ASH 2020: CRISPR-Cas9 Gene-Editing Technique May Cure Sickle Cell Disease and Beta Thalassemia

SUMMARY: Sickle Cell Disease or Sickle Cell anemia is an Autosomal Recessive disorder and affects approximately 100,000 Americans. It is estimated that it affects 1 out of every 365 African-American births and 1 out of every 16,300 Hispanic-American births. The average life expectancy for patients with Sickle Cell Disease in the United States is approximately 40-60 years. Beta thalassemia affects at least 1000 Americans and according to the WHO, more than 300,000 babies are born worldwide each year with hemoglobin disorders such as Transfusion-Dependent beta-Thalassemia (TDT) and Sickle Cell Disease (SCD). Both diseases are caused by mutations in the hemoglobin beta-globin gene.

HbSS disease or Sickle Cell anemia is the most common Sickle Cell Disease genotype and is associated with the most severe manifestations. HbSS disease is caused by a mutation substituting thymine for adenine in the sixth codon of the beta-globin chain gene. This in turn affects the hemoglobin’s ability to carry oxygen and causes it to polymerize. This results in decreased solubility thereby distorting the shape of the red blood cells, increasing their rigidity and resulting in red blood cells that are sickle shaped rather than biconcave. These sickle shaped red blood cells limit oxygen delivery to the tissues by restricting the flow in blood vessels, leading to severe pain and organ damage (Vaso-Occlusive Crises). Oxidative stress is an important contributing factor to hemoglobin polymerization with polymer formation occurring only in the deoxy state. HbS/b-0 Thalassemia (double heterozygote for HbS and b-0 Thalassemia) is clinically indistinguishable from HbSS disease. Thalassemia is an inherited hemoglobinopathy associated with an erythroid maturation defect and is characterized by ineffective erythropoiesis and impaired RBC maturation. Mutations in the hemoglobin beta-globin gene result in reduced (B+) or absent (B0) beta-globin synthesis creating an imbalance between the alpha and beta globin chains of hemoglobin, resulting in ineffective erythropoiesis. Management of Sickle Cell Disease includes pain control, transfusion support and Hydroxyurea, whereas management of beta Thalassemia include transfusion support and iron chelation therapy. None of the presently available therapies addresses the underlying cause of these diseases nor do they fully ameliorate disease manifestations. Allogeneic bone marrow transplantation can cure both these genetic disorders, but less than 20% of eligible patients have a related HLA-matched donor. There is therefore a great unmet need to find new therapies for beta-Thalassemia and Sickle Cell Disease.

Fetal hemoglobin which consists of two alpha and two gamma chains is produced in utero, but the level of gamma-globulin decreases postnatally as the production of beta-globin and adult hemoglobin which consists of two alpha and two beta chains increases. It has been noted that elevated levels of fetal hemoglobin are associated with decreased morbidity and mortality in patients with Sickle Cell Disease and Thalassemia. BCL11A gene is a repressor of gamma-globin expression and fetal hemoglobin production in adult red blood cells. Downregulating BCL11A can therefore reactivate gamma-globin expression and increase fetal hemoglobin in RBC.CRISPR-Cas9-Nuclease-Gene-Editing-Technique

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease gene editing technique cuts the DNA at the targeted location. The authors in this study used this gene-editing technique in Hematopoietic Stem and Progenitor Cells at the erythroid-specific enhancer region of BCL11A to down-regulate BCL11A expression in erythroid-lineage cells, restore gamma-globin synthesis, and reactivate production of fetal hemoglobin.

The authors reported the interim safety and efficacy data from 10 patients who received the investigational CRISPR-Cas9 nuclease gene-editing based therapy, following enrollment in CLIMB THAL-111 and CLIMB SCD-121 studies. These patients were infused with CTX001 (autologous CRISPR-Cas9-edited CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) that were genetically edited to reactivate the production of fetal hemoglobin. In the CLIMB THAL-111 and CLIMB SCD-121 open-label, PhaseI/II trials, patients with Transfusion-Dependent beta-Thalassemia and sickle cell disease , respectively, received a single intravenous infusion of CTX001. The production of CTX001 involved collection of CD34+ Hematopoietic Stem and Progenitor Cells (HSPCs) from patients by apheresis, following stem cell mobilization with either NEUPOGEN filgrastim and/or MOZOBIL® (plerixafor), after a minimum of 8 weeks of transfusions of packed red cells, to achieve a level of sickle hemoglobin of less than 30% in the patient with SCD. CTX001 was then manufactured from these CD34+ cells by editing with CRISPR-Cas9 with the use of a single-guide RNA molecule, following preclinical studies of BCL11A editing. Patients received myeloablation with pharmacokinetically adjusted, single-agent Busulfan, before the infusion of CTX001.

Eligible patients were between ages 18 and 35 years. In the CLIMB THAL-111 trial, eligible patients had a diagnosis of beta-Thalassemia (including the hemoglobin E genotype) with either homozygous or compound heterozygous mutations and had received transfusions of PRBC consisting of at least 100 ml/kg of body weight (or 10 units) per year during the previous 2 years. In the open-label CLIMB SCD-121 trial, eligible patients had a documented BS/BS or BS/B0 genotype and had a history of two or more severe vaso-occlusive episodes per year during the previous 2 years. Patients were monitored for engraftment, adverse events, total hemoglobin, hemoglobin fractions on high-performance liquid chromatography, F-cell expression (defined as the percentage of circulating erythrocytes with detectable levels of fetal hemoglobin), laboratory signs of hemolysis, requirements for transfusion support with PRBC, and occurrence of vaso-occlusive episodes in the patient with SCD. Bone marrow aspirates were obtained at 6 and 12 months after infusion, and DNA sequencing was used to measure the fraction of total DNA that was edited at the on-target site in CD34+ bone marrow cells and in nucleated peripheral-blood cells.

The Primary endpoint of the CLIMB THAL-111 trial was the proportion of patients with a transfusion reduction of 50% for at least six months, starting three months after CTX001 infusion. The Primary endpoint of CLIMB SCD-121 Sickle Cell Disease trial was the proportion of patients with fetal hemoglobin of 20% or more, sustained for at least three months, starting six months after CTX001 infusion.

CLIMB THAL-111 trial: Data was reported on 7 patients enrolled in the CLIMB THAL-111 trial, as they had reached at least three months of follow up after CTX001 infusion and therefore could be assessed for initial safety and efficacy. All seven showed a similar pattern of response, with rapid and sustained increases in total hemoglobin, fetal hemoglobin, and transfusion independence at last analysis. All 7 patients were transfusion independent with follow up ranging from 3-18 months after CTX001 infusion, with normal to near normal total hemoglobin levels at last visit. Their total hemoglobin levels ranged from 9.7 to 14.1 g/dL, and fetal hemoglobin ranged from 40.9% to 97.7%. Bone marrow allelic editing data collected from 4 patients with 6 months of follow up, and from one patient with 12 months of follow-up after CTX001 infusion showed the treatment resulted in a durable response. The safety data from all seven patients were generally consistent with an Autologous Stem Cell Transplant (ASCT) and myeloablative conditioning. There were four Serious Adverse Events (SAEs) considered related or possibly related to CTX001 reported in one patient and included headache, Hemophagocytic LymphoHistiocytosis (HLH), Acute Respiratory Distress Syndrome, and Idiopathic Pneumonia Syndrome. All four SAEs occurred in the context of HLH and resolved. Most of the non-SAEs were considered mild to moderate. CLIMB-111 is an ongoing trial and will enroll up to 45 patients and follow patients for approximately two years after infusion.

CLIMB SCD-121: Data was reported on 3 patients enrolled in the CLIMB SCD-121 sickle cell disease trial as they had reached at least three months of follow up after CTX001 infusion, and therefore could be assessed for initial safety and efficacy. Again, all 3 patients showed a similar pattern of response, with rapid and sustained increases in total hemoglobin and fetal hemoglobin, as well as elimination of Vaso-Occlusive Crises through last analysis. All 3 patients remained Vaso Occlusive Crises-free with follow up ranging from 3-15 months after CTX001 infusion and had hemoglobin levels in the normal to near normal range, including total hemoglobin from 11.5 to 13.2 g/dL and Fetal hemoglobin levels from 31.3% to 48.0%. Bone marrow allelic editing data collected from one patient with six months of follow-up and from one patient with 12 months of follow-up after CTX001 infusion demonstrated a durable response. Again the safety data were consistent with an ASCT and myeloablative conditioning. There were no Serious Adverse Events noted, thought to be related to CTX001, and the majority of non-SAEs were considered mild to moderate. CLIMB-121 is an ongoing open-label trial and will enroll up to 45 patients and follow patients for approximately two years after infusion.

It was concluded from this initial follow up that, CTX001 manufactured from Hematopoietic Stem Cells, edited of BCL11A with CRISPR-Cas9, has shown durable engraftment, with high levels of fetal hemoglobin expression, and the elimination of vaso-occlusive episodes or need for transfusion. The authors added that these preliminary results support further testing of CRISPR-Cas9 gene-editing approaches to treat other genetic diseases.

Safety and Efficacy of CTX001 in Patients with Transfusion-Dependent β- Thalassemia and Sickle Cell Disease: Early Results from the Climb THAL-111 and Climb SCD-121 Studies of Autologous CRISPR-CAS9–Modified CD34+ Hematopoietic Stem and Progenitor Cells. Frangoul H, Bobruff Y, Cappellini MD, et al. Presented at the 62nd ASH Annual Meeting and Exposition, 2020. Abstract#4

ADAKVEO® (Crizanlizumab-tmca)

The FDA on November 15, 2019 approved ADAKVEO® to reduce the frequency of Vaso-Occlusive Crises (VOCs) in adults and pediatric patients aged 16 years and older with Sickle Cell disease. ADAKVEO® is a product of Novartis Pharmaceuticals Corporation.

FDA Approves ADAKVEO®, A New Targeted Therapy for Sickle Cell Disease

SUMMARY: The FDA on November 15, 2019 approved ADAKVEO® (Crizanlizumab-tmca), a treatment to reduce the frequency of vaso-occlusive crisis, for patients age 16 years and older. Vaso-occlusive crisis is a common and painful complication of Sickle Cell Disease (SCD), that occurs when blood circulation is obstructed by sickled red blood cells. Sickle Cell Disease or Sickle Cell anemia is an Autosomal Recessive disorder and affects approximately 100,000 Americans. It is estimated that it affects 1 out of every 365 African-American births and 1 out of every 16,300 Hispanic-American births. The average life expectancy for patients with Sickle Cell Disease in the United States is approximately 40-60 years.

HbSS disease or Sickle Cell anemia is the most common Sickle Cell Disease genotype and is associated with the most severe manifestations. HbSS disease is caused by a mutation substituting thymine for adenine in the sixth codon of the beta-globin chain gene. This in turn affects the hemoglobin’s ability to carry oxygen and causes it to polymerize. This results in decreased solubility thereby distorting the shape of the red blood cells, increasing their rigidity and resulting in red blood cells that are sickle shaped rather than biconcave. These sickle shaped red blood cells limit oxygen delivery to the tissues by restricting the flow in blood vessels, leading to severe pain and organ damage (vaso-occlusive crises). Oxidative stress is an important contributing factor to hemoglobin polymerization with polymer formation occurring only in the deoxy state. HbS/b-0 thalassemia (double heterozygote for HbS and b-0 thalassemia) is clinically indistinguishable from HbSS disease.

P-Selectin is a Cell Adhesion Molecule (CAM) expressed on the surfaces of activated vascular endothelial cells and platelets. In unactivated endothelial cells and platelets, it is stored in Weibel-Palade bodies and alpha granules respectively. P-Selectin is released from endothelial cells and platelets when activated by inflammation or trauma and mediates the binding of erythrocytes and leukocytes to the vessel wall. In patients with Sickle Cell Disease (SCD), adherent masses of sickled red cells and leukocytes contribute to vaso-occlusive pain crises. ADAKVEO® is a first-in-class humanized anti-P-Selectin antibody, and the SUSTAIN study evaluated the safety and efficacy of ADAKVEO® on the frequency of Sickle Cell-related Pain Crises (SCPC) in Sickle Cell Disease patients.

The present FDA approval of ADAKVEO® was based on SUSTAIN, a multicenter, randomized, placebo controlled, double-blind clinical trial in which 198 Sickle Cell Disease patients with a history of vaso-occlusive crisis, were randomly assigned to receive ADAKVEO® at doses of 5 mg/kg, 2.5 mg/kg, or placebo, administered intravenously, 14 times over 52 weeks. Treatment groups were well balanced and patients receiving Hydroxyurea or Erythropoietin were included, if prescribed for the preceding 6 months and dose was stable for at least 3 months. The Primary end point was Sickle Cell-related Pain Crises (SCPC), defined as acute sickle cell-related pain that resulted in a visit to a medical facility and required a parenteral or oral narcotic or parenteral NSAID. Acute Chest Syndrome (ACS), priapism, hepatic and splenic sequestration were also included in this definition.

Treatment with ADAKVEO® experienced fewer health care visits for vaso-occlusive crisis annually and significantly lowered the median annual rate of vaso-occlusive crisis by 45% from 2.98 visits to 1.63 visits, compared with placebo. Reductions in the frequency of vaso-occlusive crisis were observed among patients regardless of Sickle Cell Disease genotype and/or Hydroxyurea use. More than one third (36%) of patients who received ADAKVEO® did not experience vaso-occlusive crisis during the study, compared with 17% of placebo-treated patients. ADAKVEO® delayed the time that patients first experienced vaso-occlusive crisis after starting treatment from a median of 1.4 months to 4.1 months. Common side effects associated with ADAKVEO® included back pain, nausea, fever and arthralgia. Patients should be monitored for infusion-related reactions and treatment should be discontinued for severe reactions. Patients should also be monitored for interference with automated platelet counts or platelet clumping and it is advised that CBC be performed using citrate tubes to avoid platelet activation.

It was concluded that ADAKVEO® significantly reduced Sickle Cell-related Pain Crises (SCPC) and increased the time to first and second SCPC. The authors added that chronic inhibition of P-Selectin with once a month IV dosing of ADAKVEO® represents a novel and potentially new disease-modifying, prophylactic treatment option for patients with Sickle Cell Disease. SUSTAIN: A Multicenter, Randomized, Placebo-Controlled, Double-Blind, 12-Month Study to Assess Safety and Efficacy of SelG1 with or without Hydroxyurea Therapy in Sickle Cell Disease Patients with Sickle Cell-Related Pain Crises. Ataga KI, Kutlar A, Kanter J, et al. N Engl J Med 2017; 376:429-439

SIKLOS® (Hydroxyurea)

The FDA on December 21, 2017 granted regular approval to SIKLOS® to reduce the frequency of painful crises and the need for blood transfusions in pediatric patients from 2 years of age and older with sickle cell anemia with recurrent moderate to severe painful crises. SIKLOS® is a product of Addmedica.

FDA Approves ENDARI® for Sickle Cell Disease

The FDA on July 7, 2017 approved ENDARI® (L-Glutamine oral powder) for oral administration to reduce the acute complications of Sickle Cell disease, in adult and pediatric patients 5 years and older. There is a higher L-glutamine utilization in Sickle Cell Anemia resulting in its depletion and thereby contributing to oxidative stress. This oxidative stress is an important contributing factor to hemoglobin polymerization, with polymer formation occurring only in the deoxy state. ENDARI® is the first treatment approved for patients with Sickle Cell disease in almost 20 years.

FDA Approves ENDARI®, A New Treatment for Sickle Cell Disease

SUMMARY: The FDA on July 7, 2017 approved ENDARI® (L-Glutamine oral powder) for patients age five years and older with Sickle Cell disease to reduce severe complications associated with the blood disorder. Sickle cell disease or Sickle Cell anemia is an Autosomal Recessive disorder and affects approximately 100,000 Americans. It is estimated that it affects 1 out of every 365 African-American births and 1 out of every 16,300 Hispanic-American births. The average life expectancy for patients with Sickle Cell disease in the United States is approximately 40 to 60 years.

HbSS disease or Sickle Cell anemia is the most common Sickle Cell disease genotype and is associated with the most severe manifestations. HbSS disease is caused by a mutation substituting thymine for adenine in the sixth codon of the beta-globin chain gene. This in turn affects the hemoglobin’s ability to carry oxygen and causes it to polymerize. This results in decreased solubility thereby distorting the shape of the red blood cells, increasing their rigidity and resulting in red blood cells that are sickle shaped rather than biconcave. These sickle shaped red blood cells limit oxygen delivery to the tissues by restricting the flow in blood vessels, leading to severe pain and organ damage (vaso-occlusive crises). Oxidative stress is an important contributing factor to hemoglobin polymerization with polymer formation occurring only in the deoxy state. HbS/b-0 thalassemia (double heterozygote for HbS and b-0 thalassemia) is clinically indistinguishable from HbSS disease.

L-glutamine is a precursor for the synthesis of essential metabolic Oxidation-Reduction cofactors including Nicotinamide Adenine Dinucleotide (NAD). It has been shown in previous studies that there is higher L-glutamine utilization in Sickle Cell Anemia resulting in its depletion and thereby contributing to oxidative stress. Based on a phase II study showing favorable outcomes with ENDARI® compared with placebo, a phase III, randomized trial was conducted, in which the safety and efficacy of ENDARI® was studied in 230 Sickle Cell disease or beta-0 thalassemia patients, who had at least two episodes of painful crises during the 12 months before screening. Patients were randomized in a 2:1 ratio to receive ENDARI® (N=152) or placebo (N=78). Enrolled patients were 5-58 yrs old and ENDARI® was administered orally at 0.3 mg/kg/day for 48 weeks followed by a 3 week tapering period. Two thirds of the patients were on Hydroxyurea. The effect of treatment was evaluated over 48 weeks.

Patients who were treated with ENDARI® experienced fewer hospital visits for Sickle Cell crises pain management with parenteral narcotics or Ketorolac compared to those who received a placebo, fewer hospitalizations for Sickle Cell pain , and fewer days in the hospital (median 6.5 days versus median 11 days) compared to those on placebo. Further, patients who received ENDARI® also had fewer occurrences of acute chest syndrome (a life-threatening complication of sickle cell disease), compared with patients who received a placebo (8.6% versus 23.1%). The common side effects of ENDARI® included, nausea, constipation, headache, abdominal pain, cough, pain in the extremities, back pain and chest pain.

It was concluded that the benefit with ENDARI® for patients with Sickle Cell disease, was seen in all age groups and there was a consistent advantage with ENDARI® regardless of whether the patient was on Hydroxyurea or not. ENDARI® is the first treatment approved for patients with Sickle Cell disease in almost 20 years. Phase 3 Study of L-Glutamine Therapy in Sickle Cell Anemia and Sickle ß0-Thalassemia Subgroup Analyses Show Consistent Clinical Improvement. Niihara Y, Viswanathan K, Miller ST, et al. Abstarct#1318. Presented at ASH 58th Annual Meeting & Exposition, San Diego, CA. December 3-6, 2016