Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated, site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study, we demonstrate the ability to target the integration of WAS2-12-containing constructs into intron 1 of the endogenous WAS gene of primary CD34+ hematopoietic stem and progenitor cells (HSPCs), as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore, TI restored WASp-dependent function to WAS patient T cells. Edited CD34+ HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient's CD34+ cells.

The Laparoscopy in Biliary Exploration Research and Training Initiative (LIBERTI) trial: simulator-based training for laparoscopic management of choledocholithiasis.

BACKGROUND: Laparoscopic cholecystectomy with common bile duct exploration (LCBDE) is equivalent in safety and efficacy to endoscopic retrograde cholangiopancreatography (ERCP) plus laparoscopic cholecystectomy (LC) while decreasing number of procedures and length of stay (LOS). Despite these advantages LCBDE is infrequently utilized. We hypothesized that formal, simulation-based training in LCBDE would result in increased utilization and improve patient outcomes across participating institutions. METHODS: Data was obtained from an on-going multi-center study in which simulator-based transcystic LCBDE training curricula were instituted for attending surgeons and residents. A 2-year retrospective review of LCBDE utilization prior to LCBDE training was compared to utilization up to 2 years after initiation of training. Patient outcomes were analyzed between LCBDE strategy and ERCP strategy groups using χ2, t tests, and Wilcoxon rank tests. RESULTS: A total of 50 attendings and 70 residents trained in LCBDE since November 2020. Initial LCBDE utilization rate ranged from 0.74 to 4.5%, and increased among all institutions after training, ranging from 9.3 to 41.4% of cases. There were 393 choledocholithiasis patients analyzed using LCBDE (N = 129) and ERCP (N = 264) strategies. The LCBDE group had shorter median LOS (3 days vs. 4 days, p < 0.0001). No significant differences in readmission rates between LCBDE and ERCP groups (4.7% vs. 7.2%, p = 0.33), or in post-procedure pancreatitis (0.8% v 0.8%, p > 0.98). In comparison to LCBDE, the ERCP group had higher rates of bile duct injury (0% v 3.8%, p = 0.034) and fluid collections requiring intervention (0.8% v 6.8%, p < 0.009) secondary to cholecystectomy complications. Laparoscopic antegrade balloon sphincteroplasty had the highest technical success rate (87%), followed by choledochoscopic techniques (64%). CONCLUSION: Simulator-based training in LCBDE results in higher utilization rates, shorter LOS, and comparable safety to ERCP plus cholecystectomy. Therefore, implementation of LCBDE training is strongly recommended to optimize healthcare utilization and management of patients with choledocholithiasis.

SAGES safe cholecystectomy modules improve practicing surgeons' judgment: results of a randomized, controlled trial.

BACKGROUND: Despite the advantages of laparoscopic cholecystectomy, major bile duct injury (BDI) rates during this operation remain unacceptably high. In October 2018, SAGES released the Safe Cholecystectomy modules, which define specific strategies to minimize the risk of BDI. This study aims to investigate whether this curriculum can change the knowledge and behaviors of surgeons in practice. METHODS: Practicing surgeons were recruited from the membership of SAGES and the American College of Surgeons Advisory Council for Rural Surgery. All participants completed a baseline assessment (pre-test) that involved interpreting cholangiograms, troubleshooting difficult cases, and managing BDI. Participants' dissection strategies during cholecystectomy were also compared to the strategies of a panel of 15 experts based on accuracy scores using the Think Like a Surgeon validated web-based platform. Participants were then randomized to complete the Safe Cholecystectomy modules (Safe Chole module group) or participate in usually scheduled CME activities (control group). Both groups completed repeat assessments (post-tests) one month after randomization. RESULTS: Overall, 41 participants were eligible for analysis, including 18 Safe Chole module participants and 23 controls. The two groups had no significant differences in pre-test scores. However, at post-test, Safe Chole module participants made significantly fewer errors managing BDI and interpreting cholangiograms. Safe Chole module participants were less likely to convert to an open operation on the post-test than controls when facing challenging dissections. However, Safe Chole module participants displayed a similar incidence of errors when evaluating adequate critical views of safety. CONCLUSIONS: In this randomized-controlled trial, the SAGES Safe Cholecystectomy modules improved surgeons' abilities to interpret cholangiograms and safely manage BDI. Additionally, surgeons who studied the modules were less likely to convert to open during difficult dissections. These data show the power of the Safe Cholecystectomy modules to affect practicing surgeons' behaviors in a measurable and meaningful way.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

Does accreditation matter? An analysis of complications of bariatric cases using the Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program and National Quality Improvement Program databases.

BACKGROUND: Two large nationwide databases collect data on common operations in the United States. The Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) collects bariatric data, whereas the National Quality Improvement Program (NSQIP) gathers details on a broader range of general surgical cases. OBJECTIVE: Evaluate the differences in rates of complications between both databases regarding Roux-en-Y gastric bypass and sleeve gastrectomy. SETTING: National databases, United States. METHODS: We evaluated the MBSAQIP and NSQIP from 2017 to 2019 using the procedure codes 43644 and 43775. Fifteen common complications were evaluated. Propensity-matched analyses (PMAs) were done to control for differences across databases. Significantly different variables after a PMA were included in multivariable models. The data were examined for differences between the 2 databases before and after the PMA, with and without adjustment for operation type. RESULTS: There were 483,361 cases reported in the MBSAQIP and 57,598 in the NSQIP. PMA matched 57,479 cases for each database. Seven complications were different, with higher rates reported in the NSQIP than in the MBSAQIP: myocardial infarction, sepsis, organ/space surgical site infections, deep vein thrombosis, urinary tract infections, pulmonary embolism, ventilator dependence >48 hours, and pneumonia. When adjusting for the procedure performed, sleeve gastrectomy in the NSQIP had higher rates of organ/space surgical site infections, deep vein thrombosis, sepsis, and death. Roux-en-Y gastric bypass in the NSQIP had higher rates of organ/space surgical site infections, ventilator dependence >48 hours, urinary tract infections, myocardial infarction, deep vein thrombosis, and sepsis. CONCLUSION: When compared with the MBSAQIP, the NSQIP reports higher rates of bariatric complications. Further studies are needed to confirm the reasons behind this.

Targeted Gene Insertion for Functional CFTR Restoration in Airway Epithelium.

Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the CFTR gene locus, of which at least 382 are disease-causing (CFTR2.org). Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all CFTR mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous CFTR locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how in vitro gene insertion methodologies may be adapted for direct in vivo editing.

Differentiation of human pluripotent stem cells into functional airway basal stem cells.

Airway basal cells play an essential role in the maintenance of the airway epithelium. Here, we provide a detailed directed differentiation protocol to generate ''induced basal cells (iBCs)'' from human pluripotent stem cells. iBCs recapitulate biological and functional properties of airway basal cells including mucociliary differentiation in vitro or in vivo in tracheal xenografts, facilitating the study of inherited and acquired diseases of the airway, as well as potential use in regenerative medicine. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2021).

SAGES TAVAC safety and efficacy analysis confocal laser endomicroscopy.

BACKGROUND: Confocal laser endomicroscopy (CLE) is a novel endoscopic adjunct that allows real-time in vivo histological examination of mucosal surfaces. By using intravenous or topical fluorescent agents, CLE highlights certain mucosal elements that facilitate an optical biopsy in real time. CLE technology has been used in different organ systems including the gastrointestinal tract. There has been numerous studies evaluating this technology in gastrointestinal endoscopy, our aim was to evaluate the safety, value, and efficacy of this technology in the gastrointestinal tract. METHODS: The Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) Technology and Value Assessment Committee (TAVAC) performed a PubMed/Medline database search of clinical studies involving CLE in May of 2018. The literature search used combinations of the keywords: confocal laser endomicroscopy, pCLE, Cellvizio, in vivo microscopy, optical histology, advanced endoscopic imaging, and optical diagnosis. Bibliographies of key references were searched for relevant studies not covered by the PubMed search. Case reports and small case series were excluded. The manufacturer's website was also used to identify key references. The United States Food and Drug Administration (U.S. FDA) Manufacturer And User facility and Device Experience (MAUDE) database was searched for reports regarding the device malfunction or injuries. RESULTS: The technology offers an excellent safety profile with rare adverse events related to the use of fluorescent agents. It has been shown to increase the detection of dysplastic Barrett's esophagus, gastric intraepithelial neoplasia/early gastric cancer, and dysplasia associated with inflammatory bowel disease when compared to standard screening protocols. It also aids in the differentiation and classification of colorectal polyps, indeterminate biliary strictures, and pancreatic cystic lesions. CONCLUSIONS: CLE has an excellent safety profile. CLE can increase the diagnostic accuracy in a number of gastrointestinal pathologies.

Derivation of Airway Basal Stem Cells from Human Pluripotent Stem Cells.

The derivation of tissue-specific stem cells from human induced pluripotent stem cells (iPSCs) would have broad reaching implications for regenerative medicine. Here, we report the directed differentiation of human iPSCs into airway basal cells ("iBCs"), a population resembling the stem cell of the airway epithelium. Using a dual fluorescent reporter system (NKX2-1GFP;TP63tdTomato), we track and purify these cells as they first emerge as developmentally immature NKX2-1GFP+ lung progenitors and subsequently augment a TP63 program during proximal airway epithelial patterning. In response to primary basal cell medium, NKX2-1GFP+/TP63tdTomato+ cells display the molecular and functional phenotype of airway basal cells, including the capacity to self-renew or undergo multi-lineage differentiation in vitro and in tracheal xenografts in vivo. iBCs and their differentiated progeny model perturbations that characterize acquired and genetic airway diseases, including the mucus metaplasia of asthma, chloride channel dysfunction of cystic fibrosis, and ciliary defects of primary ciliary dyskinesia.

Correction of Airway Stem Cells: Genome Editing Approaches for the Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction.

There is a strong rationale to consider future cell therapeutic approaches for cystic fibrosis (CF) in which autologous proximal airway basal stem cells, corrected for CFTR mutations, are transplanted into the patient's lungs. We assessed the possibility of editing the CFTR locus in these cells using zinc-finger nucleases and have pursued two approaches. The first, mutation-specific correction, is a footprint-free method replacing the CFTR mutation with corrected sequences. We have applied this approach for correction of ΔF508, demonstrating restoration of mature CFTR protein and function in air-liquid interface cultures established from bulk edited basal cells. The second is targeting integration of a partial CFTR cDNA within an intron of the endogenous CFTR gene, providing correction for all CFTR mutations downstream of the integration and exploiting the native CFTR promoter and chromatin architecture for physiologically relevant expression. Without selection, we observed highly efficient, site-specific targeted integration in basal cells carrying various CFTR mutations and demonstrated restored CFTR function at therapeutically relevant levels. Significantly, Omni-ATAC-seq analysis revealed minimal impact on the positions of open chromatin within the native CFTR locus. These results demonstrate efficient functional correction of CFTR and provide a platform for further ex vivo and in vivo editing.

Extended Cystogastrostomy with Hydrogen Peroxide Irrigation Facilitates Endoscopic Pancreatic Necrosectomy.

INTRODUCTION: Walled-off pancreatic necrosis (WOPN) is a major complication of acute pancreatitis. We hypothesized that an extended (2 cm) cystogastrostomy opening combined with hydrogen peroxide irrigation can increase the success of endoscopic necrosectomy and decrease the number of required endoscopic interventions. The aim of the study is to assess the safety and feasibility of the technique in the management of WOPN. METHODS: This is a retrospective chart review of all cases that underwent EUS with extended cystogastrostomy and hydrogen peroxide irrigation prior to necrosectomy in a tertiary referral medical center. Clinical success was defined as complete resolution of the cyst cavity or a cyst cavity less than 2 cm in size on follow-up imaging. RESULTS: 19 patients satisfied the inclusion criteria. The mean size of the walled-off cavity was 11 + 0.9 cm. Technical success of the procedure was 100%. The median number of necrosectomy sessions was 2 (range 1 to 7). Cavity resolution was noted in 18 out of 19 patients resulting in a clinical success of 94.7%. The median follow-up period was 12 months. The adverse events rate in our cohort was 15.7%. CONCLUSION: Extended cystogastrostomy coupled with hydrogen peroxide irrigation of WOPN cavity is safe and feasible.

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells.

Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.

Pathological Findings of the Antral and Pyloric Smooth Muscle in Patients with Gastroparesis-Like Syndrome Compared to Gastroparesis: Similarities and Differences.

BACKGROUND: Gastroparesis (GP)-like syndrome presents with the symptoms of GP but without delayed gastric emptying (GE). Whether GP-like syndrome is part of a spectrum of GP is not clear. This study aimed to compare the histopathological features of antral and pyloric smooth muscle tissue in GP and GP-like syndrome. METHODS: Full-thickness antral and/or pyloric biopsies were obtained from 37 GP and 18 GP-like syndrome patients who underwent abdominal surgery to place a gastric electrical stimulator or jejunal feeding tube and/or pyloroplasty. The tissues were stained with H&E, C-Kit, and trichrome. Based on previous control data, an interstitial cells of Cajal (ICC) count of <10 per high power field in the antrum and/or pylorus was considered depletion. Baseline total symptom score (TSS) was recorded. RESULTS: Twenty-four GP and 7 GP-like patients had pyloric biopsies. Pyloric ICC loss was observed in 20/24 (83.3%) GP and 2/7 (28.6%) GP-like patients (p < 0.01). Fibrosis was detected in the pyloric tissue of 20/24 (83.3%) GP and 2/7 (28.6%) GP-like patients who had pyloric trichrome staining (p < 0.01). Seventeen out of 24 (70.8%) GP patients with pyloric biopsies had concomitant pyloric ICC loss and fibrosis, while only one GP-like patient had ICC loss and simultaneous pyloric fibrosis. GP patients had a greater TSS compared to GP-like patients. In GP patients, those with pyloric ICC loss had a greater TSS compared to those with normal ICC. GP patients with pyloric fibrosis had a higher TSS compared to those without pyloric fibrosis. CONCLUSIONS: Compared to GP-like patients, the pyloric histopathological findings of ICC loss and fibrosis are common in GP and predict a greater symptom score. These pathological findings might be considered as markers of "pyloric dysfunction" and explain delayed GE in GP.

Case report of robotic dor fundoplication for scleroderma esophagus with aperistalsis on manometry.

Scleroderma is a systemic disease of collagen deposition resulting in fibrosis of small arteries and arterioles. It commonly affects the skin, lungs, and gastrointestinal tract. The most common site of GI tract involvement is the esophagus. We present the case report of a 44year old female with scleroderma esophagus and severe reflux which was successfully treated with robotic dor fundoplication. Because of the wide variety of symptoms with which this problem can present, a tailored approach taking into consideration the patient's symptomatology and findings during diagnostic work-up was implemented with good results. The patient exhibited complete resolution of symptoms at short term follow up. Robotic dor fundoplication is an effective option for patients with scleroderma esophagus and no evidence of hiatal hernia or esophageal shortening.

Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells.

It has been postulated that during human fetal development, all cells of the lung epithelium derive from embryonic, endodermal, NK2 homeobox 1-expressing (NKX2-1+) precursor cells. However, this hypothesis has not been formally tested owing to an inability to purify or track these progenitors for detailed characterization. Here we have engineered and developmentally differentiated NKX2-1GFP reporter pluripotent stem cells (PSCs) in vitro to generate and isolate human primordial lung progenitors that express NKX2-1 but are initially devoid of differentiated lung lineage markers. After sorting to purity, these primordial lung progenitors exhibited lung epithelial maturation. In the absence of mesenchymal coculture support, this NKX2-1+ population was able to generate epithelial-only spheroids in defined 3D cultures. Alternatively, when recombined with fetal mouse lung mesenchyme, the cells recapitulated epithelial-mesenchymal developing lung interactions. We imaged these progenitors in real time and performed time-series global transcriptomic profiling and single-cell RNA sequencing as they moved through the earliest moments of lung lineage specification. The profiles indicated that evolutionarily conserved, stage-dependent gene signatures of early lung development are expressed in primordial human lung progenitors and revealed a CD47hiCD26lo cell surface phenotype that allows their prospective isolation from untargeted, patient-specific PSCs for further in vitro differentiation and future applications in regenerative medicine.

The Long-Term Efficacy and Safety of Pyloroplasty Combined with Gastric Electrical Stimulation Therapy in Gastroparesis.

INTRODUCTION: Gastroparesis is delayed gastric emptying without mechanical obstruction. Symptomatic improvement can be 50-60% with gastric electrical stimulation. To address delayed gastric emptying, pyloroplasty was added. This study examines the long-term efficacy and safety of simultaneous gastric electrical stimulator implantation and pyloroplasty. METHODS: In this prospective single-arm trial conducted from 2012 to 2015, 27 [23 females; mean age 43 (22-63)] gastroparesis patients who underwent simultaneous gastric electrical stimulator implantation with Heineke-Mikulicz pyloroplasty were studied. Six (25%) underwent simultaneous robot-assisted pyloroplasty and gastric electrical stimulator implantation. Diagnosis of gastroparesis was based on the 4-h gastric emptying test defined as >60% retention of isotope at 2 h and >10% at 4 h. Total symptom scores assessing severity of nausea, early satiety, bloating, vomiting, post-prandial fullness, and epigastric pain were obtained at baseline and at follow-up visits, ranging from 3 to 38 months (mean: 17). RESULTS: Follow-up data from 24 patients were available for analysis. There was 71% improvement in total symptom score on follow-up. Mean retention decreased by 29.6 and 48.7% at 2 and 4 h and gastric emptying was normalized in 60%. There were no post-surgical complications. CONCLUSIONS: Combination of gastric electrical stimulator and pyloroplasty significantly accelerated gastric emptying and improved gastroparesis symptoms. Combining these two surgical therapies improves both subjective and objective endpoints in drug refractory gastroparesis.

Homology Requirements for Efficient, Footprintless Gene Editing at the CFTR Locus in Human iPSCs with Helper-dependent Adenoviral Vectors.

Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology, the frequencies of targeted recombinants were 50-64.6% after positive selection for vector integration, and 97.4-100% after negative selection against random integrations. With 14.8 kb, the frequencies were 26.9-57.1% after positive selection and 87.5-100% after negative selection. With 9.6 kb, the frequencies were 21.4 and 75% after positive and negative selection, respectively. With only 5.6 kb, the frequencies were 5.6-16.7% after positive selection and 50% after negative selection, but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore, we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However, low frequencies (≤ 1 × 10-3) necessitated negative selection for piggyBac-excision product isolation.

Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.