58; Table 25, RNP32). Cpf1 generally produces larger NHEJ deletions when compared to Cas9 at this site.

Indel analysis of the bulk CD34+ population (progenitor cells) 24-72 hours after electroporation can determine whether the editing (indel) at this site is derived from Cpf1 or Cas9. For example, for Cpf1, the most prevalent indels detected at this time point were 159_ -18 deletion (Table 25). However, the most frequent indels of SpCas9 were 157_-13 deletions with an average percentage of total indels of 31.88%, compared with an average percentage of 2.63% of total indels for Cpf1 (Table 25). There is (Table 24).

The results of the indel center point and indel length distribution are shown in FIGS. 58 and 59, respectively. The central position (peak) of the most common indel was at position TSS:-113, 6 bp away from the cleavage site. Deletion length distribution peaks were observed at 13 bp and 18 bp, corresponding mainly to MMEJ indels at 157_-13_NA and 159_-18_NA. Due to the excision and utilization of microhomology sequences during MMEJ repair, this pathway tends to generate larger size deletions than those mediated by the NHEJ pathway. In addition to those, the most commonly observed deletion length was 5 bp at 9.30% (Figure 60). Few insertions were detected (0.50% total contribution). Deletions of 1 bp to 25 bp have a total contribution of 92.03% among all indels (deletions of 3 bp to 25 bp have a total contribution of 85.85% among all indels, 4 bp Deletions of ~25 bp had a total contribution of 80.35% among all indels, and deletions of 5 bp to 25 bp had a total contribution of 72.04% among all indels (Table 25). Overall, the distributions of both indel centerpoints and indel lengths were very similar in normal and SCD samples.As shown in Figure 41C above, the production of indels with lengths of 4 bp or longer was the most Resulting in high HbF induction.

Results for the percentage of bases deleted (deletion profile) along the target region are shown in Figures 61 and 62 (normalized to the same maximum value of 1 for comparison between samples). The shapes of the profiles are very similar for all samples, despite the different sums of edits (Table 23). No significant differences were observed between normal donor (Normal) and sickle cell donor (SCD) samples or between different mobilization regimens. The deletion profile shows that the peak of the distribution (the most commonly deleted base) is at TSS:-113, 6 bp from the cleavage site towards the TSS.

Of the total 385 indels detected at 0.1% in any of the samples evaluated, 108 indels were detected in all 14 samples (Figure 63 and Table 25). All indels present with an average percentage of indels greater than 0.22% were detected in all 14 samples (55 indels total, above the gray line in Figure 64, and Table 25), giving a total indel contribution of was 69.90%. Overall, the reproducibility of indel detection was very high.

To examine in more detail the consistency of individual indel frequencies among the indels detected in all samples, we calculated their pairwise correlation plots and ^R2 . To avoid sampling bias, only indels with a percentage (among all indels) greater than or equal to 0.1% in at least one of the samples were considered when comparing two samples. All ^R2 values were above 0.8, except for 3 samples where editing was less than 60% due to the low number of cells used for electroporation (see Table 22). (data not shown). Overall, the correlation between indel percentages among samples with high editing rates was very high.

In summary, a total of 385 unique indels were detected at a percentage greater than 0.1% in at least one of the 14 edited samples tested in this example. The most abundant indel, 159_-18_NA, was present among all indels, averaging 15.15% in all samples. Indel 157_-13_NA, more commonly known as 13bpHPFH deletion, was detected in an average of 2.63%. The shapes of the indel profiles of all edited samples were very similar in all studies despite having different sums of editing values. No significant differences were observed between normal and SCD donor samples or between different mobilization regimens.

Evaluation of the positions of individual indels showed that the indels generated by RNP32 were primarily centered at position TSS:-113, which was also the most commonly deleted base observed. rice field. Evaluation of the distribution of indel lengths was primarily based on MMEJ indels, 159_-18_NA (most abundant indels, TSS: 18 bases long starting at -104) and 157_-13_NA (TSS: starting at -102, 13 bp HPFH deletion). showed peaks at -18 and -13, corresponding to . In addition to these, the most commonly observed deletion length was 5 bp in 9.30%. Few insertions were detected (0.50% total contribution). Deletions from 1 bp to 25 bp contributed 92.03% among all indels. Overall, the distribution of indel lengths and center positions was very similar between normal and SCD samples. Out of a total of 385 unique indels, 108 indels were detected in all 14 samples. All indels present at an average percentage greater than 0.22% in the indels were detected in all 14 samples (55 indels total), with a total indel contribution of 69.90%. Pairwise correlations between detected indels (except for 3 samples with less than 60% editing) had ^R2 values above 0.8.

This data indicates that RNP32 editing at the HBG1/2 distal CCAAT box results in a unique indel signature that is reproducible across different samples and levels of editing.

Example 19 Analysis of RNP32 Editing Efficiency of Normal Donor CD34+ Cells Containing Deletion of the 4.9 kb Fragment On-target editing efficiency of RNP32 in mobilized peripheral blood CD34+ cells obtained from 4 independent healthy adult donors, and 2 The frequency of deletion of the 4.9 kb fragment between the two RNP32 cleavage sites was assessed. In addition, on-target indel levels and frequencies of the 4.9 kb deletion were also measured in several subpopulations of HSPCs sorted from total CD34+ cells, phenotypically long-term hematopoietic stem cells (LT-HSCs) We investigated whether it could be efficiently edited using RNP32.

Leukopaks from 4 normal donors treated with granulocyte colony stimulating factor (G-CSF) and Mozobil or G-CSF alone were obtained from HemaCare. CD34+ cells (lots: CEL045-002, CEL046-004, CEL047-002, and CEL021-021) were enriched using the CliniMACS system (Miltenyi), aliquoted and cryopreserved in Cryostor CS10. Cells were thawed, washed and added at 1 x ¹⁰⁶ cells/mL with 1 x Glutamax, 100 ng/mL stem cell factor (SCF), 100 ng/mL thrombopoietin (TPO), 100 ng/mL FMS-like tyrosine kinase 3 ligand ( The cells were cultured in a complete medium consisting of X-Vivo 10 supplemented with Flt3L) at 37° C. and 5% carbon dioxide (CO 2 ) in a wet incubator for 2 days. After 2 days of culture, cells were harvested and resuspended in Maxcyte electroporation buffer. RNP32 (gRNA/protein molar ratio of 2) was added to the cell suspension to a final concentration of 6 μM. The mixture was transferred to a Maxcyte OC-100 cartridge and electroporated using a Maxcyte GT electroporator according to the manufacturer's instructions.

RNP32 recognizes both the HBG1 and HBG2 promoters. Truncation at both sites can lead to deletion of the 4.9 kb intervening sequence. To assess the frequency of 4.9 kb fragment deletions, two ddPCR assays, reference amplicon and on-target amplicon, were performed as described in Example 17 (see Figures 48A-48D). . Briefly, cells 1 or 2 days after electroporation with RNP32 and sorted cells were resuspended in Quick Extract at a concentration of 2,000-4,000 cells/μL. Crude genomic deoxyribonucleic acid (gDNA) extraction was performed by exposing the lysate to the following conditions in a thermocycler: 65°C for 15 minutes followed by 95°C for 10 minutes. The crude gDNA was then analyzed for indels (insertions and deletions) by next generation sequencing (using primers described in Figure 55A) and a 4.9 kb fragment deletion by digital droplet polymerase chain reaction (ddPCR). was evaluated.

RNP32 edited the HBG1 and HBG2 promoters of normal donor CD34+ cells in an RNP concentration- and time-dependent manner without significantly affecting cell viability (Figures 65A, 65B, 66, and 67). At 8 μM, the highest concentration of RNP32 tested, an average on-target indel level of 86% was achieved in normal donor CD34+ cells by day 1 post-electroporation, rising to 91% by day 2 (FIG. 68). ). Mean indel levels at 1 μM increased from 62% to 77% by days 1 and 2 after electroporation, with the increase being more pronounced at lower RNP concentrations. By day 2 post-electroporation, on-target indel levels of 85% or greater were routinely achieved when CD34+ cells were transfected with 3 μM or greater of RNP32.

Editing of CD34+ cells with RNP32 resulted in frequent deletions of an intervening fragment of 4.9 kb between the two RNP32 cleavage sites in the HBG1 and HBG2 gene promoters, respectively. On average approximately 35% of the β-globin locus in CD34+ cells had a 4.9-kb fragment deleted both 1 and 2 days after electroporation with 2 μM or more RNP32 (FIG. 69A, 69B and 70). After electroporation with ≧2 μM RNP32, the ratio of deletions to indels of the 4.9 kb fragment in CD34+ cells was approximately 0.47 on day 1 and decreased to approximately 0.40 on day 2.

CD34+ cells are heterogeneous and include lineage-restricted progenitor cells, MPPs, and self-renewing LT-HSCs. Because LT-HSCs are responsible for providing long-term reconstitution of the patient's hematopoietic system, high-level editing in this population is relevant to treatment persistence. To investigate whether different subpopulations of CD34+ cells are similarly edited, we evaluated on-target indel levels in CMP, MPP, and phenotypical LT-HSCs, as defined by surface immunophenotype, and analyzed multiple Compared to on-target indel levels in total CD34+ cells across RNP concentrations.

Briefly, CD34+ cells were sorted 2 days after electroporation with RNP32 and phenotypic LT-HSCs were sorted using a BD fluorescence-activated cell sorting (FACS) Aria Fusion cell sorter (BD Biosciences). , multipotent progenitor cells (MPP), and common myeloid progenitor cells (CMP) were obtained. Gating was set to pick the following enriched CD34+ cell subpopulations: phenotypic LT-HSC (P7, CD34 bright, CD38 low/negative, CD90+, CD45RA−), MPP (P6, CD34 bright, CD38 low/negative, CD90-, CD45RA-) and CMP (P9, CD34 bright, CD38 high, CD123+, CD45RA-).

Of the three subpopulations, CMP consistently showed the highest on-target indel levels and phenotypic LT-HSCs showed the lowest on-target indel levels across all RNP concentrations evaluated (shown in Figure 55B). analyzed via NGS using the primers provided) (Figures 71A, 71B, and 72). (Study MAX072). Nevertheless, MRNP32 ≧3μ achieved on-target indel levels of ≧85% in phenotypic LT− HSCs, similar to those detected in all CD34+ cells, indicating self-renewing LT− It was suggested that HSCs could be efficiently edited using RNP32 (Figures 71A, 71B, and 72).

RNP32 editing resulted in the deletion of a 4.9 kb fragment in all three subpopulations of hematopoietic stem and progenitor cells tested (FIGS. 73A and 74). Of the three subpopulations, CMP consistently had the highest deletion levels of the 4.9 kb fragment, and phenotypic LT-HSCs had the lowest deletion levels of the 4.9 kb fragment across all RNP concentrations evaluated. . The ratio of 4.9 kb fragment to indels averaged across all RNP concentrations was approximately 0.43, 0.49, 0.33, and 0 for total CD34+ cells, CMPs, MPPs, and phenotype LT-HSCs, respectively. .25 (FIG. 73B), demonstrating that LT-HSCs had a lower deletion frequency of the 4.9 kb fragment than their short-term counterparts as a result of RNP32 editing. Retention of the 4.9 kb interediting region is beneficial, as higher deletions of the 4.9 kb fragment result in lower production of HbF. Therefore, lower level deletion of the 4.9 kb fragment of LT-HSC edited by RNP32 is advantageous. The results herein show that editing with RNP32 can generate cells with clinically relevant levels of healthy HbF.

In summary, deletion of a 4.9 kb fragment associated with on-target indel levels between the HBG1 and HBG2 promoters was assessed following electroporation of normal human donor CD34+ cells with RNP32. RNP32 was highly efficient in editing CD34+ cells, achieving consistent editing across multiple RNP batches and cell donor lots. Greater than 85% on-target indels were routinely achieved in CD34+ cells when electroporated with 3 μM or higher RNP32. Comparable levels of indels were also observed between total CD34+ cells and sorted phenotypic LT hematopoietic stem cells when electroporated with 3 μM or higher RNP32. Deletions of the 4.9 kb fragment between the two RNP32 cleavage sites occurred at cell subpopulation-dependent frequencies. All CD34+ cells deleted the 4.9 kb fragment at a frequency of approximately 0.4 per indel. This frequency was reduced to approximately 0.25 per indel in the phenotypic LT-HSC subset of CD34+ cells.

Example 20 Treatment of β-Hemoglobinopathies Using Edited Hematopoietic Stem Cells - can be used in the treatment of hemoglobinopathies; For example, in autologous treatment, genome editing can be performed on patient-derived cells. Ex vivo modification of the patient's cells and reintroduction of the cells into the patient can result in increased HbF expression and treatment of β-hemoglobinopathies.

For example, HSCs can be extracted from the bone marrow of patients with β-hemoglobinopathies using techniques well known to those skilled in the art. HSCs can be modified using the methods disclosed herein for genome editing. For example, RNPs containing guide RNAs (gRNAs) targeting one or more regions of the HBG gene, complexed with RNA-guided nucleases, can be used to edit HSCs. In certain embodiments, the RNA-guided nuclease can be the Cpf1 protein. In certain embodiments, the Cpf1 protein can be a modified Cpf1 protein. In certain embodiments, the modified Cpf1 protein comprises SEQ ID NOs: 1000, 1001, 1008-1018, 1032, 1035-39, 1094-1097, 1107-09 (Cpf1 polypeptide sequences) or SEQ ID NOs: 1019-1021, 1110-17 (Cpf1 Polynucleotide Sequences). For example, a modified Cpf1 protein can be encoded by the sequence set forth in SEQ ID NO:1097. In certain embodiments, the gRNA can be modified or unmodified gRNA. In certain embodiments, the gRNA may comprise a sequence listed in Table 6, Table 12 or Table 13. For example, in certain embodiments, the gRNA can comprise the sequence set forth in SEQ ID NO:1051. In certain embodiments, the RNP complexes may comprise RNP complexes listed in Table 15. For example, an RNP complex can comprise a gRNA comprising the sequence set forth in SEQ ID NO: 1051 and a modified Cpf1 protein encoded by the sequence set forth in SEQ ID NO: 1097 (RNP32, Table 15). In certain embodiments, modified HSCs have an increased frequency or level of indels in the human HBG1 gene, HBG2 gene, or both compared to unmodified HSCs. In certain embodiments, modified HSCs can differentiate into red blood cells that express increased levels of HbF. A population of modified HSCs can be selected for reintroduction into the patient via transfusion or other methods known to those of skill in the art. A population of modified HSCs for reintroduction can be selected based on, for example, increased HbF expression in erythroid progeny of modified HSCs or increased frequency of indels in modified HSCs. In some embodiments, any form of ablation prior to cell reintroduction may be used to enhance engraftment of modified HSCs. In other embodiments, peripheral blood stem cells (PBSCs) can be extracted from a patient with β-hemoglobinopathy using techniques well known to those skilled in the art (e.g., apheresis or leukoapheresis), and the stem cells are can be removed. The genome editing methods described above can be performed on stem cells and the modified stem cells can be reintroduced into the patient as described above.

Sequence Genome editing system components according to the present disclosure, including but not limited to RNA-guided nucleases, guide RNAs, donor template nucleic acids, nucleic acids encoding nucleases or guide RNAs, and portions or fragments of any of the foregoing ) are exemplified by the nucleotide and amino acid sequences presented in the sequence listing. The sequences presented in the sequence listing are not intended to be limiting, but rather to illustrate certain principles of the genome editing system and its component parts, which, in combination with the present disclosure, are within the scope of the present disclosure. It will inform those skilled in the art about additional implementations and modifications within.

INCORPORATION BY REFERENCE All publications, patents and patent applications referred to in this specification are hereby incorporated by reference, as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. The entire contents of which are incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims (44)

a plurality of modified CD34+ or hematopoietic stem cells;
one or more of said plurality of modified cells having indels in the HBG gene promoter;
HBG1/2 in HBG1 promoter, HBG2 promoter, or combinations thereof c. said plurality of modified cells as a whole comprising deletions of -104 to -121;
Said HBG1/2c. A first population of modified cells, wherein -104 to -121 deletions constitute 2% or more of said indels in said plurality of modified cells as a whole. Said HBG1/2c. 2. The first population of modified cells of claim 1, wherein -104 to -121 deletions constitute less than 25% of said indels in said plurality of modified cells as a whole. a plurality of modified CD34+ or hematopoietic stem cells;
one or more of said plurality of modified cells comprising an indel in the HBG gene promoter;
A first population of modified cells, wherein 60% or more of said indels in said plurality of modified cells as a whole are deletions of at least 4 base pairs. delivering a first RNP complex comprising a first guide RNA (gRNA) and a Cpf1 RNA guide nuclease or a modified Cpf1 RNA guide nuclease to a first population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells and said first gRNA comprises a first gRNA targeting domain . a plurality of modified CD34+ or hematopoietic stem cells;
one or more of said plurality of modified cells comprising an indel in the HBG gene promoter;
A first population of modified cells, wherein 60% or more of said indels in said plurality of modified cells as a whole are deletions of at least 4 base pairs;
wherein the indel is a first RNP complex comprising a first gRNA and a Cpf1 RNA-guided nuclease or a modified Cpf1 RNA-guided nuclease to a first population of unmodified cells comprising a plurality of unmodified CD34+ cells or hematopoietic stem cells; A first population of modified cells produced by delivery, wherein said first gRNA comprises a first gRNA targeting domain. Claims 1-, wherein 25% or more of said indels in said plurality of modified cells as a whole are deletions of at least 4 base pairs and are introduced by a repair mechanism other than microhomology-mediated end joining (MMEJ) repair. 6. A first population of modified cells according to any one of 5. 6. Any one of claims 1-5, wherein 25% or more of said indels in said plurality of modified cells as a whole are deletions of at least 4 base pairs and are introduced by non-homologous end joining (NHEJ) repair. A first population of modified cells according to paragraph. 8. The first modified cell of any one of claims 1-7, wherein 50% or more of said indels in said plurality of modified cells as a whole are deletions of between 1 and 25 base pairs. a group of 8. The first modified cell of any one of claims 1-7, wherein 50% or more of said indels in said plurality of modified cells as a whole are deletions of between 3 and 25 base pairs. a group of 8. The first modified cell of any one of claims 1-7, wherein 50% or more of said indels in said plurality of modified cells as a whole are deletions of between 4 and 25 base pairs. a group of 8. The first modified cell of any one of claims 1-7, wherein 50% or more of said indels in said plurality of modified cells as a whole are deletions of between 5 and 25 base pairs. a group of The plurality of modified cells as a whole
HBG1/2c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -104 to -121 deletions, wherein the HBG1/2c. -104 to -121 deletions collectively constitute 2% or more of the indels in the plurality of modified cells;
HBG1/2c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -110 to -115 deletion (the HBG1/2c. -110 to -115 deletion constitutes 2% or more of the indels in the plurality of modified cells as a whole). A first population of modified cells according to any one of 1-11. The plurality of modified cells as a whole
HBG1/2c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -104 to -121, wherein said HBG1/2c. -104 to -121 deletions collectively constitute 1% to 15.5% of said indels in said plurality of modified cells;
HBG1/2c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -110 to -115, wherein said HBG1/2c. -110 to -115 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG110/115 in HBG1 promoter, HBG2 promoter, or combinations thereof c. -112 to -115, wherein said HBG1/2c. -110 to -115 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/115c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -113 to -115, wherein said HBG1/2c. -113 to -115 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/115c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -111 to -115, wherein said HBG1/2c. -111 to -115 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/117c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -111 to -117, wherein said HBG1/2c. -111 to -117 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/114c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -102 to -114, wherein said HBG1/2c. -102 to -114 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/118c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -114 to -118, wherein said HBG1/2c. -114 to -118 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells;
HBG2/116c in the HBG1 promoter, HBG2 promoter, or combinations thereof. -112 to -116, wherein said HBG1/2c. -112 to -116 deletions collectively constitute 1% to 6% of said indels in said plurality of modified cells, and HBG2/117c in HBG1 promoter, HBG2 promoter, or combinations thereof . -113 to -117, wherein said HBG1/2c. 13. The method of any one of claims 1-12, wherein the deletion of -113 to -117 comprises deletions that collectively constitute 1% to 6% of said indels in said plurality of modified cells. A first population of modified cells. 14. The first modified cell of any one of claims 1-13, wherein said plurality of modified cells as a whole comprises 108 deletions present in all 14 samples set forth in Figure 56A. a group of 14. The modified cells of any one of claims 1-13, wherein the plurality of modified cells as a whole comprises indels identified in Table 25 as having an average % in indels of 0.1% or greater. First group. 16. The first population of modified cells of any one of claims 1-15, wherein said plurality of modified cells as a whole comprises an indel listed in Table 25. said plurality of modified cells as a whole comprises at least 4 base pair deletions that are at least 10% greater than a second population of modified cells comprising a plurality of modified CD34+ or hematopoietic stem cells;
one or more modified cells in the second population of modified cells have an indel in the HBG gene promoter;
wherein said indels in said second population of modified cells transduce a second RNP complex comprising a second gRNA and a Cas9 RNA-guided nuclease into a second population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells; 17. The first modified cell of any one of claims 1-16, wherein said second gRNA comprises a second gRNA targeting domain comprising SEQ ID NO:339, produced by delivery to a population. group. said plurality of modified cells as a whole comprises at least 10% more deletions of at least 4 base pairs introduced by the NHEJ repair machinery than a second population of modified cells comprising a plurality of modified CD34+ or hematopoietic stem cells;
one or more modified cells in the second population of modified cells comprise an indel at the HBG gene promoter;
wherein said indels in said second population of modified cells transduce a second RNP complex comprising a second gRNA and a Cas9 RNA-guided nuclease into a second population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells; 18. The first modified cell of any one of claims 1-17, wherein said second gRNA comprises a second gRNA targeting domain comprising SEQ ID NO:339, produced by delivery to a population. group. 19. The first population of modified cells of any one of claims 4-18, wherein said first RNP complex is delivered to said first population of unmodified cells by electroporation. 19. The first population of modified cells of claim 17 or 18, wherein said second RNP complex is delivered to said second population of unmodified cells by electroporation. 21. The first population of modified cells of any one of claims 1-20, wherein the first population of unmodified cells is from a subject with sickle cell disease. 19. The first population of modified cells of claim 17 or 18, wherein said second population of unmodified cells is from a subject with sickle cell disease. 23. The first population of modified cells of any one of claims 4-22, wherein the first population of modified cells comprises higher HbF levels than the first population of unmodified cells. 19. The first population of modified cells of claim 17 or 18, wherein said first population of modified cells comprises higher HbF levels than said second population of modified cells. 25. Any of claims 4-24, wherein the first gRNA comprises 5' and 3' ends, a DNA extension at the 5' end, and a 2'-O-methyl-3'-phosphorothioate modification at the 3' end. A first population of modified cells according to paragraph 1. 26. The first population of modified cells of claim 25, wherein said DNA stretches comprise sequences set forth in SEQ ID NOS:1235-1250. 27. The first population of modified cells of any one of claims 4-26, wherein said first gRNA targeting domain comprises SEQ ID NO:1254. 28. The first population of modified cells of any one of claims 4-27, wherein said gRNA comprises SEQ ID NO:1051. 29. The first population of modified cells of any one of claims 4-28, wherein said modified Cpf1 comprises SEQ ID NO:1097. 30. The first population of modified cells of any one of claims 1-29, wherein said indel in the HBG gene promoter is within the CCAAT box target region. A method of inducing expression of fetal hemoglobin (HbF) in a first population of engineered cells comprising a plurality of engineered CD+34 or hematopoietic stem cells, said method comprising a first induction RNA (gRNA) and a Cpf1 RNA induction delivering a first RNP complex comprising a nuclease or a modified Cpf1 RNA-inducing nuclease to a first population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells to generate indels; 1 gRNA comprises a first gRNA targeting domain;
each modified CD34+ or hematopoietic stem cell contains an indel in the HBG gene promoter;
60% or more of said indels in said plurality of modified cells as a whole are deletions of at least 4 base pairs;
The method, wherein the first population of modified cells comprises higher HbF levels than the first population of unmodified cells. 32. A method of inducing expression of HbF according to claim 31, wherein said first population of modified cells comprises the first population of modified cells according to any one of claims 1-30. A method of reducing sickling in a first population of red blood cells (RBCs) cultured from a first population of modified cells comprising a plurality of modified CD34+ cells from a subject with sickle cell disease, said method but,
a) delivering a first RNP complex comprising a first gRNA and a Cpf1 RNA-guided nuclease or a modified Cpf1 RNA-guided nuclease to a first population of unmodified cells comprising a plurality of unmodified CD34+ cells from the subject; , generating an indel; and
b) culturing a first population of RBCs from said first population of cells comprising said plurality of modified CD34+ cells;
wherein said first gRNA comprises a first gRNA targeting domain, each modified CD34+ cell contains an indel at the HBG gene promoter, and 60% or more of said indels in said plurality of modified CD34+ cells as a whole are a deletion of at least 4 base pairs;
wherein said first population of RBCs exhibits significantly reduced sickle cells upon deoxygenation than a second population of RBCs comprising a plurality of RBCs cultured from said first population of unmodified cells. . 34. The method of reducing sickling of claim 33, wherein the first population of RBCs sickles at a significantly lower partial pressure of oxygen than the second population of RBCs. 35. The method of reducing sickling of claim 34, wherein the oxygen partial pressure is measured by relative oxygen pressure. 36. A method of reducing sickling according to any one of claims 33 to 35, wherein the first population of RBCs has a significantly higher minimum elongation index upon deoxygenation than the second population of RBCs. . Claims 33-35, wherein said first population of RBCs has a significantly faster rate upon deoxygenation than a second population of RBCs comprising a plurality of RBCs cultured from said first population of unmodified cells. A method of reducing sickling according to any one of the preceding claims. 36. Any one of claims 33-35, wherein the first population of RBCs comprises a higher HbF level than a second population of RBCs comprising a plurality of RBCs cultured from the first population of unmodified cells. 13. A method of reducing sickling according to . Reducing sickling according to any one of claims 33-38, wherein said first population of modified cells comprises a first population of modified cells according to any one of claims 1-30 Method. a)
delivering a first RNP complex comprising a first gRNA and a Cpf1 RNA-guided nuclease or a modified Cpf1 RNA-guided nuclease to a first population of unmodified cells comprising a plurality of unmodified CD34+ or hematopoietic stem cells to induce indels; a step of generating;
b) administering to said subject a first population of modified cells comprising said plurality of modified CD34+ or hematopoietic stem cells to alleviate one or more symptoms of sickle cell disease; A method of alleviating one or more symptoms of sickle cell disease in a subject, comprising:
The method, wherein each modified CD34+ or hematopoietic stem comprises an indel at the HBG gene promoter, and 60% or more of said indels in said plurality of modified CD34+ or hematopoietic stem cells as a whole are deletions of at least 4 base pairs. detecting a population of modified red blood cell progeny cells comprising a plurality of modified red blood cell progeny cells cultured from the first population of modified cells at one or more time periods between one week and five years after administration. 41. The method of claim 40, further comprising: 42. The method of claim 40 or 41, wherein said method results in long-term engraftment of multiple HSC clones in bone marrow. 43. The method of any of claims 40-42, wherein said method results in long-term expression of at least 50% of total hemoglobin compared to healthy subjects. 44. The method of any one of claims 40-43, wherein said first population of modified cells comprises the first population of modified cells of any of claims 1-30.

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