CRISPR screen identifies CEBPB as contributor to dyskeratosis congenita fibroblast senescence via augmented inflammatory gene response.

Aging is the consequence of intra- and extracellular events that promote cellular senescence. Dyskeratosis congenita (DC) is an example of a premature aging disorder caused by underlying telomere/telomerase-related mutations. Cells from these patients offer an opportunity to study telomere-related aging and senescence. Our previous work has found that telomere shortening stimulates DNA damage responses (DDRs) and increases reactive oxygen species (ROS), thereby promoting entry into senescence. This work also found that telomere elongation via TERT expression, the catalytic component of the telomere-elongating enzyme telomerase, or p53 shRNA could decrease ROS by disrupting this telomere-DDR-ROS pathway. To further characterize this pathway, we performed a CRISPR/Cas9 knockout screen to identify genes that extend life span in DC cells. Of the cellular clones isolated due to increased life span, 34% had a guide RNA (gRNA) targeting CEBPB, while gRNAs targeting WSB1, MED28, and p73 were observed multiple times. CEBPB is a transcription factor associated with activation of proinflammatory response genes suggesting that inflammation may be present in DC cells. The inflammatory response was investigated using RNA sequencing to compare DC and control cells. Expression of inflammatory genes was found to be significantly elevated (P < 0.0001) in addition to a key subset of these inflammation-related genes [IL1B, IL6, IL8, IL12A, CXCL1 (GROa), CXCL2 (GROb), and CXCL5]. which are regulated by CEBPB. Exogenous TERT expression led to downregulation of RNA/protein CEBPB expression and the inflammatory response genes suggesting a telomere length-dependent mechanism to regulate CEBPB. Furthermore, unlike exogenous TERT and p53 shRNA, CEBPB shRNA did not significantly decrease ROS suggesting that CEBPB's contribution in DC cells' senescence is ROS independent. Our findings demonstrate a key role for CEBPB in engaging senescence by mobilizing an inflammatory response within DC cells.

Retrospective comparison of operative technique for chest wall injuries.

BACKGROUND: Surgical management of chest wall injuries is a common procedure. However, operative techniques are diverse, and no universal guidelines exist. There is a lack of studies comparing the outcome with different operative techniques for chest wall surgery. The aim of this study was to compare hospital outcomes between patients operated for chest wall injuries with a conventional method with large incisions and often a thoracotomy or a minimally invasive, muscle sparing method. PATIENTS AND METHODS: A retrospective study was carried out including patients ≥18 years operated for chest wall injuries 2010-2020. Patients were divided into two groups based on the surgery performed: conventional surgery (C-group) and minimally invasive surgery (M-group). Data on demographics, trauma, surgery, and outcomes were extracted from patient records. Primary outcome was length of stay on mechanical ventilator (MV-LOS). Secondary outcomes were length of stay in intensive care (ICU-LOS) and in hospital (H-LOS), and complications such as re-operation, incidence of empyema, tracheostomy, pneumonia, and mortality. RESULTS: Of 311 included patients, 220 were in the C-group and 91 in the M-group. The groups were similar in demographics and injury pattern. MV-LOS was 0 (0-65) in the C-group vs 0 (0-34) in the M-group (p < 0.001). ICU-LOS and H-LOS were significantly shorter in the M-group as compared to the C-group (p < 0.001), however with a large overlap. Tracheostomy was performed in 22.3% of patients in the C-group vs 5.4% in the M-group (p < 0.001). Pneumonia was diagnosed in 32.3% of patients in the C-group vs 16.1% in the M-group (p = 0.004). In-hospital mortality was lower in the M-group compared to the C-group but there was no difference in mortality within 30 days or a year. CONCLUSIONS: Our study indicates that a minimally invasive technique was favorable regarding clinical outcomes for patients operated for chest wall injuries.

Generation of inducible pluripotent stem cell lines from Alzheimer's disease patients with APOE e3/e3 genotype.

iPSCs were generated from B lymphocytes of two Alzheimer's disease (AD) patients homozygous for the APOE e3 allele. The iPSCs express pluripotency-specific markers and have the capacity to differentiate into all three embryonic germ layers. Karyotyping analyses confirmed the iPSCs have normal karyotypes. These iPSCs can be utilized as an in vitro model to study AD and to evaluate efficacies of new treatments.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

Quality of life and gastric acid-suppression medication 20 years after laparoscopic fundoplication.

BACKGROUND: Laparoscopic fundoplication is an effective treatment for gastro-oesophageal reflux disease (GERD). We aimed to assess quality of life (QoL), long-term residual symptoms, patient satisfaction and use of acid-suppression medication at 5, 10 and 20 years after surgery. METHODS: We identified a cohort of 100 patients who underwent laparoscopic fundoplication between 1993 and 1998. The validated QoL questionnaires Short Form health survey (SF-36), and Quality-of-Life in Reflux and Dyspepsia (QOLRAD), as well as a specific questionnaire regarding post-fundoplication symptoms, were sent to the patients at 5, 10 and 20 years after surgery. Furthermore, patients who reported using the acid-suppression medication after 20 years were interviewed by telephone regarding their reason for taking it. RESULTS: Eighty-eight percent of the patients responded at 5 and 10 years post-surgery. Twenty years following fundoplication, 68 (84% of those still alive) patients completed the questionnaires. The patients had equivalent health-related QoL scores in both the QOLRAD and SF-36 questionnaires after 10 and 20 years, and those scores were in line with a Swedish age-matched population. After 20 years, 87% were satisfied with the results, and 84% of the patients would recommend reflux surgery to a relative or a friend. At the telephone interview, 32% (22/68) confirmed using acid-suppression medication, but only half (11/68) used it because of reflux symptoms. CONCLUSION: The long-term, satisfying outcomes in GERD symptoms and QoL 5 and 10 years after surgery were maintained at a 20-year follow-up. Half of the patients used acid-suppression medication for reasons other than GERD symptoms.

Bone Marrow Transplantation after Nonmyeloablative Treosulfan Conditioning Is Curative in a Murine Model of Sickle Cell Disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative for patients with sickle cell disease (SCD). However, morbidity associated with myeloablative conditioning and graft-versus-host disease has limited its utility. To this end, autologous HSCT for SCD using lentiviral gene-modified bone marrow (BM) or peripheral blood stem cells has been undertaken, although toxicities of fully ablative conditioning with busulfan and incomplete engraftment have been encountered. Treosulfan, a busulfan analog with a low extramedullary toxicity profile, has been used successfully as part of a myeloablative conditioning regimen in the allogeneic setting in SCD. To further minimize toxicity of conditioning, noncytotoxic monoclonal antibodies that clear stem cells from the marrow niche, such as anti-c-Kit (ACK2), have been considered. Using a murine model of SCD, we sought to determine whether nonmyeloablative conditioning followed by transplantation with syngeneic BM cells could ameliorate the disease phenotype. Treosulfan and ACK2, in a dose-dependent manner, decreased BM cellularity and induced cytopenia in SCD mice. Conditioning with treosulfan alone at nonmyeloablative dosing (3.6 g/kg), followed by transplantation with syngeneic BM donor cells, permitted long-term mixed-donor chimerism. Level of chimerism correlated with improvement in hematologic parameters, normalization of urine osmolality, and improvement in liver and spleen pathology. Addition of ACK2 to treosulfan conditioning did not enhance engraftment. Our data suggests that pretransplant conditioning with treosulfan alone may allow sufficient erythroid engraftment to reverse manifestations of SCD, with clinical application as a preparative regimen in SCD patients undergoing gene-modified autologous HSCT.

Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction.

CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (ß(A)/[ß(S)+ß(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications.

Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells.

Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism whereby telomerase deficiency and subsequent shortened telomeres initiate a DDR and create a pro-oxidant environment, especially in cells carrying the TINF2 mutations. Finally, the ameliorative effects of antioxidants in vitro suggest this could translate to therapeutic benefits in DC patients.

Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting.

Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

DNA damage responses and oxidative stress in dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21(WAF). N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.

The p53/p21(WAF/CIP) pathway mediates oxidative stress and senescence in dyskeratosis congenita cells with telomerase insufficiency.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal growth arrest, leading to senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in genes for telomere binding proteins or components of telomerase give rise to the premature aging disorder dyskeratosis congenita (DC), which is characterized by extremely short telomeres and an aging phenotype. The current study demonstrates that DC cells signal a DNA damage response through p53 and its downstream mediator, p21(WAF/CIP), which is accompanied by an elevation in steady-state levels of superoxide and percent glutathione disulfide, both indicators of oxidative stress. Poor proliferation of DC cells can be partially overcome by reducing O(2) tension from 21% to 4%. Further, restoring telomerase activity or inhibiting p53 or p21(WAF/CIP) significantly mitigated growth inhibition as well as caused a significant decrease in steady-state levels of superoxide. Our results support a model in which telomerase insufficiency in DC leads to p21(WAF/CIP) signaling, via p53, to cause increased steady-state levels of superoxide, metabolic oxidative stress, and senescence.

Telomere restoration and extension of proliferative lifespan in dyskeratosis congenita fibroblasts.

Dyskeratosis congenita (DC), an inherited bone marrow failure syndrome, is caused by defects in telomerase. Somatic cells from DC patients have shortened telomeres and clinical symptoms are most pronounced in organs with a high cell turnover, including those involved in hematopoiesis and skin function. We previously identified an autosomal dominant (AD) form of DC that is caused by mutations in the telomerase RNA component (TER). In this study, we evaluated whether retroviral expression of TER and/or telomerase reverse transcriptase (TERT), the catalytic component of telomerase, could extend telomere length and rescue AD DC cells from a phenotype characteristic of early senescence. Exogenous TER expression, without TERT, could not activate telomerase in AD DC skin fibroblasts. Transduction of TERT alone, however, provided AD DC cells with sufficient telomerase activity to extend average telomere length and proliferative capacity. Interestingly, we found that expression of TER and TERT together resulted in extension of lifespan and higher levels of telomerase and longer telomeres than expression of TERT alone in both AD DC and normal cells. Our results provide evidence that AD DC cells can be rescued from defects in telomere maintenance and proliferation, and that coexpression of TERT and TER together provides a more efficient means to elongate telomeres than expression of TERT alone. Similar strategies may be useful for ameliorating the detrimental effects of telomere shortening in AD DC and other diseases associated with telomerase or telomere defects.

Systematic screening for subtelomeric anomalies in a clinical sample of autism.

High-resolution karyotyping detects cytogenetic anomalies in 5-10% of cases of autism. Karyotyping, however, may fail to detect abnormalities of chromosome subtelomeres, which are gene rich regions prone to anomalies. We assessed whether panels of FISH probes targeted for subtelomeres could detect abnormalities beyond those identified by karyotyping in 104 individuals with Pervasive Developmental Disorders (PDDs) drawn from a general clinical population. Four anomalies were detected by karyotyping, while no additional anomalies were detected by subtelomere FISH or by probes targeted for 15q11.2q13 or 22q11.2 in subgroups of our sample. We conclude that while karyotyping may be more broadly indicated for autism than previously supposed, subtelomere FISH appears less likely to be a useful screening tool for unselected PDD populations.