Development of ß-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the ß-globin gene (HBB). Ex vivo ß-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

Gene replacement of α-globin with ß-globin restores hemoglobin balance in ß-thalassemia-derived hematopoietic stem and progenitor cells.

ß-Thalassemia pathology is due not only to loss of ß-globin (HBB), but also to erythrotoxic accumulation and aggregation of the ß-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in ß-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize ß-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing ß-thalassemia.

Metabolic engineering generates a transgene-free safety switch for cell therapy.

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.

Macrophage Subpopulation Dynamics Shift following Intravenous Infusion of Mesenchymal Stromal Cells.

Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, we show that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9+ macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.

Cas9-AAV6-engineered human mesenchymal stromal cells improved cutaneous wound healing in diabetic mice.

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly efficient editing of the ß-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease.

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 ß-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.

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.

Gene correction for SCID-X1 in long-term hematopoietic stem cells.

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.

Rapid Phenotypic and Genotypic Diversification After Exposure to the Oral Host Niche in Candida albicans.

In vitro studies suggest that stress may generate random standing variation and that different cellular and ploidy states may evolve more rapidly under stress. Yet this idea has not been tested with pathogenic fungi growing within their host niche in vivo Here, we analyzed the generation of both genotypic and phenotypic diversity during exposure of Candida albicans to the mouse oral cavity. Ploidy, aneuploidy, loss of heterozygosity (LOH), and recombination were determined using flow cytometry and double digest restriction site-associated DNA sequencing. Colony phenotypic changes in size and filamentous growth were evident without selection and were enriched among colonies selected for LOH of the GAL1 marker. Aneuploidy and LOH occurred on all chromosomes (Chrs), with aneuploidy more frequent for smaller Chrs and whole Chr LOH more frequent for larger Chrs. Large genome shifts in ploidy to haploidy often maintained one or more heterozygous disomic Chrs, consistent with random Chr missegregation events. Most isolates displayed several different types of genomic changes, suggesting that the oral environment rapidly generates diversity de novo In sharp contrast, following in vitro propagation, isolates were not enriched for multiple LOH events, except in those that underwent haploidization and/or had high levels of Chr loss. The frequency of events was overall 100 times higher for C. albicans populations following in vivo passage compared with in vitro These hyper-diverse in vivo isolates likely provide C. albicans with the ability to adapt rapidly to the diversity of stress environments it encounters inside the host.

Precise Correction of Disease Mutations in Induced Pluripotent Stem Cells Derived From Patients With Limb Girdle Muscular Dystrophy.

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes, respectively. Using patient-derived induced pluripotent stem cells (iPSC), we corrected the dysferlin nonsense mutation c.5713C>T; p.R1905X and the most common alpha-sarcoglycan mutation, missense c.229C>T; p.R77C, by single-stranded oligonucleotide-mediated gene editing, using the CRISPR/Cas9 gene-editing system to enhance the frequency of homology-directed repair. We demonstrated seamless, allele-specific correction at efficiencies of 0.7-1.5%. As an alternative, we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22, using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination, and DICE also utilized site-specific recombinases. With DICE and THRIP, we obtained targeting efficiencies after selection of ~20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization, as shown by immunoblot and immunocytochemistry. In summary, we demonstrate for the first time precise correction of LGMD iPSC and validation of expression, opening the possibility of cell therapy utilizing these corrected iPSC.