Manufacturing Cell and Gene Therapies: Challenges in Clinical Translation

The safety and efficacy of both cell and gene therapies have been demonstrated in numerous preclinical and clinical trials. Chimeric antigen receptor T (CAR-T) cell therapy, which leverages the technologies of both cell and gene therapies, has also shown great promise for treating various cancers. Advancements in pertinent fields have also highlighted challenges faced while manufacturing cell and gene therapy products. Potential problems and obstacles must be addressed to ease the clinical translation of individual therapies. Literature reviews of representative cell-based, gene-based, and cell-based gene therapies with regard to their general manufacturing processes, the challenges faced during manufacturing, and QC specifications are limited. We review the general manufacturing processes of cell and gene therapies, including those involving mesenchymal stem cells, viral vectors, and CAR-T cells. The complexities associated with the manufacturing processes and subsequent QC/validation processes may present challenges that could impede the clinical progression of the products. This article addresses these potential challenges. Further, we discuss the use of the manufacturing model and its impact on cell and gene therapy.

Nervonic Acid Inhibits Replicative Senescence of Human Wharton’s Jelly-Derived Mesenchymal Stem Cells

Cellular senescence causes cell cycle arrest and promotes permanent cessation of proliferation. Since the senescence of mesenchymal stem cells (MSCs) reduces proliferation and multipotency and increases immunogenicity, aged MSCs are not suitable for cell therapy. Therefore, it is important to inhibit cellular senescence in MSCs. It has recently been reported that metabolites can control aging diseases. Therefore, we aimed to identify novel metabolites that regulate the replicative senescence in MSCs. Using a fecal metabolites library, we identified nervonic acid (NA) as a candidate metabolite for replicative senescence regulation. In replicative senescent MSCs, NA reduced senescence-associated β-galactosidase positive cells, the expression of senescence-related genes, as well as increased stemness and adipogenesis. Moreover, in non-senescent MSCs, NA treatment delayed senescence caused by sequential subculture and promoted proliferation. We confirmed, for the first time, that NA delayed and inhibited cellular senescence.Considering optimal concentration, duration, and timing of drug treatment, NA is a novel potential metabolite that can be used in the development of technologies that regulate cellular senescence.

Contemporary Utilization and Outcomes of Resuscitative Endovascular Balloon Occlusion of the Aorta for Traumatic Noncompressible Torso Hemorrhage in Korea: A Retrospective Multi-Center Study

Purpose@#This study aimed to evaluate the utilization and outcomes of resuscitative endovascular balloon occlusion of the aorta (REBOA) in managing noncompressible torso hemorrhage (NCTH) among trauma patients in Korea. The evolution of REBOA and its impact on patient survival was investigated as well as predictors of mortality. @*Methods@#This retrospective study included 234 post-REBOA patients from 5 leading regional trauma centers across Korea between 2016 and 2021. Primary outcomes were in-hospital mortality, and secondary outcomes were various clinical parameters regarding REBOA, overall treatment flow, and complications. For comparative analyses, patients were dichotomized into in-hospital non-survivors or survivors. Then, generalized additive and linear regression models were used to evaluate the trend of in-hospital mortality. @*Results@#The overall in-hospital mortality was 65.4%. The survivors had a higher proportion of responders following REBOA (87.7% vs 62.7%, p < 0.001). Key variables influencing outcomes included total occlusion time, red blood cell transfusion volume within the first 24 hours, revised trauma score, and systolic blood pressure gap. These factors significantly correlated with mortality rates in multivariate logistic regression. @*Conclusion@#Over 6 years, survival rates for NCTH patients undergoing REBOA in Korea have shown improvement. Despite diverse REBOA protocols across institutions, the results underscore the need for continued research, standardized practices, and national quality control measures to further optimize patient outcome and establish more effective treatment protocols for NCTH.

Transsplenic Ultrasound-Guided Balloon Positioning During a Zone 1 Resuscitative Endovascular Balloon Occlusion of the Aorta: A Case Report

Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an effective resuscitative modality to temporize noncompressible truncal hemorrhage. Confirming the proper position of the balloon catheter in the target aortic zone is vital. Currently, there is a need for nonradiographical methods. This would overcome the drawbacks of conventional imaging modalities, such as fluoroscopy. Several studies have suggested ultrasound-guided visualization via subxiphoid, transperitoneal, or transesophageal views as an alternative to conventional imaging methods. However, such views are easily obscured in emergency settings. Herein, we report the case of a 70-year-old patient who was successfully resuscitated by REBOA under the guidance of transsplenic ultrasound. REBOA was safely performed using transsplenic visualization without fluoroscopy.

A course on endovascular training for resuscitative endovascular balloon occlusion of the aorta: a pilot study for residents and specialists

Purpose@#Resuscitative endovascular balloon occlusion of the aorta (REBOA) has emerged as a salvage technique changing the paradigm in the management of noncompressible torso hemorrhage. However, training for the REBOA procedure is rarely performed. The endovascular training for REBOA (ET-REBOA) course was conducted to develop the endovascular skills of participants. @*Methods@#Sixteen residents and 12 specialists participated in this educational course. All participants were provided with precourse learning materials. The ET-REBOA course consisted of 2 sections; an ultrasound-guided sheath insertion on the puncture model, and a balloon manipulation on the vascular circuit model. A 13-item procedure checklist and the time required to perform the procedure were examined. Pre/post self-reported confidence score and course satisfaction questionnaire were obtained. @*Results@#Twenty-eight participants performed the 56 REBOA procedures. On the first attempt, the median total time for REBOA from ultrasound-guided vascular access to balloon inflation was 1,139 ± 250 seconds in the resident group and 828 ± 280 seconds in the specialist group. The median shortened time for completion was 273 seconds and 290 seconds respectively. A significant decrease in procedure task time was observed between first and second attempts in the resident group (P = 0.016), specialist group (P = 0.004), and in total among all participants (P < 0.001). @*Conclusion@#The ET-REBOA course significantly decreased the time taken to perform the REBOA procedure with high satisfaction of the participants. The course could be an effective curriculum for the development of endovascular skills for performing REBOA.

Clinical Outcomes of Arteriovenous Graft in End-Stage Renal Disease Patients with an Unsuitable Cephalic Vein for Hemodialysis Access

Background@#As the population of patients with end-stage renal disease has grown older, the proportion of patients with poorly preserved vasculature has concomitantly increased. Thus, arteriovenous grafts (AVG) have been used more frequently to access blood vessels for hemodialysis. Despite this increasing demand, studies of AVG are limited. In this study, we examined the surgical outcomes of upper-limb AVG creation. @*Methods@#Among the arteriovenous fistula formation procedures performed between January 2014 and March 2019 at Dankook University Hospital, 42 cases involved AVG creation. We compared patients in whom the axillary vein was used (group A; brachioaxillary AVG [B-Ax AVG]; n=20) with those in whom upper limb veins were used (group B; brachiobasilic AVG or brachioantecubital AVG; n=22). @*Results@#The 1-year primary patency rate was higher in group A than in group B (57.9% vs. 41.7%; p=0.262). The incidence of postoperative complications was not significantly different between groups. @*Conclusion@#AVG using the axillary vein showed no major differences in safety or functionality compared to AVG using other veins. Therefore, accounting for age, underlying disease, and expected patient lifespan, B-Ax AVG can be considered an acceptable surgical method.

Impacts of Social Distancing During the COVID-19 Outbreaks in Korea: Level 1 Trauma Center Data of Domestic Incidents and Intentional Injury

Objectives@#As a protective measure to slow down the transmission of coronavirus disease 2019 in Korea, social distancing was implemented from February 29th , 2020. This study aimed to evaluate the prevalence of domestic incidents and intentional injury during March 2020 when social distancing was in effect. @*Methods@#There were 12,638 patients who visited the Level 1 trauma center of Chungnam province with injuries from domestic incidents, familial discord, and intentional injury. The prevalence of injuries during March 2020 was compared with the average of the previous 5 years, and the average for every March between 2015 and 2019. @*Results@#The prevalence of domestic incidents in March 2020 was significantly higher than the 5-year average, and the average for every March from 2015 to 2019 (p < 0.001). Familial discord (p = 0.002) and intentional injury (p = 0.031) were more frequently observed in March 2020. Adolescents showed a markedly higher level of intentional injury in March 2020 than in both the 5-year average (p = 0.031), and average for every March over the previous 5 years (p = 0.037). @*Conclusion@#The prevalence of domestic incidents and intentional injury were significantly higher during the period of social distancing in Korea. There is a need for social consensus, better policies, and psychological support services, especially if faced with a second or third wave of coronavirus disease.

Impacts of Social Distancing During the COVID-19 Outbreaks in Korea: Level 1 Trauma Center Data of Domestic Incidents and Intentional Injury

Objectives@#As a protective measure to slow down the transmission of coronavirus disease 2019 in Korea, social distancing was implemented from February 29th , 2020. This study aimed to evaluate the prevalence of domestic incidents and intentional injury during March 2020 when social distancing was in effect. @*Methods@#There were 12,638 patients who visited the Level 1 trauma center of Chungnam province with injuries from domestic incidents, familial discord, and intentional injury. The prevalence of injuries during March 2020 was compared with the average of the previous 5 years, and the average for every March between 2015 and 2019. @*Results@#The prevalence of domestic incidents in March 2020 was significantly higher than the 5-year average, and the average for every March from 2015 to 2019 (p < 0.001). Familial discord (p = 0.002) and intentional injury (p = 0.031) were more frequently observed in March 2020. Adolescents showed a markedly higher level of intentional injury in March 2020 than in both the 5-year average (p = 0.031), and average for every March over the previous 5 years (p = 0.037). @*Conclusion@#The prevalence of domestic incidents and intentional injury were significantly higher during the period of social distancing in Korea. There is a need for social consensus, better policies, and psychological support services, especially if faced with a second or third wave of coronavirus disease.

FGF-17 from Hypoxic Human Wharton's Jelly-Derived Mesenchymal Stem Cells Is Responsible for Maintenance of Cell Proliferation at Late Passages

BACKGROUND AND OBJECTIVES: Although it is well known that hypoxic culture conditions enhance proliferation of human mesenchymal stem cells, the exact mechanism is not fully understood. In this study, we investigated the effect of fibroblast growth factor (FGF)-17 from hypoxic human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on cell proliferation at late passages. METHODS AND RESULTS: hWJ-MSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum (FBS) in normoxic (21% O₂) and hypoxic (1% O₂) conditions. Protein antibody array was performed to analyze secretory proteins in conditioned medium from normoxic and hypoxic hWJ-MSCs at passage 10. Cell proliferation of hypoxic hWJ-MSCs was increased compared with normoxic hWJ-MSCs from passage 7 to 10, and expression of secretory FGF-17 was highly increased in conditioned medium from hypoxic hWJ-MSCs at passage 10. Knockdown of FGF-17 in hypoxic and normoxic hWJ-MSCs decreased cell proliferation, whereas treatment of hypoxic and normoxic hWJ-MSCs with recombinant protein FGF-17 increased their proliferation. Signal transduction of FGF-17 in hypoxic and normoxic hWJ-MSCs involved the ERK1/2 pathway. Cell phenotypes were not changed under either condition. Differentiation-related genes adiponectin, Runx2, and chondroadherin were downregulated in normoxic hWJ-MSCs treated with rFGF-17, and upregulated by siFGF-17. Expression of alkaline phosphatase (ALP), Runx2, and chondroadherin was upregulated in hypoxic hWJ-MSCs, and this effect was rescued by transfection with siFGF-17. Only chondroadherin was upregulated in hypoxic hWJ-MSCs with rFGF-17. CONCLUSIONS: In hypoxic culture conditions, FGF-17 from hypoxic hWJ-MSCs contributes to the maintenance of high proliferation at late passages through the ERK1/2 pathway.

The First Generation of iPSC Line from a Korean Alzheimer's Disease Patient Carrying APP-V715M Mutation Exhibits a Distinct Mitochondrial Dysfunction

Alzheimer's Disease (AD) is a progressive neurodegenerative disease, which is pathologically defined by the accumulation of amyloid plaques and hyper-phosphorylated tau aggregates in the brain. Mitochondrial dysfunction is also a prominent feature in AD, and the extracellular Aβ and phosphorylated tau result in the impaired mitochondrial dynamics. In this study, we generated an induced pluripotent stem cell (iPSC) line from an AD patient with amyloid precursor protein (APP) mutation (Val715Met; APP-V715M) for the first time. We demonstrated that both extracellular and intracellular levels of Aβ were dramatically increased in the APP-V715M iPSC-derived neurons. Furthermore, the APP-V715M iPSC-derived neurons exhibited high expression levels of phosphorylated tau (AT8), which was also detected in the soma and neurites by immunocytochemistry. We next investigated mitochondrial dynamics in the iPSC-derived neurons using Mito-tracker, which showed a significant decrease of anterograde and retrograde velocity in the APP-V715M iPSC-derived neurons. We also found that as the Aβ and tau pathology accumulates, fusion-related protein Mfn1 was decreased, whereas fission-related protein DRP1 was increased in the APP-V715M iPSC-derived neurons, compared with the control group. Taken together, we established the first iPSC line derived from an AD patient carrying APP-V715M mutation and showed that this iPSC-derived neurons exhibited typical AD pathological features, including a distinct mitochondrial dysfunction.

Heparin-free extracorporeal membrane oxygenation in a patient with severe pulmonary contusions and bronchial disruption

Pulmonary contusion complicated with endobronchial hemorrhage is potentially life-threatening, particularly in patients with tracheobronchial tree disruption and severe airway bleeding after blunt trauma, and pose a high mortality risk. In such cases, extracorporeal membrane oxygenation (ECMO) can be used as a salvage treatment modality. However, the use of ECMO for moribund trauma patients with respiratory failure may be limited for several reasons, such as intractable bleeding. In this case report, we describe a patient with severe bilateral pulmonary contusions with tracheobronchial tree disruption that was successfully treated using heparin-free venovenous ECMO.

iPSC Modeling of Presenilin1 Mutation in Alzheimer's Disease with Cerebellar Ataxia

Disease modeling of Alzheimer's disease (AD) has been hampered by the lack of suitable cellular models while animal models are mainly based on the overexpression of AD-related genes which often results in an overemphasis of certain pathways and is also confounded by aging. In this study, we therefore developed and used induced pluripotent stem cell (iPSC) lines from a middle-aged AD patient with a known presenilin 1 (PSEN1) mutation (Glu120Lys; PS1-E120K) and as a control, an elderly normal subject. Using this approach, we demonstrated that the extracellular accumulation of Aβ was dramatically increased in PS1-E120K iPSC-derived neurons compared with the control iPSC line. PS1-E120K iPSC-derived neurons also exhibited high levels of phosphorylated tau, as well as mitochondrial abnormalities and defective autophagy. Given that the effect of aging is lost with iPSC generation, these abnormal cellular features are therefore indicative of PSEN1-associated AD pathogenesis rather than primary changes associated with aging. Taken together, this iPSC-based approach of AD modeling can now be used to better understand AD pathogenesis as well as a tool for drug discovery.

Delayed massive hemothorax requiring surgery after blunt thoracic trauma over a 5-year period: complicating rib fracture with sharp edge associated with diaphragm injury

Delayed massive hemothorax requiring surgery is relatively uncommon and can potentially be life-threatening. Here, we aimed to describe the nature and cause of delayed massive hemothorax requiring immediate surgery. Over 5 years, 1,278 consecutive patients were admitted after blunt trauma. Delayed hemothorax is defined as presenting with a follow-up chest radiograph and computed tomography showing blunting or effusion. A massive hemothorax is defined as blood drainage >1,500 mL after closed thoracostomy and continuous bleeding at 200 mL/hr for at least four hours. Five patients were identified all requiring emergency surgery. Delayed massive hemothorax presented 63.6±21.3 hours after blunt chest trauma. All patients had superficial diaphragmatic lacerations caused by the sharp edge of a broken rib. The mean preoperative chest tube drainage was 3,126±463 mL. We emphasize the high-risk of massive hemothorax in patients who have a broken rib with sharp edges.

Effect of Single and Double Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Following Focal Cerebral Ischemia in Rats

Stem cell therapies are administered during the acute phase of stroke to preserve the penumbral tissues from ischemic injury. However, the effect of repeated cell therapy during the acute phase remains unclear. In this study, we investigated and compared the functional outcome of single (two days post-injury) and repeated (two and nine days post-injury) treatment with human umbilical cord derived mesenchymal stem cells (hUCB-MSCs) after middle cerebral artery occlusion (MCAO). The rotarod and limb placement tests were utilized to investigate functional outcomes, while infarct volume and tissue damage were measured by immunofluorescent staining for neovascularization, neurogenesis, apoptosis, and inflammation in the penumbral zones. We observed notable motor dysfunction and a significant decrease in infarcted brain volume, as well as increases in neurons and vessels in both single and repeated hUCB-MSC treatments compared to the control group. Interestingly, repeated administration of hUCB-MSCs was not found to elicit additional or synergistic improvements over monotherapy. This study suggests that a clearer understanding of the therapeutic window after stroke will facilitate the development of more efficient treatment protocols in the clinical application of stem cell therapy.

Emergency Department Thoracotomy with Clamshell Incision for Traumatic Cardiac Tamponade

The emergency department thoracotomy (EDT) is a bold and challenging procedure, which may be the only chance of survival for some moribund trauma patients. EDT provides ample exposure to the injury site of the heart, enabling an effective open cardiac massage. Clamshell thoracotomy is a rapid and simple procedure that provides excellent exposure to internal structures. Because EDT has more favorable outcomes for penetrating injuries than for blunt injuries, the indication for EDT in patients with blunt trauma should be well established. Cardiac tamponade is a life-threatening condition that requires emergent pericardial decompression. EDT has been associated with successful initial resuscitation for traumatic cardiac arrest with cardiac tamponade. To date, there has not been any reports of clamshell incision via EDT for trauma patients in South Korea. Hence, herein, we describe two cases in which EDT with clamshell incision was implemented for cardiac tamponade with cardiac arrest after blunt trauma.

Extracorporeal Membrane Oxygenation for the Support of a Potential Organ Donor with a Fatal Brain Injury before Brain Death Determination / 대한중환자의학회지

The shortage of available organ donors is a significant problem and various efforts have been made to avoid the loss of organ donors. Among these, extracorporeal membrane oxygenation (ECMO) has been introduced to help support and manage potential donors. Many traumatic brain injury patients have healthy organs that might be eligible for donation for transplantation. However, the condition of a donor with a fatal brain injury may rapidly deteriorate prior to brain death determination; this frequently results in the loss of eligible donors. Here, we report the use of venoarterial ECMO to support a potential donor with a fatal brain injury before brain death determination, and thereby preserve donor organs. The patient successfully donated his liver and kidneys after brain death determination.

Extracorporeal Membrane Oxygenation for the Support of a Potential Organ Donor with a Fatal Brain Injury before Brain Death Determination / 대한구급학회지

The shortage of available organ donors is a significant problem and various efforts have been made to avoid the loss of organ donors. Among these, extracorporeal membrane oxygenation (ECMO) has been introduced to help support and manage potential donors. Many traumatic brain injury patients have healthy organs that might be eligible for donation for transplantation. However, the condition of a donor with a fatal brain injury may rapidly deteriorate prior to brain death determination; this frequently results in the loss of eligible donors. Here, we report the use of venoarterial ECMO to support a potential donor with a fatal brain injury before brain death determination, and thereby preserve donor organs. The patient successfully donated his liver and kidneys after brain death determination.

Case of Acute Myeloid Leukemia with Mass of Eyelid and Conjunctiva in the Early Stage

PURPOSE: To report a case of an 82-year-old male with acute myeloid leukemia presenting with bilateral isolated conjunctival and eyelid masses. CASE SUMMARY: An 82-year-old male presented with a bilateral conjunctival mass and right eyelid mass occurring 10 days prior. He was diagnosed with prostate cancer 8 years ago and complete recovery was achieved using selective mass chemotherapy. He experienced a stroke 4 years ago and was treated using a carotid artery stent insertion and medication. In the initial laboratory test, hemoglobin was 13.7 g/dL and leukocyte count 5,530/mm3(neutrophil 74.4%, lymphocyte 10%, monocyte 11.8%). Light reflex, movement of extraocular muscle and fundus examination were all normal. Biopsy was performed 1 week after the first visit. Seven days after biopsy, he complained of sudden dyspnea and febrile sense and was admitted to the intensive care unit via the emergency room (ER). The laboratory tests performed in the ER showed hemoglobin was 9.6 g/dL and leukocyte count was 78,020/mm3(neutrophil 0%, lymphocyte 7%, monocyte 5%, promyelocyte 1%, metamyelocyte 4%, myelocyte 6%, blast 67%). The biopsy revealed diffuse proliferation of atypical plasmacytoid cells, consistent with leukemic infiltration. Under the diagnosis of acute myeloid leukemia, chemotherapy was administered. However, the patient died due to aggravated pneumonia. CONCLUSIONS: Even if non-specific findings appear on the peripheral blood tests, eyelid and conjunctival masses should be considered as possible tumors in acute myeloid leukemia.

Delayed Diagnosis of Pulmonary Artery Injury, Due to Blunt Trauma, which Mimicked Traumatic Lung Pseudocyst

A 67-year-old female presented to the emergency department with complaints of dyspnea and chest wall pain after a fall from a cultivator. Initial chest CT showed multiple left rib fractures, a loculated hematoma without active bleeding, and hemothorax. On the third day of admission, the chest X-ray showed an abrupt aggravation of haziness and the chest CT showed that the size of the hematoma had increased with active bleeding from the pulmonary artery. In cases of loculated hematomas adjacent to the hilum on CT scan, the diagnosis of pulmonary artery injury should be considered.

Surgical Treatment for Occlusion of Graft Arteriovenous Fistula in Patients Undergoing Hemodialysis

BACKGROUND: Maintenance of adequate vascular access for hemodialysis is important in patients with end-stage renal disease. Once arteriovenous fistula (AVF) occlusion occurs, the patient should be treated with rescue therapy. This study was performed to evaluate the results of a rescue therapy for AVF occlusion. METHODS: From January 2008 to December 2012, 47 patients who underwent surgical rescue therapy for AVF occlusion after graft AVF formation, were enrolled in this study. The patients were divided into two groups, namely the graft repair group (group A, n=19) and the thrombectomy group (group B, n=28). Postoperative results of both groups were analyzed retrospectively. RESULTS: There were no statistically significant differences in the clinical characteristics between the two groups. In terms of the duration of AVF patency after the first rescue therapy, group A showed a longer AVF patency duration than group B (24.5+/-21.9 months versus 17.7+/-13.6 months), but there was no statistically significant difference (p=0.310). In terms of the annual frequency of AVF occlusion after the rescue therapy of group A was lower than that of group B (0.59 versus 0.71), but there was no statistically significant difference (p=0.540). The AVF patency rates at 1, 2, 3, and 5 years after the first rescue therapy in group A were 52.6%, 31.5%, 21.0%, and 15.7%, respectively, and those in group B, they were 32.1%, 25.0%, 17.8%, and 7.14%, respectively. There was no statistically significant difference (p=0.402). CONCLUSION: Graft repair revealed comparable results. Although there was no statistically significant difference, the patent duration and annual frequency of AVF occlusion of group A were better than those of group B. Therefore, graft repair is considered as a safe and useful procedure for maintaining graft AVF.