Query: gene therapy for sickle cell crispr

title: CRISPR/Cas9 Gene Edited Hematopoietic Stem Cell Therapy for Sickle Cell Disease
abstract: Sickle cell disease (SCD) is a life threatening hereditary disorder that affects thirty million individuals worldwide. The disease results from a single amino acid change in the b globin gene, which causes polymerization of hemoglobin and deformation of red blood cells, leading to vaso occlusion, severe pain crisis, multi organ dysfunction, and ultimately reduction of lifespan in affected individuals. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for SCD, yet its success is limited by donor availability and high risk of graft versus host disease. Increasing expression of fetal g globin in adults is an accepted therapeutic strategy for treating SCD. Supporting this approach are GWAS studies that have identified genetic loci, including within the BCL11A gene, that are associated with elevated fetal hemoglobin (HbF) and mitigated disease severity. Leveraging CRISPR/Cas9 genome editing technology, we aim to develop a safe and curative therapy by autologous, genome modified HSCT leading to sustained generation of HbF containing, functional red blood cells. Guide RNAs (gRNAs) targeting the erythroid specific enhancer region of the BCL11A gene were assayed in CD34+ hematopoietic stem and progenitor cells (HSPCs) and one gRNA was selected for in vivo evaluation based on editing efficiency and functional potency. We achieved approximately 80% target site editing in human CD34+ HSPCs and 65% in CD34+CD38 CD90+CD45RA CD49f+ defined LT HSCs. Potential off target editing sites were identified by two approaches: in silico prediction and unbiased oligo insertion method. These sites were then directly interrogated in HSPCs by targeted PCR amplification and NGS analysis, and to date our selected gRNA had zero validated off target sites. In addition, HSPC composition was not altered following electroporation of ribonucleoprotein complex, and cells were capable of in vitro expansion and multi lineage differentiation as measured by CFU assays. Upon in vitro differentiation into the erythroid lineage, edited cells demonstrated ~40% reduction in BCL11A mRNA with a corresponding two fold increase in g globin transcript, translating to a 30 40% increase in HbF positive cells. Most importantly, when CD34+ cells derived from SCD patients were gene edited and differentiated into erythroid cells, a similar editing efficiency and increase in HbF positive cells was observed. To evaluate whether gene editing alters the self renewal, engraftment or lineage differentiation capacity of HSCs, gene edited HSPCs were transplanted into NOD scid IL2rgnull (NSG) mice to assess reconstitution of the hematopoietic system. Mice displayed 30 40% human CD45+ cell engraftment and multi lineage reconstitution at 16 weeks following transplantation. When long term engrafted bone marrow cells were harvested from the recipients and differentiated into erythroid cells in vitro, gene edited bone marrow cells produced significantly higher numbers of HbF positive cells compared to mock edited bone marrow cells. These data support the development of autologous genome modified HSPC transplantation as a novel cell therapy for SCD. Disclosures Yu: Novartis Institutes for BioMedical Research: Employment.  Novartis Institutes for BioMedical Research: Employment.  Novartis Institutes for BioMedical Research:  Novartis:   Novartis: Employment.  Novartis Institutes for BioMedical Research: Employment.  Novartis Institutes for BioMedical Research:  Novartis Institutes for BioMedical Research: Employment.  Novartis:  Novartis:  Novartis:     Employment.  Novartis:  Novartis:  Novartis: Employment.

title: Gene therapy for sickle cell disease: An update.
abstract: Sickle cell disease (SCD) is one of the most common life threatening monogenic diseases affecting millions of people worldwide. Allogenic hematopietic stem cell transplantation is the only known cure for the disease with high success rates, but the limited availability of matched sibling donors and the high risk of transplantation related side effects force the scientific community to envision additional therapies. Ex vivo gene therapy through globin gene addition has been investigated extensively and is currently being tested in clinical trials that have begun reporting encouraging data. Recent improvements in our understanding of the molecular pathways controlling mammalian erythropoiesis and globin switching offer new and exciting therapeutic options. Rapid and substantial advances in genome engineering tools, particularly CRISPR/Cas9, have raised the possibility of genetic correction in induced pluripotent stem cells as well as patient derived hematopoietic stem and progenitor cells. However, these techniques are still in their infancy, and safety/efficacy issues remain that must be addressed before translating these promising techniques into clinical practice.

title: In Vivo HSC Gene Therapy Using a Bi modular HDAd5/35+ Vector Cures Sickle Cell Disease in a Mouse Model.
abstract: We have recently reported that, after in vivo hematopoietic stem cell/progenitor (HSPC) transduction with HDAd5/35+ vectors, SB100x transposase mediated g globin gene addition achieved 10% 15% g globin of adult mouse globin, resulting in significant but incomplete phenotypic correction in a thalassemia intermedia mouse model. Furthermore, genome editing of a g globin repressor binding site within the g globin promoter by CRISPR Cas9 results in efficient reactivation of endogenous g globin. Here, we aimed to combine these two mechanisms to obtain curative levels of g globin after in vivo HSPC transduction. We generated a HDAd5/35+ adenovirus vector (HDAd combo) containing both modules and tested it in vitro and after in vivo HSPC transduction in healthy CD46/b YAC mice and in a sickle cell disease mouse model (CD46/Townes) Compared to HDAd vectors containing either the g globin addition or the CRISPR Cas9 reactivation units alone, in vivo HSC transduction of CD46/Townes mice with the HDAd combo resulted in significantly higher g globin in red blood cells, reaching 30% of that of adult human a and bS chains and a complete phenotypic correction of sickle cell disease.

title: Gene therapy in sickle cell disease: Possible utility and impact
abstract: CRISPR Cas9 makes it possible to edit the patient's own genes, but questions remain.

title: Gene therapy for sickle cell disease.
abstract: Sickle cell disease is a commonly occurring genetic disorder that can be diffi cult to manage and for which stem cell transplantation is the only potential curative therapy. It is estimated that around 25 million people worldwide have the disease, with 300 000 new patients born each year, the majority living in low income and middleincome countries. Although treatment can relieve pain and help manage symptoms, sickle cell disease can have a substantial burden on an individual's quality of life, particularly during pain crises. As a typically monogenetic disorder, sickle cell disease has been long considered a logical candidate for gene therapy. Substantial progress has been made towards this goal, although these techniques are still far from being ready to roll out to the everyday patient worldwide. Elizabeth Traxler and collaborators have used the CRISPR/Cas9 editing system to promote the expression of fetal haemoglobin in blood cells taken from patients with sickle cell disease. Since disorders such as sickle cell disease and b thalassaemia become symptomatic when haemoglobin expression switches from the fetal g globin to mutated adult b globin, promoting expression of the fetal form could potentially elevate the sickling phenotype of the cells. Traxler and colleagues showed that by mutating a 13 nucleotide sequence that is present in the promoters of the HBG1 and HBG2 genes, they could replicate a naturally occurring hereditary persistence of fetal haemoglobin associated mutation. Also aiming to increase fetal haemoglobin expression, but by diff erent means, Christian Brendel and colleagues reduced the expression of the fetal haemoglobin repressor BCL11A by lineage specifi c short hairpin RNA in mice. Other studies of gene therapy for sickle cell disease and b thalassaemia have attempted to insert a functional b globin into a patient's haemopoietic stem cells ex vivo via a lentivirus vector. The cells were then returned to patients by autologous stemcell transplantation. Early clinical trial data presented at last year's American Society of Hematology annual meeting indicated that for some patients, the introduced functional b globin was expressed and resulted in transfusion independence but not for all patients treated. Effi cient and safe delivery to target cells and tissues has been the long standing challenge to successful gene therapy strategies. Viral vectors are often used to deliver gene therapy components, however, in the case of CRISPR/Cas9 technology, this delivery could lead to persistent expression of the DNA cleaving enzyme Cas9, which could result in off target cleavage. A recent murine study by Hao Yin and colleagues used a mix of viral and non viral delivery methods to deliver gene therapy. The CRISPR guide RNA and genetic repair template were introduced via an adenovirus vector, which allowed them to be expressed over a prolonged period of time. Cas9 was delivered by a lipid nanoparticle, which allows for short term expression of Cas9, and could reduce the risk of potential off target cutting by the CRISPR/Cas9 system. Off target eff ects are a major concern for the CRISPR/Cas9 technology; mutations could occur when an unintended section of DNA is cleaved. Even if Cas9 expression is limited, off target binding by CRISPR can be a concern. No two people's genomes are identical, and methods to identify potential off target binding tend to be based on composite or reference genomes. These methods might indicate that there no off target binding sites, but this may not be the case in reality. Despite these concerns, the fi rst US clinical trial involving CRISPR technology has passed an initial safety review by the National Institutes of Health in June of this year. The trial, which will begin in 2017, pending approval from the US Food and Drug Administration and local ethics committees, will use CRISPR technology to edit out PD 1 and TCR from T cells from patients with multiple myeloma, melanoma, or sarcoma. In murine experiments, the CRISPR technique resulted in some residual PD 1 and TCR expression, but at low enough levels to make engineered T cells target cancer cells more eff ectively than those with full expression. These edited T cells will then be returned to the patient with the aim of targeting and clearing the cancer. Gene therapy might one day provide a cure for sickle cell disease, but the technology is still in very early clinical and preclinical stages. Nonetheless, there have also been advances in non gene therapy treatment of sickle cell disease, such as better pain management, prophylactic transfusions, hydroxyurea, and continued research into experimental drugs like anti sickling haemoglobin modifi ers. Until gene therapy reaches fruition, these treatments represent the best opportunity for improved global management of sickle cell disease. The Lancet Haematology For the study by ElizabethTraxler and colleagues see Nat Med 2016; 22: 987 90

title: A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta Thalassemia and Sickle Cell Disease
abstract: Beta thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with b thalassemia (b thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD) Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence specific nuclease to enhance correction of a specific HBB mutation by homology directed repair (HDR) is becoming straightforward, targeting various HBB mutations of b thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two b thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in b thal and SCD. Stem Cells Translational Medicine 2018;7:87 97

title: [Progress in Gene Therapy of Sickle Cell Disease Based on Hemoglobin F Review]
abstract: Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF) the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF 1, c MYB and SOX6, as well as the application of CRISPR Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for b like hemoglobin diseases, such as sickle cell disease and b thalassemia.

title: Hemoglobinopathy correction with CRISPR or not; gene therapy is the solution
abstract: Hemoglobin (Hb) disorders or hemoglobinopathies are groups of blood conditions involving inherited genetic diseases mostly as single gene autosomal recessive that lead to the formation of abnormal Hb structure or inadequate to no production of globin chains in Hbs. Disorders of Hb are a global concern since these diseases can cause severe morbidity and early mortality of the affected populations. Treatments vary between chemicals and molecular approaches. The most promising approach is Hb correction. However, the stability of the correction faces a big challenge along with safety concerns. It is worth noting that most of the inherited hemoglobinopathies share common clinical presentations and laboratory findings, although some have distinct features. Hemoglobinopathies with emphasis on recent advances in gene therapy targeting sickle cell disease and thalassemia are discussed in this review.

title: Cas9 AAV6 Gene Correction of Beta Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice
abstract: CRISPR/Cas9 mediated beta globin (HBB) gene correction of Sickle Cell Disease (SCD) patient derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9 based HBB correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin cluster SCD mouse model to study Cas9 AAV6 mediated HBB correction in functional HSCs within the context of autologous transplantation. We discover that long term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin A production can be achieved in vivo from HBB corrected HSCs following autologous transplantation. We observed a direct correlation between increased HBB corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

title: Targeted deletion of BCL11A gene by CRISPR Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease.
abstract: Hemoglobinopathies, such as b thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of b chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous b thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of b thalassemia and SCD. Using CRISPR Cas9 genome editing strategy, we deleted a 200bp genomic region within the human erythroid specific BCL11A (B cell lymphoma/leukemia 11A) enhancer in KU 812, KG 1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of g hemoglobin expression in K562 cells, but not in KU 812 and KG 1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR Cas9 as a precision genome editing tool for treating b thalassemia. In addition, our data indicate that KU 812 and KG 1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.