Browsing by Author "Bao, Gang"

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    A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
    (Elsevier, 2019) Li, Ang; Lee, Ciaran M.; Hurley, Ayrea E.; Jarrett, Kelsey E.; De Giorgi, Marco; Lu, Weiqi; Balderrama, Karol S.; Doerfler, Alexandria M.; Deshmukh, Harshavardhan; Ray, Anirban; Bao, Gang; Lagor, William R.
    Adeno-associated viral (AAV) vectors packaging the CRISPR-Cas9 system (AAV-CRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editizng applications, permanent expression of non-human proteins such as the bacterially derived Cas9 nuclease is undesirable. Methods are needed to achieve efficient genome editing in vivo, with controlled transient expression of CRISPR-Cas9. Here, we report a self-deleting AAV-CRISPR system that introduces insertion and deletion mutations into AAV episomes. We demonstrate that this system dramatically reduces the level of Staphylococcus aureus Cas9 protein, often greater than 79%, while achieving high rates of on-target editing in the liver. Off-target mutagenesis was not observed for the self-deleting Cas9 guide RNA at any of the predicted potential off-target sites examined. This system is efficient and versatile, as demonstrated by robust knockdown of liver-expressed proteins in vivo. This self-deleting AAV-CRISPR system is an important proof of concept that will help enable translation of liver-directed genome editing in humans.
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    A Trace Theorem for Solutions of Linear Partial Differential Equations
    (1989-10) Bao, Gang; Symes, William W.
    The main goal of this work is to determine circumstances under which the trace of the solution of a linear partial differential equation is as smooth as the solution itself. Clearly, if the linear partial differential equation is strictly hyperbolic with smooth coefficients, standard energy estimates will yield the fact that the solution along any spacelike trace is as smooth as itself locally, provided a sufficiently smooth right-hand side. Unfortunately, for more general equations or even a strictly hyperbolic differential equation but this time along a nonspacelike trace, the same idea will not work, essentially because one does not know how to apply energy estimates to a nonhyperbolic problem directly. In this paper, we shall investigate the trace regularities of solutions to linear P.D.E. Our result shows that the difficulties discussed above may be cured by imposing some additional microlocal smoothness.
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    Accurate Quantification of Disease Markers in Human Serum Using Iron Oxide Nanoparticle-linked Immunosorbent Assay
    (Ivyspring, 2016) Zhang, Linlin; Tong, Sheng; Zhou, Jun; Bao, Gang
    Accurate and reliable quantification of biomarkers in the blood is essential in disease screening and diagnosis. Here we describe an iron oxide nanoparticle (IONP)-linked immunosorbent assay (ILISA) for detecting biomolecules in human serum. Sandwich ILISA was optimized for the detection of four important serological markers, IgA, IgG, IgM, and C-reactive protein (CRP), and assessed with normal sera, simulated disease-state sera and the serum samples from patients infected with West Nile virus (WNV) or human herpes virus (HHV). Our study shows that using the detection assay formulated with 18.8 nm wüstite nanocrystals, ILISA can achieve sub-picomolar detection sensitivity, and all four markers can be accurately quantified over a large dynamic range. In addition, ILISA is not susceptible to variations in operating procedures and shows better linearity and higher stability compared with ELISA, which facilitates its integration into detection methods suitable for point of care. Our results demonstrate that ILISA is a simple and versatile nanoplatform for highly sensitive and reliable detection of serological biomarkers in biomedical research and clinical applications.
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    CAGE sequencing reveals CFTR-dependent dysregulation of type I IFN signaling in activated cystic fibrosis macrophages
    (AAAS, 2023) Gillan, Jonathan L.; Chokshi, Mithil; Hardisty, Gareth R.; Clohisey Hendry, Sara; Prasca-Chamorro, Daniel; Robinson, Nicola J.; Lasota, Benjamin; Clark, Richard; Murphy, Lee; Whyte, Moira K. B.; Baillie, J. Kenneth; Davidson, Donald J.; Bao, Gang; Gray, Robert D.
    An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5′ end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.
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    Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung
    (Springer Nature, 2019) Lee, Yu-Cheng; Kurtova, Antonina V.; Xiao, Jing; Nikolos, Fotis; Hayashi, Kazukuni; Tramel, Zoe; Jain, Antrix; Chen, Fengju; Chokshi, Mithil; Lee, Ciaran; Bao, Gang; Zhang, Xiang; Shen, Jianjun; Mo, Qianxing; Jung, Sung Yun; Rowley, David; Chan, Keith Syson
    Metastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)—a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.
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    Collagen-rich airway smooth muscle cells are a metastatic niche for tumor colonization in the lung
    (Springer Nature, 2019) Lee, Yu-Cheng; Kurtova, Antonina V.; Xiao, Jing; Nikolos, Fotis; Hayashi, Kazukuni; Tramel, Zoe; Jain, Antrix; Chen, Fengju; Chokshi, Mithil; Lee, Ciaran; Bao, Gang; Zhang, Xiang; Shen, Jianjun; Mo, Qianxing; Jung, Sung Yun; Rowley, David; Chan, Keith Syson
    Metastases account for the majority of cancer deaths. While certain steps of the metastatic cascade are well characterized, identification of targets to block this process remains a challenge. Host factors determining metastatic colonization to secondary organs are particularly important for exploration, as those might be shared among different cancer types. Here, we showed that bladder tumor cells expressing the collagen receptor, CD167a, responded to collagen I stimulation at the primary tumor to promote local invasion and utilized the same receptor to preferentially colonize at airway smooth muscle cells (ASMCs)—a rich source of collagen III in lung. Morphologically, COL3-CD167a-driven metastatic foci are uniquely distinct from typical lung alveolar metastatic lesions and exhibited activation of the CD167a-HSP90-Stat3 axis. Importantly, metastatic lung colonization could be abrogated using an investigational drug that attenuates Stat3 activity, implicating this seed-and-soil interaction as a therapeutic target for eliminating lung metastasis.
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    Comprehensive analysis and accurate quantification of unintended large gene modifications induced by CRISPR-Cas9 gene editing
    (AAAS, 2022) Park, So Hyun; Cao, Mingming; Pan, Yidan; Davis, Timothy H.; Saxena, Lavanya; Deshmukh, Harshavardhan; Fu, Yilei; Treangen, Todd; Sheehan, Vivien A.; Bao, Gang
    Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.
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    Computation of Pseudo-Differential Operators
    (1992-11) Bao, Gang; Symes, William W.
    A simple algorithm is described for computing general pseudo-differential operator actions. Our approach is based on the asymptotic expansion of the symbol together with the Fast Fourier Transform (FFT). The idea is motivated by the characterization of pseudo-differential operator algebra. We show that the algorithm is efficient through analyzing its complexity. Some numerical experiments are also presented.
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    Controlled delivery of β-globin-targeting TALENs and CRISPR/Cas9 into mammalian cells for genome editing using microinjection
    (Macmillan Publishers Limited, 2015) Cottle, Renee N.; Lee, Ciaran M.; Archer, David; Bao, Gang
    Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents.
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    Development and Characterization of Viral-Based Gene Editing In Vivo
    (2020-08-12) Li, Ang; Bao, Gang
    Adeno-Associated Viral (AAV) vectors packaging the CRISPR/Cas9 system (AAVCRISPR) can efficiently modify disease-relevant genes in somatic tissues with high efficiency. AAV vectors are a preferred delivery vehicle for tissue-directed gene therapy because of their ability to achieve sustained expression from largely non-integrating episomal genomes. However, for genome editing applications, permanent expression of non-human proteins such as the bacterially-derived Cas9 nuclease is undesirable. Recent studies indicate a high prevalence of neutralizing antibodies and T-cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. Additionally, persistent expression of CRISPR/Cas9 has the potential to increase the chances of off-target cutting. There is a need for efficient genome editing in vivo, with controlled transient expression of CRISPR/Cas9. The topic of my thesis covers the development of a self-deleting AAVCRISPR system that introduces insertion and deletion mutations into AAV episomes, understanding the effects AAV-CRISPR editing in vivo in a Cas9 immunized mouse model, and the characterization of AAV integrations into the genome in the context of CRISPR-based gene editing.
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    Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation
    (Cell Press, 2020) Asthana, Vishwaratn; Stern, Brett S.; Tang, Yuqi; Bugga, Pallavi; Li, Ang; Ferguson, Adam; Asthana, Anantratn; Bao, Gang; Drezek, Rebekah A.
    Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2′-deoxyuridine (2′-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.
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    Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule
    (Frontiers Media S.A., 2017) Butt, Haroon; Eid, Ayman; Ali, Zahir; Atia, Mohamed A.M.; Mokhtar, Morad M.; Hassan, Norhan; Lee, Ciaran M.; Bao, Gang; Mahfouz, Magdy M.
    The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.
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    Fine-mapping within eQTL credible intervals by expression CROP-seq
    (Oxford University Press, 2020) Pan, Yidan; Tian, Ruoyu; Lee, Ciaran; Bao, Gang; Gibson, Greg
    The majority of genome-wide association study (GWAS)-identified SNPs are located in noncoding regions of genes and are likely to influence disease risk and phenotypes by affecting gene expression. Since credible intervals responsible for genome-wide associations typically consist of ≥100 variants with similar statistical support, experimental methods are needed to fine map causal variants. We report here a moderate-throughput approach to identifying regulatory GWAS variants, expression CROP-seq, which consists of multiplex CRISPR-Cas9 genome editing combined with single-cell RNAseq to measure perturbation in transcript abundance. Mutations were induced in the HL60/S4 myeloid cell line nearby 57 SNPs in three genes, two of which, rs2251039 and rs35675666, significantly altered CISD1 and PARK7 expression, respectively, with strong replication and validation in single-cell clones. The sites overlap with chromatin accessibility peaks and define causal variants for inflammatory bowel disease at the two loci. This relatively inexpensive approach should be scalable for broad surveys and is also implementable for the fine mapping of individual genes.
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    Gene correction for SCID-X1 in long-term hematopoietic stem cells
    (Springer Nature, 2019) Pavel-Dinu, Mara; Wiebking, Volker; Dejene, Beruh T.; Srifa, Waracharee; Mantri, Sruthi; Nicolas, Carmencita E.; Lee, Ciaran; Bao, Gang; Kildebeck, Eric J.; Punjya, Niraj; Sindhu, Camille; Inlay, Matthew A.; Saxena, Nivedita; DeRavin, Suk See; Malech, Harry; Roncarolo, Maria Grazia; Weinberg, Kenneth I.; Porteus, Matthew H.
    Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.
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    Genome editing for inborn errors of metabolism: advancing towards the clinic
    (BioMed Central, 2/27/2017) Schneller, Jessica L; Lee, Ciaran M; Bao, Gang; Venditti, Charles P
    Inborn errors of metabolism (IEM) include many disorders for which current treatments aim to ameliorate disease manifestations, but are not curative. Advances in the field of genome editing have recently resulted in the in vivo correction of murine models of IEM. Site-specific endonucleases, such as zinc-finger nucleases and the CRISPR/Cas9 system, in combination with delivery vectors engineered to target disease tissue, have enabled correction of mutations in disease models of hemophilia B, hereditary tyrosinemia type I, ornithine transcarbamylase deficiency, and lysosomal storage disorders. These in vivo gene correction studies, as well as an overview of genome editing and future directions for the field, are reviewed and discussed herein.
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    Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: Optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation
    (Ferrata-Storti Foundation, 2021) Wiebking, Volker; Lee, Ciaran M.; Mostrel, Nathalie; Lahiri, Premanjali; Bak, Rasmus; Bao, Gang; Roncarolo, Maria Grazia; Bertaina, Alice; Porteus, Matthew H.
    Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of αβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population that allogeneic hematopoietic stem cell transplantation can be used in. Leukemic relapse, however, remains a problem. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including in the central nervous system. We hypothesized that by modifying donor αβ T cells to simultaneously express a CD19-specific chimeric antigen receptors and inactivating the T cell receptor by genome editing, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrate a CD19-specific chimeric antigen receptor in-frame into the TRAC locus. Greater than 90% of cells lost TCR expression, while >75% expressed the CAR. The product was further purified to ultimately have less than 0.05% residual TCR+ cells. In vitro, the CAR T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene modified T cells eliminated leukemia without causing xenogeneic graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of αβ T cell-derived, genome edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of αβ T cell-depleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.
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    Genome editing strategies for treating β-hemoglobinopathies
    (2020-04-23) Park, So Hyun; Bao, Gang
    β-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia are debilitating, painful diseases and a major cause of global mortality and health disparities. Currently, there is no cure for the majority of patients with β-hemoglobinopathies and therapeutic options are limited. We have developed novel approaches to curing β-hemoglobinopathies using CRISPR/Cas9 based ex vivo genome editing of β-globin (HBB) gene in patients’ hematopoietic stem and progenitor cells (HSPCs). Although gene-editing strategies, including correction of the sickle mutation, targeted insertion of the β-globin gene and induction of fetal hemoglobin are very promising in curing β-hemoglobinopathies, significant safety concerns exist, including off-target effects, large deletions and insertions in HBB, and chromosomal rearrangements. In Aim 1, we optimized the design of CRISPR gRNA and short single-strand oligonucleotide donor template to correct the sickle mutation, and demonstrated high rates of gene correction in SCD HSPCs. Erythrocytes derived from gene-edited cells showed high levels of normal hemoglobin expression and a marked reduction of sickle cells. We found that gene-corrected HSPCs retained the ability to engraft in mouse models, and the off-target effects could be significantly reduced by using HiFi-Cas9. In Aim 2, we developed and validated two next-generation sequencing-based assays, LongAmp-seq (long-range PCR amplification based sequencing) and NEW-seq (nuclease-activity identified by gEnome-wide sequencing), to comprehensively investigate the gene-editing outcomes and the potential consequences of unexpected mutations. We performed a thorough analysis of gene-editing outcomes, including large deletions and insertions at the HBB cut site not previously reported. The LongAmp-seq and NEW-seq also have the potential to detect and quantify chromosomal rearrangements including inversions, translocations and large chromosomal deletions. To aid the development of new therapies for β-hemoglobinopathies, in Aim 3, we applied genome editing to establish erythroid cell models for SCD and β-thalassemia that exhibit disease phenotypes. These cell models are reliable, reproducible and low-cost in performing disease studies, including validation of genome editing based therapies and screening of pharmacological drugs. The systematic studies of the efficiency and safety of the gene-editing approaches, and the cell models developed for discovery of therapeutic agents may significantly facilitate the clinical translation of gene editing based therapies for β-hemoglobinopathies.
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    Genome Editing Strategies to Cure Cystic Fibrosis
    (2022-06-01) Chokshi, Mithil; Bao, Gang
    Cystic Fibrosis (CF) is a recessive genetic disorder with fatal consequences caused by over 1,700 mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. We studied gene editing in patient-derived induced pluripotent stem cells (iPSCs) by correcting the most prevalent disease-causing mutation, F508del, a 3-bp deletion that prevents CFTR protein to fold properly. The challenges in editing primary and stem cells have caused current approaches to focus on using drug-based selection and fluorescence-based enrichment of the edited cells. We systematically optimized different parameters to obtain highest reported repair rate of 20% allelic HDR at the F508del locus in the iPSCs and conducted an extensive search for off-target activity of the tested gRNA. The cells were then differentiated to epithelia to show recovery of CFTR expression and function. We further optimized the CRISPR / Cas9 machinery to increase the repair rates to above 50%, which is comparable or higher than virus-based editing approaches. To translate our approaches in vivo we are further engineering AAVs to target lung cell types of interest for delivery of the optimized gene editing machinery for an in vivo therapy. These approaches were tested in novel gene-edited human bronchial epithelia-based cell models containing a halide-sensitive fluorescence reporter to conduct high-throughput assays for the CFTR channel activity. We have made such cell models for multiple classes of CF to enable faster discovery of new therapies to cure CF. Finally, CF is a multifaceted disease with fatal lung inflammation and infection cycles; we use gene edited iPSCs developed above to test the hypothesis that CF lung macrophages are hyperinflammatory and poorly bactericidal to perpetuate the inflammation and infection.
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    Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease
    (Oxford University Press, 2019) Park, So Hyun; Lee, Ciaran M.; Dever, Daniel P.; Davis, Timothy H.; Camarena, Joab; Srifa, Waracharee; Zhang, Yankai; Paikari, Alireza; Chang, Alicia K.; Porteus, Matthew H.; Sheehan, Vivien A.; Bao, Gang
    Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the β-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16–19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.