sickle cell anemia crispr
149943127
In vitro correction of hbb v6g mutation in lymphocyte cells of patients with sickle cell anemia using genome editing crispr cas9 technique
Bu arastirma Selcuk Universitesi Bilimsel Arastirma Projeleri Koordinatorlugu tarafindan proje (17202004) numarasi ile desteklenmistir.
Hasanain Akram Zainalbden Zainalbden
80323244
Target Site Mutagenesis during Crispr/ Cas 9/Single Stranded Oligonucleotide Directed Gene Editing for Sickle Cell Anemia
Introduction Sickle Cell Disease (SCD) results from a simple substitution of valine for glutamic acid at codon 6 in the b globin gene, resulting in an AaT transition in the third position. The mutation results in the production of hemoglobin HbS which differs from the normal HbAin that it tends to polymerize into long strands that deform the erythrocyte. While a variety of traditional treatment regimens or reagents, such as hydroxyurea and chronic transfusions have been used widely, these therapies are wrought with short and long term side effects that limit efficacy. There is great interest in the hypothesis that the repair of a single nucleotide is facilitated by the combined action of CRISPR/Cas 9 and single stranded oligonucleotides (ssODNs) could prove a significant therapeutic advance for sickle cell disease. CRISPR/Cas 9 induces a site specific double stranded break while the single stranded oligonucleotide provides a DNA template to improve the rate of accurate genetic correction. There is a great effort to understand off site mutagenesis caused by editing of DNA regions remote to the target sequence. It is critical that investigators understand the frequency and types of DNA alterations induced by the gene editing reaction and affecting the region surrounding the targeted nucleotide site (herein termed on site mutagenesis) Methods We targeted beta globin genes with a CRISPR/Cas9 system designed to cleave 2 bases to the 59side of the targeted (A) nucleotide (the PAM site is located 2 bases to the 39 side on the complimentary strand) coupled to the electroporation of a single stranded oligonucleotide (72 mer) designed to convert the wild type (A) to the mutant (T) nucleotide. To evaluate the rate of on site mutagenesis among individual alleles, we clonally expanded populations of edited K562 cells. We hypothesize that the evaluation of individual clones will permit a clear identification of intended and un intended on site DNA alteration. Allelic heterogeneity is analyzed using Tracking of Indels by DEcompositon (TIDE) methodology combined with Sanger sequencing. Results We isolated 26 clonal lines for continued expansion after evidence of CRISPR/Cas 9 plasmid uptake and activity. Sanger sequencing with TIDE analysis revealed that the DNA sequence surrounding the targeted base is altered significantly as a result of the CRISPR/Cas9 gene editing process. Twenty three percent of the clones contain at least one corrected allele but one hundred percent of the clones exhibited mutagenicity of the DNA sequence surrounding the targeted base. All clones analyzed displayed varying degrees of sequence alteration (Figure 1) Interestingly, one clone contained a DNA insertion homologous to a region of the delta globin gene, suggesting that gene editing of the beta globin gene may have been repaired, in part, by delta globin DNA. The sequence of the delta globin locus was not changed. Conclusions Taken together, our data suggest that combinatorial approaches to beta globin gene editing using CRISPR/Cas 9 and single stranded oligonucleotides induce significant onsite mutagenesis and potential genetic swapping between related members of the same gene family. These observations provide insight into the type of molecular activity that accompanies combinatorial gene editing, particularly surrounding the target site. This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM109021 Disclosures No relevant conflicts of interest to declare.
Dula Man Brett M Sansbury Pawel Bialk Kevin Bloh Edward Anders Kolb Eric B Kmiec
235516765
The Fabulous Impact of CRISPR Method in Sickle Cell Disease Treatment
Sickle cell diseases are the most prevalent monogenic blood diseases with complications such as severe end organ harm, pain, and reduced life expectancy. Dealing options for sickle cell diseases are inadequate, as there are just two FDA approved drugs to decrease acute manifestation. The only sickle cell diseases curative procedure is bone marrow transplantation, frequently from a harmonized, related donor. Ex vivo manipulation of autologous hematopoietic stem and progenitor cells and subsequent transplantation of genetically altered cells theoretically offer an everlasting therapy appropriate to all sickle cell anemia patients, regardless of the accessibility of fit donors and graft versus host disease. In this review, we emphasize applying CRISPR gene editing strategies for sickle cell anemia treatment, containing the genetic modification and rectification of sickle cell disease mutation in b globin and the stimulation of fetal hemoglobin to protect cells against sickling. We summarize the importance of stem cell manipulation to cure sickle cell anemia having likely lifetime efficacy for cell and gene therapies. HIGHLIGHTS* Sickle cell disease (SCD) is a type of monogenic blood disorders. The CRISPR/Cas9 technology can be used to treat SCD. RBC sickling is reversed by mutation correction or fetal hemoglobin induction.
Vida Ebrahimi Atieh Hashemi
236100580
Cell Based Therapy for Disease Using CRISPR
CRISPR has accelerated basic research and therapeutic development. Its modular nature means it can be adapted to study and potentially treat nearly any genetic disease, including major killers like cancer and sickle cell anemia. The potential for CRISPR to transform medicine with highly precise, potentially one off treatment options has given hope to those suffering from chronic and debilitating illness. Here we discuss some of the newest methods of using CRISPR in a clinical environment. In particular, we explore promising ex vivo and in vivo results, as well as active clinical trials.
Soren H Hough
90816092
343. Repair of the Sickle Cell Disease Mutation in Human Stem Cells Using CRISPR/Cas9 and Single Stranded Oligodeoxynucleotides
Sickle cell disease is a hereditary anemia caused by a homozygous point mutation in the b globin gene resulting in a glutamic acid to valine change at position 6 in the globin b chain. Association of the mutated b chain with a chain results in the formation of hemoglobin S, which polymerizes intracellularly thus causing erythrocyte sickling. One promising therapy relies on the transplantation of autologous hematopoietic stem cells transduced with a lentiviral vector encoding a wild type or mutant b globin gene. However, one of the main concerns associated with the use of recombinant retroviruses to deliver therapeutic genes in stem cells is the risk of insertional mutagenesis following semi random retroviral DNA integration. Alternatively, gene repair by homology directed repair (HDR) is recognized as a potentially less genotoxic approach to repair mutations. Although HDR is intrinsically inefficient in human cells, enzymes that create specific DNA double strand breaks can efficiently increase HDR frequency. Our long term goal is to develop an efficient HDR approach to repair the sickle cell anemia mutation in stem cells. We have previously evaluated meganucleases and TALEN based approaches to edit the b globin gene in a human induced pluripotent stem (iPS) cell line carrying a homozygous sickle cell mutation. Here, we investigate the use of CRISPR/Cas9 system in combination with single stranded oligodeoxynucleotides (ssODNs) to repair the sickle mutation in the above mentioned sickle iPS line (sciPS) Based on gene alignment analysis with the highly homologous D globin gene, we have designed two b globin gene specific CRISPR guide RNAs (gRNAs) Using these gRNAs, along with Cas9 and ssODNs, we show repair of the sickle cell mutation in sciPS cells. In the short term, we expect to translate these genome editing technologies to repair the sickle mutation in patient specific cells, such as CD34+ hematopoietic cells, an approach that holds promise for future autologous stem cell therapies.
Jorge Mansilla-Soto Yan Leifman Michel Sadelain
197361699
Detection of Sickle Cell Disease associated Single Nucleotide Polymorphism Using a Graphene Field Effect Transistor
Sickle Cell Disease (SCD) is a hereditary monogenic disorder that affects millions of people worldwide and is associated with symptoms such as stroke, lethargy, chronic anemia, and increased mortality. SCD can be quickly detected and diagnosed using a simple blood test as an infant, but as of now, there is currently limited treatment to cure an individual of sickle cell disease. Recently, there have been several promising developments in CRISPR Cas associated gene editing therapeutics; however, there have been limitations in gene editing efficiency monitoring, which if improved, could be beneficial to advancing CRISPR based therapy, especially in SCD. The CRISPR Chip, a three terminal graphene based field effect transistor (gFET) was used to detect genomic samples of individuals with SCD, with and without amplificati on. With the dRNP HTY3' complex, CRISPR Chip was able to specifically detect its target sequence with and without pre amplification. With the dRNP MUT3' complex, CRISPR Chip was only able to specifically detect one of its two target sequences. Fa cile detection, analysis, and editing of sickle cell disease using CRISPR based editing and monitoring would be beneficial for simple diagnostic and gene editing therapeutic treatment of other single nucleotide polymorphisms as well, such as beta thalassemia and cystic fibrosis.
Kandace Fung
91862353
In vivo selection for corrected ss globin alleles after CRISPR/Cas9 editing in human sickle hematopoietic stem cells enhances therapeutic potential
Sickle Cell Disease (SCD) one of the world's most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP mediated gene editing with an ssDNA oligonucleotide donor yields more than 20% correction of the sickle mutation in long term engrafting human HSCs. Using RNA seq, we further find that in vivo erythroid differentiation markedly enriches for cells carrying corrected ss globin alleles. Adoption of a high fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off target events. These findings indicate that the sickle mutation can be corrected in human HSCs at levels that are likely to be curative if translated into a therapy.
Wendy Magis Mark A DeWitt Stacia K Wyman Jonathan T Vu S J Heo Shirley J Shao Fiona Hennig Zulema Romero Beatriz Campo-Fernandez Matthew S McNeill Garrett R Rettig Yongming Sun Patrick J Lau Yu Wang Mark A Behlke Donald B Kohn Dario Boffelli Mark C Walters Jacob E Corn David I K Martin
3317702
A Comprehensive, Ethnically Diverse Library of Sickle Cell Disease Specific Induced Pluripotent Stem Cells
Summary Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle cell disease specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, b globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype specific polymorphisms that affect disease severity, as well as for the development of patient specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell and gene based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.
Stem cell reports
Seonmi Park Andreia Gianotti-Sommer F Javier Molina-Estevez Kim Vanuytsel Nicholas Skvir Amy Leung Sarah S Rozelle Elmutaz M Shaikho Isabelle R Weir Zhihua Jiang Hong-yuan Luo David H K Chui Maria Stella Figueiredo Abdulraham Alsultan Amein K Al-Ali Paola Sebastiani Martin H Steinberg Gustavo Mostoslavsky George J Murphy
201616293
CRISPR comes to the silver screen
Slick visuals and compelling interviews render gene editing intelligible to nonexperts If you could cure a child suffering from sickle cell anemia, would you? This is the question at the center of Human Nature, a new documentary directed by Adam Bolt about the revolutionary gene editing technology known as CRISPR. The documentary is visually stunning, thought provoking, informative, and tightly focused on human health which means that there are a few pieces missing from the overall picture.
Science
Taylor Winkleman
90040220
C&EN talks with Jennifer Doudna, CRISPR pioneer
It's been less than five years since University of California, Berkeley, biochemist Jennifer Doudna and colleagues unveiled the gene editing tool CRISPR in Science (2012, DOI: 10.1126/science.1225829) but its use among researchers across the globe has already exploded. Last April, for example, Sichuan University scientists announced that they had used CRISPR to program T cells from the immune system of a person with lung cancer to kill tumor cells (Nature 2016, DOI: 10.1038/nature.2016.20988) And late last year, a UC Berkeley team reported harnessing the technology to correct the genetic mutation responsible for sickle cell anemia in stem cells from patients with the disease (Sci. Transl. Med. 2016, DOI: 10.1126/scitranslmed.aaf9336) Doudna didn't set out to develop a far reaching gene editing tool years ago. Initially, her team wanted to uncover the role of CRISPRs short for "clustered regularly interspaced short palindromic repeats" which are repetitive sequences of RNA hidden in the genomes of many
Melissa Pandika