Association of muscle disorders in late postmenopausal women according to the type of experienced menopause.

OBJECTIVE: Musculoskeletal disorders frequently affect postmenopausal women. This study aims to compare muscle disorders between women according to the type of experienced menopause: premature (PM) or normal age of menopause (NAM). METHODS: This was a cross-sectional study conducted in nine Latin American countries in which late postmenopausal women (55 to 70 years) were surveyed with a general questionnaire, the Menopause Rating Scale (MRS: item #4 exploring musculoskeletal discomfort), and the trength, assistance with walking, rising from a chair, climbing stairs, and falling questionnaire (risk of sarcopenia). RESULTS: A total of 644 women were included: 468 who had NAM, and 176 who had PM (116 spontaneous and 60 surgical). The overall mean age of the participants was 60.9 ± 4.2 years. Women who had PM experienced more musculoskeletal discomfort (33.5% vs 20.9%, P < 0.001) and a higher likelihood of sarcopenia (35.2% vs 19.9%, P < 0.001) than women who had a NAM. Women who had surgical PM exhibited a higher prevalence of severe musculoskeletal discomfort (46.7% vs 29.3%, P < 0.02) and a higher likelihood of sarcopenia (45.0% vs 27.6%, P < 0.02) than women who had a NAM. After adjusting for covariates (age, body mass index, menopausal hormone therapy use, physical activity, education, cigarette consumption, use of antidepressants, sexual activity, comorbidities, and having a partner), our logistic regression model determined that spontaneous PM was not associated with higher odds of musculoskeletal discomfort and higher odds of sarcopenia. On the other hand, women who had surgical PM were more likely to experience musculoskeletal discomforts (odds ratio: 2.26; 95% confidence interval: 1.22-4.17) and higher odds for sarcopenia (odds ratio: 2.05; 95% confidence interval: 1.16-3.65) as compared to women who experienced a NAM. CONCLUSIONS: Women experiencing surgical PM have a higher likelihood of developing muscle disorders. This underscores the potential significance of hormonal levels in influencing musculoskeletal health during postmenopause.

Anxiety but not menopausal status influences the risk of long-COVID-19 syndrome in women living in Latin America.

OBJECTIVE: To study sociodemographic and clinical factors associated with the long-COVID-19 syndrome among women living in Latin American countries using undirected and directed methods. METHOD: We studied 347 patients with COVID-19 (confirmed by polymerase chain reaction) living in nine Latin American countries between May 2021 and July 2022, including 70 premenopausal, 48 perimenopausal, and 229 postmenopausal women. We compared the sociodemographic and general health information of women with (n = 164) and without (n = 183) the long-COVID-19 syndrome. They also completed the Connor-Davidson Resilience Scale, the Fear of COVID-19 Scale, the Jenkins Sleep Scale, and the Menopause Rating Scale to define the minimum set of variables for adjustment. We designed a directed acyclic graph (DAG) to identify factors related to the long-COVID-19 syndrome. Data were submitted to categorical logistic regression analyses. Results are reported as means and standard deviations or ß-coefficients and 95 % confidence intervals. RESULTS: Women with long-COVID-19 syndrome had a poor lifestyle, severe menopause symptoms, hypertension, insomnia, depression, anxiety, chronic diseases/conditions, risk of hospitalization, sleep disturbance, and low menopause-related quality of life compared to women without the syndrome. The DAG identified the following long-COVID-19 covariates: age, obesity, anxiety, depression, cancer, lifestyle, smoking, and menstrual status. A multivariable logistic model with these covariates indicated that anxiety is the only factor to be significantly associated with long-COVID-19 syndrome, whereas other covariates were confounding factors. There was no significant influence of menopausal status on the long-COVID-19 syndrome. CONCLUSION: Among factors selected by the DAG, only anxiety was significantly associated with the long-COVID-19. There was no significant influence of the menopause status on the long-COVID-19 syndrome in the studied population.

Generation of a heterozygous and a homozygous CSF1R knockout line from iPSC using CRISPR/Cas9.

Mutations in Colony-stimulating factor 1 receptor (CSF1R) lead to CSF1R-related leukoencephalopathy, also known as Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a rapidly progressing neurodegenerative disease with severe cognitive and motor impairment. In this study, a homozygous and a heterozygous CSF1R knockout induced pluripotent stem cell (iPSC) line were generated by CRISPR/Cas9-based gene editing. These in vitro models will provide a helpful tool for investigating the still largely unknown pathophysiology of CSF1R-related leukoencephalopathy.

Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of ß-Hemoglobinopathies.

Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for ß-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.

Post-COVID-19 syndrome in a sample of climacteric women living in Latin America.

OBJECTIVE: The aim of this study was to assess resilience, fear of COVID-19, sleep disorders, and menopause-related symptoms after the acute phase of COVID-19 in middle-aged women with positive reverse transcription-polymerase chain reaction and noninfected women. METHODS: This is a cross-sectional, analytical study of climacteric women from 9 Latin American countries, aged 40-64 years, attending a routine health checkup. We evaluated clinical characteristics and used the Connor-Davidson Resilience Scale, the Fear of COVID-19 Scale, the Jenkins Sleep Scale, and the Menopause Rating Scale to evaluate their health. RESULTS: A total of 1,238 women were studied, including 304 who were positive for COVID-19 reverse transcription-polymerase chain reaction. The median (interquartile range) age was 53 (12) years; years of studies, 16 (6); body mass index, 25.6 (5.1) kg/m 2 ; and time since first COVID-19 symptom, 8 (6) months. COVID-19 patients reported fatigability (18.8%), joint and muscular discomfort (14.1%), and anosmia (9.5%). They had a significantly lower resilience score (26.87 ± 8.94 vs 29.94 ± 6.65), higher Fear of COVID-19 score (17.55 ± 7.44 vs 15.61 ± 6.34), and a higher Jenkins Scale score (6.10 ± 5.70 vs 5.09 ± 5.32) compared with control women. A logistic regression model confirmed these results. There was not a significant difference in the total Menopause Rating Scale score, although the odds ratios for both severe menopausal symptoms (1.34; 95% confidence interval, 1.02-1.76) and the use of hypnotics were higher in women with COVID-19 (1.80; 95% confidence interval, 1.29-2.50) compared with those without infection. We found no decrease in studied outcomes between the initial 7 months versus those reported after 8 to 18 months since first COVID-19 symptoms. CONCLUSIONS: COVID-19 climacteric women have sleep disorders, lower resilience and higher fear of COVID-19.

Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.

X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.

Factors affecting climacteric women with SARS-CoV-2 infection: A multinational Latin America study (REDLINC XI).

OBJECTIVE: To evaluate the association between factors, especially those linked to the climacteric, and a history of COVID-19 infection. METHODS: This was an observational, cross-sectional, and analytical study in which women from ten Latin American countries, aged 40-64, who attended a routine health check-up were invited to participate. A positive history for COVID-19 was based on reverse transcription-polymerase chain reaction reports. We evaluated sociodemographic, clinical, lifestyle, anthropometric variables, and menopausal symptoms using the Menopause Rating Scale (MRS). RESULTS: A total of 1238 women were included for analysis, of whom 304 (24.6 %) had a positive history for COVID-19. The median [interquartile range: IQR] age of participants was 53 [IQR 12] years, duration of formal education was 16 [6] years, body mass index 25.6 [5.1] kg/m2, and total MRS score 10 [13]. In a logistic regression model, factors positively associated with COVID-19 included postmenopausal status and having a family history of dementia (OR: 1.53; 95 % CI: 1.13-2.07, and 2.40; 1.65-3.48, respectively), whereas negatively associated were use of menopausal hormone therapy (current or past), being a housewife, and being nulliparous (OR: 0.47; 95 % CI: 0.30-0.73; 0.72; 0.53-0.97 and 0.56; 0.34-0.92, respectively). Smoking, being sexually active, and use of hypnotics were also factors positively associated with COVID-19. CONCLUSION: Postmenopausal status and a family history of dementia were more frequent among women who had had COVID-19, and the infection was less frequent among current or past menopause hormone therapy users and in those with less physical contact.

Female orgasmic dysfunction and severe climacteric symptomatology in women aged 40 to 59 years: an independent association from an analysis of a multicenter Latin American study.

OBJECTIVE: To evaluate the association between the severity of climacteric symptoms (CS) and orgasmic dysfunction (OD), controlled by demographic, clinical, and partner variables. METHODS: We carried out a secondary analysis of a multicenter Latin American cross-sectional study that surveyed sexually active women 40 to 59 years old. We assessed CS (global, somatic, psychological, or urogenital domains) and OD. Also, we explored clinical variables and partner sexual conditions. We performed logistic regression models with nonparametric bootstrap resampling to estimate crude and adjusted odds ratios (aOR) with 95% confidence intervals (CI). RESULTS: We included data of 5,391 women in the analysis. Regarding CS, 24.8%, 10.8%, 28.4%, and 32.9% had respectively severe symptoms according to total, somatic, psychological, and urogenital domain scores of the Menopause Rating Scale. OD was found in 25.4% of women. The adjusted model (including menopausal status and partner sexual dysfunction) showed that severe CS increased the odds of OD (aOR = 2.77; 95% CI: 2.41-3.19 [total Menopause Rating Scale score]; aOR = 1.65; 95% CI: 1.37-2.00 [somatic domain]; aOR = 2.02; 95% CI: 1.76-2.32 [psychological domain] and aOR = 3.89; 95% CI: 3.40-4.45 [urogenital]). CONCLUSIONS: Severe CS were associated with OD independently of demographic, clinical, and partner variables. Severe urogenital symptoms had the strongest association.

Tracing the connections between international business and communicable diseases.

We posit that international business and the emergence and spread of communicable diseases are intrinsically connected. To support our arguments, we first start with a historical timeline that traces the connections between international business and communicable diseases back to the sixth century. Second, following the epidemiology of communicable diseases, we identify two crucial transitions related to international business: the emergence of epidemics within a host country and the shift from epidemics to global pandemics. Third, we highlight international business contextual factors (host country regulatory quality, urbanization, trade barriers, global migration) and multinationals' activities (foreign direct investment, corporate political activity, global supply chain management, international travel) that could accelerate each transition. Finally, building on public health insights, we suggest research implications for business scholars on how to integrate human health challenges into their studies and practical implications for global managers on how to help prevent the emergence and spread of communicable diseases.

Nous postulons que les affaires internationales et l'émergence et la propagation des maladies transmissibles sont intrinsèquement liées. Afin d'étayer nos arguments, nous commençons par une chronologie historique qui retrace les liens entre les affaires internationales et les maladies transmissibles jusqu'au sixième siècle. Deuxièmement, en suivant l'épidémiologie des maladies transmissibles, nous identifions deux transitions cruciales liées aux affaires internationales: l'émergence des épidémies dans un pays d'accueil et le passage des épidémies aux pandémies mondiales. Troisièmement, nous mettons en lumière les facteurs contextuels des affaires internationales (qualité de la réglementation du pays d'accueil, urbanisation, barrières commerciales, migration au niveau mondial) et les activités des multinationales (investissement direct à l'étranger, activité politique des entreprises, gestion de la chaîne d'approvisionnement mondiale, voyages internationaux) qui pourraient accélérer chaque transition. Enfin, nous appuyant sur la littérature de la santé publique, nous suggérons, d'une part, des implications de recherche pour les chercheurs en management sur la manière d'intégrer des défis liés à la santé humaine dans leurs travaux et, d'autre part, des implications pratiques pour les managers internationaux sur la manière d'aider à prévenir l'émergence et la propagation des maladies transmissibles.

Planteamos que los negocios internacionales y el surgimiento y propagación de enfermedades transmisibles están conectados intrínsecamente. Para apoyar nuestros argumentos, primero comenzamos con un cuadro cronológico que rastrea las conexiones entre negocios internacionales y enfermedades transmisibles hasta el siglo VI. Segundo, siguiendo la epidemiologia de las enfermedades transmisibles, identificamos dos transiciones cruciales relacionados con los negocios internacionales: el surgimiento de epidemia en un país anfitrión y el cambio de epidemia a pandemia global. Tercero, resaltamos los factores contextuales de negocios internacionales (calidad regulatoria del país anfitrión, urbanización, barreras comerciales, migración global) y las actividades de las multinacionales (inversión extranjera directa, actividad política corporativa, gestión de la cadena de abastecimiento, viajes internacionales) que podrían acelerar cada transición. Finalmente, sobre la base de los aportes de la salud pública, sugerimos implicaciones de investigación para académicos de negocios internacionales sobre cómo integrar los retos de la salud pública en sus estudios y las repercusiones prácticas para los gerentes globales sobre cómo ayudar a prevenir el surgimiento y propagación de enfermedades transmisibles.

Postulamos que negócios internacionais e o surgimento e disseminação de doenças transmissíveis estão intrinsecamente conectados. Para apoiar nossos argumentos, começamos com uma linha do tempo histórica que traça as conexões entre negócios internacionais e doenças transmissíveis até o século VI. Em segundo lugar, seguindo a epidemiologia de doenças transmissíveis, identificamos duas transições cruciais relacionadas a negócios internacionais: o surgimento de epidemias em um país anfitrião e a mudança de epidemias para pandemias globais. Em terceiro lugar, destacamos fatores contextuais de negócios internacionais (qualidade regulatória do país anfitrião, urbanização, barreiras comerciais, migração global) e atividades de multinacionais (investimento estrangeiro direto, atividade política corporativa, gestão da cadeia de suprimentos global, viagens internacionais) que poderiam acelerar cada transição. Por fim, com base em insights de saúde pública, sugerimos implicações de pesquisa para acadêmicos de negócios sobre como integrar desafios de saúde humana em seus estudos e implicações práticas para gerentes globais sobre como ajudar a prevenir o surgimento e a disseminação de doenças transmissíveis.

A Mutation-Agnostic Hematopoietic Stem Cell Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is a rare genetic disorder caused by mutations in the Arylsulfatase-A (ARSA) gene. The enzyme plays a key role in sulfatide metabolism in brain cells, and its deficiency leads to neurodegeneration. The clinical manifestations of MLD include stagnation and decline of motor and cognitive function, leading to premature death with limited standard treatment options. Here, we describe a mutation-agnostic hematopoietic stem and progenitor cell (HSPC) gene therapy using CRISPR-Cas9 and AAV6 repair template as a prospective treatment option for MLD. Our strategy achieved efficient insertions and deletions (>87%) and a high level of gene integration (>47%) at the ARSA locus in human bone marrow-derived HSPCs, with no detectable off-target editing. As a proof of concept, we tested our mutation-agnostic therapy in HSPCs derived from two MLD patients with distinct mutations and demonstrated restoration of ARSA enzyme activity (>30-fold improvement) equivalent to healthy adults. In summary, our investigation enabled a mutation-agnostic therapy for MLD patients with proven efficacy and strong potential for clinical translation.

Universal Gene Correction Approaches for ß-hemoglobinopathies Using CRISPR-Cas9 and Adeno-Associated Virus Serotype 6 Donor Templates.

Mutations in the human ß-globin gene are the cause of ß-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (ß0/ß+) and homozygous (ß0/ß0, ß+/ß+) ß-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of ß-hemoglobinopathies.

Autoantibodies against the immunodominant sCha epitope discriminate the risk of sudden death in chronic Chagas cardiomyopathy.

In Chagas disease (ChD) caused by Trypanosoma cruzi, new biomarkers to predict chronic cardiac pathology are urgently needed. Previous studies in chagasic patients with mild symptomatology showed that antibodies against the immunodominant R3 epitope of sCha, a fragment of the human basic helix-loop-helix transcription factor like 5, correlated with cardiac pathology. To validate sCha as a biomarker and to understand the origin of anti-sCha antibodies, we conducted a multicenter study with several cohorts of chagasic patients with severe cardiac symptomatology. We found that levels of antibodies against sCha discriminated the high risk of sudden death, indicating they could be useful for ChD prognosis. We investigated the origin of the antibodies and performed an alanine scan of the R3 epitope. We identified a minimal epitope MRQLD, and a BLAST search retrieved several T. cruzi antigens. Five of the hits had known or putative functions, of which phosphonopyruvate decarboxylase showed the highest cross-reactivity with sCha, confirming the role of molecular mimicry in the development of anti-sCha antibodies. Altogether, we demonstrate that the development of antibodies against sCha, which originated by molecular mimicry with T. cruzi antigens, could discriminate electrocardiographic alterations associated with a high risk of sudden death.

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of ß-hemoglobinopathies.

ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.

Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.

ß-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the ß-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of ß-hemoglobinopathies.

CRISPR/Cas9 technology: towards a new generation of improved CAR-T cells for anticancer therapies.

Chimeric antigen receptor (CAR)-modified T cells have raised among other immunotherapies for cancer treatment, being implemented against B-cell malignancies. Despite the promising outcomes of this innovative technology, CAR-T cells are not exempt from limitations that must yet to be overcome in order to provide reliable and more efficient treatments against other types of cancer. The purpose of this review is to shed light on the field of CAR-T cell gene editing for therapy universalization and further enhancement of antitumor function. Several studies have proven that the disruption of certain key genes is essential to boost immunosuppressive resistance, prevention of fratricide, and clinical safety. Due to its unparalleled simplicity, feasibility to edit multiple gene targets simultaneously, and affordability, CRISPR/CRISPR-associated protein 9 system has been proposed in different clinical trials for such CAR-T cell improvement. The combination of such powerful technologies is expected to provide a new generation of CAR-T cell-based immunotherapies for clinical application.

Hematopoietic stem cell gene therapy: The optimal use of lentivirus and gene editing approaches.

Due to pioneering in vitro investigations on gene modification, gene engineering platforms have incredibly improved to a safer and more powerful tool for the treatment of multiple blood and immune disorders. Likewise, several clinical trials have been initiated combining autologous hematopoietic stem cell transplantation (auto-HSCT) with gene therapy (GT) tools. As several GT modalities such as lentivirus and gene editing tools have a long developmental path ahead to diminish its negative side effects, it is hard to decide which modality is optimal for treating a specific disease. Gene transfer by lentiviruses is the platform of choice for loss-of-mutation diseases, whereas gene correction/addition or gene disruption by gene editing tools, mainly CRISPR/Cas9, is likely to be more efficient in diseases where tight regulation is needed. Therefore, in this review, we compiled pertinent information about lentiviral gene transfer and CRISPR/Cas9 gene editing, their evolution to a safer platform for HSCT, and their applications on other types of gene disorders based on the etiology of the disease and cell fitness.

CRISPR/Cas9-modified hematopoietic stem cells-present and future perspectives for stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a standard therapeutic intervention for hematological malignancies and several monogenic diseases. However, this approach has limitations related to lack of a suitable donor, graft-versus-host disease and infectious complications due to immune suppression. On the contrary, autologous HSCT diminishes the negative effects of allogeneic HSCT. Despite the good efficacy, earlier gene therapy trials with autologous HSCs and viral vectors have raised serious safety concerns. However, the CRISPR/Cas9-edited autologous HSCs have been proposed to be an alternative option with a high safety profile. In this review, we summarized the possibility of CRISPR/Cas9-mediated autologous HSCT as a potential treatment option for various diseases supported by preclinical gene-editing studies. Furthermore, we discussed future clinical perspectives and possible clinical grade improvements of CRISPR/cas9-mediated autologous HSCT.

Mitochondria and Coenzyme Q10 in the Pathogenesis of Preeclampsia.

Hypertensive disorders during pregnancy constitute one of the main causes of maternal and perinatal morbidity and mortality across the world and particularly in developing countries such as Ecuador. However, despite its impact on public health, the primary pathophysiological processes involved are yet to be elucidated. It has been proposed, among other theories, that an abnormal placentation may induce an endothelial dysfunction, which is ultimately responsible for the final clinical manifestations. Mitochondria, particularly from trophoblastic cells, are responsible for the production of energy, which is extremely important for normal placentation. The malfunction in this supply of energy may produce higher levels of free radicals. In both production of energy and free radicals, coenzyme Q10 (CoQ10) plays a crucial role in electron transport. As such, the role of CoQ10 in the genesis and prevention of preeclampsia has become the focus of a number of research groups, including that of the authors. Developing an in-depth understanding of these mechanisms might allow us to design new and feasible strategies with which we can reduce preeclampsia, particularly in the Latin-American countries.

[Evolution and spatial analysis of leishmaniasis cases in a rural area bordering a community outbreak in Madrid: 2001-2017]. / Evolución y análisis espacial de los casos de leishmaniasis en una zona rural colindante al brote comunitario de Madrid: 2001-2017.

OBJECTIVE: Leishmaniasis is a zoonotic disease, widely spread all over the world, and an endemic disease in some Spanish regions. Within the Autonomous Region of Madrid, some south populations were affected by an outbreak from 2009. This outbreak had special features and implications related to epidemiology, reservoir and environment intervention. An increased rate in rural areas bordering the outbreak area was detected by epidemiological surveillance. This area has the same environment characteristics. The objective of this study was to research and analyze the evolution of leishmaniasis cases declared in a rural area and their comparison with the cases of the outbreak area. METHODS: The cases declared to Epidemiology Surveillance Network have been used. Kulldorff´s tools were used for the cluster analysis. A hot spot analysis (Getis-Ord Gi*) was made. Leporidae and vector information of the area was related to the location of cases. RESULTS: The number of observed cases exceeded the number of expected cases in this area (epidemic index 7.8 in 2013), after the outbreak. This showed a relation between both incidence rates. It seemed to be spatial correlation when the number of cases was analyzed by census sections and grids (Moran´s I 0,208; z= 9,336). Using the hot-spot analysis, a higher incidence of the study area could be observed, and within Health Basic Area of Griñón after the outbreak years. A spatial relation between cases and a greater presence of vectors and leporidae was found. CONCLUSIONS: The distribution of leishmaniasis cases, in the period and study area suggests a link between the community outbreak and the increase of cases in the study area last years, overall in the rural area. It would be useful to strengthen surveillance and it should apply effective measures used in the bordering area if they were necessary. These measures can help to control the spread of the outbreak.

OBJETIVO: La leishmaniasis en una enfermedad zoonótica muy extendida a nivel mundial y es endémica en algunas regiones de España. Desde 2009 un brote de leishmaniasis ha afectado a algunos municipios del sur de Madrid con características e implicaciones especiales en cuanto a epidemiología, reservorio e intervención medioambiental. Se ha observado un aumento de casos en las zonas rurales adyacentes con características ambientales similares. El objetivo de este trabajo fue estudiar y analizar la evolución de los casos de leishmaniasis declarados en una zona rural colindante y compararlos con el brote comunitario. METODOS: Se utilizó la información de los casos declarados a la Red de Vigilancia Epidemiológica de la Comunidad de Madrid en la zona de estudio desde 2001-2017 y se compararon las tasas de incidencia con las del área del brote. Se realizó un análisis espacial de los casos y de los conglomerados para cuatro unidades espaciales. Se realizó análisis de conglomerados según la técnica de Kulldorff y análisis de puntos calientes según Gi* de Getis-Ord. Se relacionó la información disponible de lepóridos y vectores con la ubicación de los casos. RESULTADOS: Los casos observados en el área de estudio superaron a los esperados tras al inicio del brote (índice epidémico 7,8 en 2013) mostrándose una relación gráfica entre las tasas de incidencia. Existió una autocorrelación espacial cuando se analizaron el número de casos por secciones censales y por cuadrículas (índice de Moran de 0,208; z= 9,336). En el análisis de puntos calientes se pudo apreciar una mayor incidencia en el área de estudio en el periodo posterior al brote, en particular en la Zona Básica de Salud de Griñón. Se constató una relación espacial entre casos y zonas de mayor presencia de lepóridos y vectores. CONCLUSIONES: La distribución de casos de leishmaniasis en el periodo y área de estudio sugiere que puede existir una asociación entre el brote comunitario y el aumento de casos de los últimos años en el área de estudio, específicamente en la zona rural, por lo que sería necesario reforzar la vigilancia y aplicar medidas de control ambiental en caso necesario, lo cual puede contribuir a limitar la extensión del brote.

Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors.

BACKGROUND: ß-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate ß-globin expression. Although lentiviral-mediated expression of ß-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control. METHODS: We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common ß-thalassemia splicing variant HBBIVS1-110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs). RESULTS: Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1-110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells-HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs. CONCLUSION: Our approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for ß-thalassemia.