Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

The Clinical and Laboratory Profiles of Immunocompetent Patients With Short-Duration Fever With Neutropenia in a Tertiary Care Hospital in Pune, India.

Background Management of a febrile patient is based on understanding the pathophysiology of an abnormal temperature and temperature regulation, impacts of fever, and its treatment. In the current study, we aimed to characterize and compare the epidemiological, etiologic, microbiological, serological, clinical, and outcome traits of febrile patients with acute neutropenia admitted to a tertiary care center in Western Maharashtra. Methods Adult patients with a history of fever of less than two weeks' duration and without any immunosuppressive state were screened with predefined inclusion and exclusion criteria. General and demographic information (age and gender), and clinical examinations (type and duration of fever) were recorded. Biochemical, hematologic (total and differential cell counts), and immunologic measurements (rapid malaria, dengue, Leptospira, and viral hepatitis antigen antibodies) were performed. Data were analyzed using an appropriate statistical package. Results A total of 403 (214 males) young adults (aged: 29±11 years) with clinical presentation of fever were studied. The majority (n=361, 89.6%) had low-grade continuous fever with an average duration of 3±1 (mean±standard deviation (SD)) days. Headache and myalgia were the common symptoms present, and patients had an average hospital stay of 4±1 days. Dengue (55%) was the most common cause of febrile neutropenia, and all patients recovered well without antibiotics and granulocyte colony-stimulating factor. The mean C-reactive protein (CRP) level was 61.4±4.4 mg/L. CRP and procalcitonin (PCT) were directly correlated with the degree of neutropenia and inversely correlated with total leucocyte count (TLC). Conclusions It was highlighted from this study that antibiotics are not necessary for viral infections that have been diagnosed to stop the development of secondary bacterial infections. A clinician should be aware of "when not to use antibiotics," or the world will soon have to deal with superbugs.

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the ß-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.