Caroline Y. Kuo

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells

Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation

Background: X‐linked hyper‐IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients. Objectives: We sought to collect data on the clinical presentation, treatment, and follow‐up of a large sample of patients with XHIGM to (1) compare long‐term overall survival and general well‐being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT. Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan‐Meier method compared with log‐rank tests and modeled by using proportional hazards regression. Results: Twenty‐eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow‐up and vital status information. Sixty‐seven (38%) patients received HCT. The average follow‐up time was 8.5 ± 7.2 years (range, 0.1‐36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987‐1995; the hazard ratio was significantly less than 1 for diagnosis years 1995‐1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2‐10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft‐versus‐host disease, and most deaths occurred within 1 year of transplantation. Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964‐2013). However, survivors treated with HCT experienced somewhat greater well‐being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates.

Gene Therapy for the Treatment of Primary Immune Deficiencies.

The use of gene therapy in the treatment of primary immune deficiencies (PID) has advanced significantly in the last decade. Clinical trials for X-linked severe combined immunodeficiency, adenosine deaminase deficiency (ADA), chronic granulomatous disease, and Wiskott-Aldrich syndrome have demonstrated that gene transfer into hematopoietic stem cells and autologous transplant can result in clinical improvement and is curative for many patients. Unfortunately, early clinical trials were complicated by vector-related insertional mutagenic events for several diseases with the exception of ADA-deficiency SCID. These results prompted the current wave of clinical trials for primary immunodeficiency using alternative retro- or lenti-viral vector constructs that are self-inactivating, and they have shown clinical efficacy without leukemic events thus far. The field of gene therapy continues to progress, with improvements in viral vector profiles, stem cell culturing techniques, and site-specific genome editing platforms. The future of gene therapy is promising, and we are quickly moving towards a time when it will be a standard cellular therapy for many forms of PID.

Newborn screening for severe combined immunodeficiency does not identify bare lymphocyte syndrome.

V staining (Fig 2, A, top row) and elevated caspase activity (Fig 2, B). In culture for 24 hours, B cells from patient III.2 demonstrated even greater Annexin Vand considerable 7-AAD staining, evidence of accelerated cell death (Fig 2, A, middle row). The patient’s B cells were only partially rescued by the stimulation of their B-cell receptors (Fig 2, A, bottom row). We also found enhanced caspase activity in B cells from the 2 additional patients who were heterozygous for either the E353K or the I156T missense STAT1 mutation (Fig 2, B). B-cell lymphopenia was a significant finding in the former patient (39 cells/mL) but not the latter (335 cells/mL). Altogether our data reveal that gainof-function STAT1 mutations increase B-cell apoptosis. Over time, this may result in B-cell lymphopenia and antibody deficiency. In summary, we identified individuals heterozygous for gainof-function STAT1 mutation with 2 unappreciated features. The first is the overexpression of PD-L1 on naive T cells, which provides a general mechanism for how constitutively active STAT1 blocks the development of the TH17 lineage. The second feature, accelerated B-cell apoptosis that may result in progressive B-cell lymphopenia and humoral immunodeficiency, further broadens the clinical phenotype associated with gain-of-function STAT1 mutations.

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs.

Profound T-cell lymphopenia associated with prenatal exposure to purine antagonists detected by TREC newborn screening

Recognition of non-SCID and secondary causes of T cell lymphopenia detected by TREC newborn screening is important in directing subsequent care and identifying those who would not benefit from more invasive interventions. Here, we report two infants with low TRECs and severe, but self-resolving, T cell lymphopenia identified by SCID NBS that were caused by in utero exposure to purine antimetabolites.

Characterization of Gene Alterations following Editing of the β-Globin Gene Locus in Hematopoietic Stem/Progenitor Cells

The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human β- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in β-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the β- and δ-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34+ cells and colony-forming units, the intergenic β-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target β-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.

Gene Therapy for Primary Immune Deficiency Diseases

Abstract Many severe primary immune deficiencies (PIDs) are due to defects in lymphoid or hematopoietic cells that can be reconstituted by transplantation of normal hematopoietic stem cells (HSCs). Allogeneic hematopoietic stem cell transplantation (HSCT) has been successfully applied to more than a dozen human PID, limited by the need for a well-matched donor and the potential to develop graft-versus-host disease (GvHD). Gene therapy using autologous HSCs has been developed as a new treatment for these PIDs, with potential advantages from the absence of GvHD and the less intense conditioning and immune suppression needed. In the early years (ca. 1990 – 1999), insufficient gene transfer to HSC was achieved and no efficacy was seen. Subsequently (ca. 2000 – 2010), several clinical trials of gene therapy for PIDs using gamma-retroviral vectors showed effective immune reconstitution, but some of these patients developed myelodysplasia or leukemia due to genotoxicity from the retroviral vectors. Currently, improved vectors modified to minimize risks of genotoxicity are being used safely and effectively in clinical trials for a growing list of PIDs. More recently, methods are being established for direct genome editing to correct the specific gene underlying a PID in the genome of autologous HSC. Precise gene editing may have broader applications to the PIDs where precise gene expression regulation is needed for safety and efficacy. The advances in gene therapy will be compared to those ongoing for allogeneic HSCT approaches to define optimal treatments for severe PID.

Outcome of domino hematopoietic stem cell transplantation in human subjects: An international case series

This multicenter cohort study demonstrates that at a median follow up of ten years, domino-HSCT results in overall survival of 80%, event-free survival of 49%, and low risk of GvHD. Domino-HSCT can be considered, weighed against other available transplantation strategies.

The PTH/PTHrP-SIK3 pathway affects skeletogenesis through altered mTOR signaling

Disrupting the PTH/PTHrP-SIK3 pathway impairs degradation of DEPTOR, affects mTOR signaling, and leads to abnormalities in skeletal development. Skeletal signaling sleuthing Skeletal dysplasias are rare genetic disorders affecting bone and cartilage growth during development. Csukasi et al. identified two patients with developmental delay and a skeletal phenotype similar to Jansen metaphyseal chondrodysplasia, a disorder caused by altered parathyroid hormone signaling. They identified a genetic mutation in SIK3 as the cause of the of the patients’ dysplasia. The SIK3 mutation altered mTOR signaling, and parathyroid hormone signaling was found to regulate SIK3 activity. This study identifies a common signaling pathway underlying two distinct skeletal disorders, suggesting it plays an important role during skeletal development. Studies have suggested a role for the mammalian (or mechanistic) target of rapamycin (mTOR) in skeletal development and homeostasis, yet there is no evidence connecting mTOR with the key signaling pathways that regulate skeletogenesis. We identified a parathyroid hormone (PTH)/PTH-related peptide (PTHrP)–salt-inducible kinase 3 (SIK3)–mTOR signaling cascade essential for skeletogenesis. While investigating a new skeletal dysplasia caused by a homozygous mutation in the catalytic domain of SIK3, we observed decreased activity of mTOR complex 1 (mTORC1) and mTORC2 due to accumulation of DEPTOR, a negative regulator of both mTOR complexes. This SIK3 syndrome shared skeletal features with Jansen metaphyseal chondrodysplasia (JMC), a disorder caused by constitutive activation of the PTH/PTHrP receptor. JMC-derived chondrocytes showed reduced SIK3 activity, elevated DEPTOR, and decreased mTORC1 and mTORC2 activity, indicating a common mechanism of disease. The data demonstrate that SIK3 is an essential positive regulator of mTOR signaling that functions by triggering DEPTOR degradation in response to PTH/PTHrP signaling during skeletogenesis.