Challenges of Robust RNAi-Mediated Gene Silencing in Aedes Mosquitoes.

In this study, we report the complexities and challenges associated with achieving robust RNA interference (RNAi)-mediated gene knockdown in the mosquitoes Aedes aegypti and Aedes albopictus, a pivotal approach for genetic analysis and vector control. Despite RNAi's potential for species-specific gene targeting, our independent efforts to establish oral delivery of RNAi for identifying genes critical for mosquito development and fitness encountered significant challenges, failing to reproduce previously reported potent RNAi effects. We independently evaluated a range of RNAi-inducing molecules (siRNAs, shRNAs, and dsRNAs) and administration methods (oral delivery, immersion, and microinjection) in three different laboratories. We also tested various mosquito strains and utilized microorganisms for RNA delivery. Our results reveal a pronounced inconsistency in RNAi efficacy, characterized by minimal effects on larval survival and gene expression levels in most instances despite strong published effects for the tested targets. One or multiple factors, including RNase activity in the gut, the cellular internalization and processing of RNA molecules, and the systemic dissemination of the RNAi signal, could be involved in this variability, all of which are barely understood in mosquitoes. The challenges identified in this study highlight the necessity for additional research into the underlying mechanisms of mosquito RNAi to develop more robust RNAi-based methodologies. Our findings emphasize the intricacies of RNAi application in mosquitoes, which present a substantial barrier to its utilization in genetic control strategies.

Delivery of Experimental mRNA Vaccine Encoding the RBD of SARS-CoV-2 by Jet Injection.

We studied a needle-free jet injection delivery of an experimental mRNA vaccine encoding the receptor-binding domain of the SARS-CoV-2 S protein (mRNA-RBD). Immunization of BALB/c mice with mRNA-RBD by a needle-free jet injector induced high levels of antibodies with virus-neutralizing activity and a virus-specific T-cell response. The immune response was low in the group of mice that received intramuscular injection of mRNA-RBD. The effectiveness of this simple and safe method of mRNA delivering has been demonstrated. Thus, jet injection of mRNA vaccine can be a good alternative to lipid nanoparticles.

Mosquito gene targeted RNAi studies for vector control.

Vector-borne diseases are serious public health concern. Mosquito is one of the major vectors responsible for the transmission of a number of diseases like malaria, Zika, chikungunya, dengue, West Nile fever, Japanese encephalitis, St. Louis encephalitis, and yellow fever. Various strategies have been used for mosquito control, but the breeding potential of mosquitoes is such tremendous that most of the strategies failed to control the mosquito population. In 2020, outbreaks of dengue, yellow fever, and Japanese encephalitis have occurred worldwide. Continuous insecticide use resulted in strong resistance and disturbed the ecosystem. RNA interference is one of the strategies opted for mosquito control. There are a number of mosquito genes whose inhibition affected mosquito survival and reproduction. Such kind of genes could be used as bioinsecticides for vector control without disturbing the natural ecosystem. Several studies have targeted mosquito genes at different developmental stages by the RNAi mechanism and result in vector control. In the present review, we included RNAi studies conducted for vector control by targeting mosquito genes at different developmental stages using different delivery methods. The review could help the researcher to find out novel genes of mosquitoes for vector control.

mRNA Vaccine Platform: mRNA Production and Delivery.

Vaccination is the most efficient way to prevent infectious diseases. mRNA-based vaccines is a new approach to vaccine development, which have several very useful advantages over other types of vaccines. Since mRNA encodes only the target antigen there is no potential risk of infection as in the case with attenuated or inactivated pathogens. The mode of action of mRNA-vaccines implies that their genetic information is expressed only in the cytosol, leaving very little possibility of mRNA integration into the host's genome. mRNA-vaccines can induce specific cellular and humoral immune responses, but do not induce the antivector immune response. The mRNA-vaccine platform allows for easy target gene replacement without the need to change the production technology, which is important to address the time lag between the epidemic onset and vaccine release. The present review discusses the history of mRNA vaccines, mRNA vaccine production technology, ways to increase mRNA stability, modifications of the cap, poly(A)-tail, coding and noncoding parts of mRNA, target mRNA vaccine purification from byproducts, and delivery methods.

Preferences of Persons With or at Risk for Hepatitis C for Long-Acting Treatments.

BACKGROUND: Whereas safe, curative treatments for hepatitis C virus (HCV) have been available since 2015, there are still 58 million infected persons worldwide, and global elimination may require new paradigms. We sought to understand the acceptability of approaches to long-acting HCV treatment. METHODS: A cross-sectional, 43-question survey was administered to 1457 individuals with or at risk of HCV at 28 sites in 9 countries to assess comparative interest in a variety of long-acting strategies in comparison with oral pills. RESULTS: Among HCV-positive participants, 37.7% most preferred an injection, 5.6% an implant, and 6% a gastric residence device, as compared with 50.8% who stated they would most prefer taking 1-3 pills per day. When compared directly to taking pills, differences were observed in the relative preference for an injection based on age (P<.001), location (P<.001), and prior receipt of HCV treatment (P=.005) but not sex. When an implant was compared with pills, greater preference was represented by women (P=.01) and adults of younger ages (P=.01 per 5 years). Among participants without HCV, 49.5% believed that injections are stronger than pills and 34.7% preferred taking injections to pills. Among those at-risk participants who had received injectable medications in the past, 123 of 137 (89.8%) expressed willingness to receive one in the future. CONCLUSIONS: These data point to high acceptability of long-acting treatments, which for a substantial minority might even be preferred to pills for the treatment of HCV infection. Long-acting treatments for HCV infection might contribute to global efforts to eliminate hepatitis C.

miR-124: A Promising Therapeutic Target for Central Nervous System Injuries and Diseases.

Central nervous system injuries and diseases, such as ischemic stroke, spinal cord injury, neurodegenerative diseases, glioblastoma, multiple sclerosis, and the resulting neuroinflammation often lead to death or long-term disability. MicroRNAs are small, non-coding, single-stranded RNAs that regulate posttranscriptional gene expression in both physiological and pathological cellular processes, including central nervous system injuries and disorders. Studies on miR-124, one of the most abundant microRNAs in the central nervous system, have shown that its dysregulation is related to the occurrence and development of pathology within the central nervous system. Herein, we review the molecular regulatory functions, underlying mechanisms, and effective delivery methods of miR-124 in the central nervous system, where it is involved in pathological conditions. The review also provides novel insights into the therapeutic target potential of miR-124 in the treatment of human central nervous system injuries or diseases.

Gene therapy: A promising approach for breast cancer treatment.

Breast cancer (BC) is the most prevalent malignancy and the second leading cause of death among women worldwide that is caused by numerous genetic and environmental factors. Hence, effective treatment for this type of cancer requires new therapeutic approaches. The traditional methods for treating this cancer have side effects, therefore so much research have been performed in last decade to find new methods to alleviate these problems. The study of the molecular basis of breast cancer has led to the introduction of gene therapy as an effective therapeutic approach for this cancer. Gene therapy involves sending genetic material through a vector into target cells, which is followed by a correction, addition, or suppression of the gene. In this technique, it is necessary to target tumour cells without affecting normal cells. In addition, clinical trial studies have shown that this approach is less toxic than traditional therapies. This study will review various aspects of breast cancer, gene therapy strategies, limitations, challenges and recent studies in this area.

Differential effects of different delivery methods on progression to severe postpartum hemorrhage between Chinese nulliparous and multiparous women: a retrospective cohort study.

BACKGROUND: Delivery methods are associated with postpartum hemorrhage (PPH) both in nulliparous and multiparous women. However, few studies have examined the difference in this association between nulliparous and multiparous women. This study aimed to explore the difference of maternal and neonatal characteristics and delivery methods between Chinese nulliparous and multiparous women, and then examine the differential effects of different delivery methods on PPH between these two-type women. METHODS: Totally 151,333 medical records of women who gave birth between April 2013 to May 2016 were obtained from the electronic health records (EHR) in a northern province, China. The severity of PPH was estimated and classified into blood loss at the level of < 900 ml, 900-1500 ml, 1500-2100 ml, and > 2100 ml. Neonatal and maternal characteristics related to PPH were derived from the same database. Multiple ordinal logistic regression was used to estimate associations. RESULTS: Medical comorbidities, placenta previa and accreta were higher in the nulliparous group and the episiotomy rate was higher in the multiparous group. Compared with spontaneous vaginal delivery (SVD), the adjusted odds (aOR) for progression to severe PPH due to the forceps-assisted delivery was much higher in multiparous women (aOR: 9.32; 95% CI: 3.66-23.71) than in nulliparous women (aOR: 1.70; 95% CI: 0.91-3.18). The (aOR) for progression to severe PPH due to cesarean section (CS) compared to SVD was twice as high in the multiparous women (aOR: 4.32; 95% CI: 3.03-6.14) as in the nulliparous women (aOR: 2.04; 95% CI: 1.40-2.97). However, the (aOR) for progression to severe PPH due to episiotomy compared to SVD between multiparous (aOR: 1.24; 95% CI: 0.96-1.62) and nulliparous women (aOR: 1.55; 95% CI: 0.92-2.60) was not significantly different. The (aOR) for progression to severe PPH due to vacuum-assisted delivery compared to SVD in multiparous women (aOR: 2.41; 95% CI: 0.36-16.29) was not significantly different from the nulliparous women (aOR: 1.05; 95% CI: 0.40-2.73). CONCLUSIONS: Forceps-assisted delivery and CS methods were found to increase the risk of severity of the PPH. The adverse effects were even greater for multiparous women. Episiotomy and the vacuum-assisted delivery, and SVD were similar to the risk of progression to severe PPH in either nulliparous or multiparous women. Our findings have implications for the obstetric decision on the choice of delivery methods, maternal and neonatal health care, and obstetric quality control.

Delivery of a Genetically Marked Serratia AS1 to Medically Important Arthropods for Use in RNAi and Paratransgenic Control Strategies.

Understanding how arthropod vectors acquire their bacteria is essential for implementation of paratransgenic and RNAi strategies using genetically modified bacteria to control vector-borne diseases. In this study, a genetically marked Serratia AS1 strain expressing the mCherry fluorescent protein (mCherry-Serratia) was used to test various acquisition routes in six arthropod vectors including Anopheles stephensi, Culex pipiens, Cx. quinquefaciatus, Cx. theileri, Phlebotomus papatasi, and Hyalomma dromedarii. Depending on the species, the bacteria were delivered to (i) mosquito larval breeding water, (ii) host skin, (iii) sugar bait, and (iv) males (paratransgenic). The arthropods were screened for the bacteria in their guts or other tissues. All the hematophagous arthropods were able to take the bacteria from the skin of their hosts while taking blood meal. The mosquitoes were able to take up the bacteria from the water at larval stages and to transfer them transstadially to adults and finally to transfer them to the water they laid eggs in. The mosquitoes were also able to acquire the bacteria from male sperm. The level of bacterial acquisition was influenced by blood feeding time and strategies (pool or vessel feeding), dipping in water and resting time of newly emerged adult mosquitoes, and the disseminated tissue/organ. Transstadial, vertical, and venereal bacterial acquisition would increase the sustainability of the modified bacteria in vector populations and decrease the need for supplementary release experiments whereas release of paratransgenic males that do not bite has fewer ethical issues. Furthermore, this study is required to determine if the modified bacteria can be introduced to arthropods in the same routes in nature.

Progress in Gene Therapy for Chronic Heart Failure.

Chronic heart failure (CHF) is still the leading cause of morbidity and mortality worldwide, and carries with it large economic and social burdens. Although steady and substantial progress has been made in reducing mortality from heart failure using conventional treatments, novel pharmacologic and surgical interventions have not been effective in extending five year survival rates. Therefore, it is necessary to explore new therapies. Gene therapy was introduced in 1970s with the development of recombinant DNA technology. Due to recent progress in the understanding of myocardial metabolism and application of vector based gene transfer strategies in animal models and initial clinical trials, gene therapy possibly affords an ideal treatment alternative for CHF. In last 2 decades, much research has been done on gene therapy, using various genes, signal transduction passages and delivery methods to treat advanced heart failure. Current research in ischemic heart disease (IHD) mainly focuses on stimulating angiogenesis, modifying the coronary vascular environment, and improving the vascular endothelial function with localized gene coated catheters and stents. Compared with standard ischemic heart disease treatment, the main goal of gene therapy for CHF is to inhibit apoptosis, reduce the undesirable remodeling and increase contractility through the most efficient cardiomyocyte transfection [Katz 2012a]. In this paper, we review various gene transfer technologies in ischemic heart disease and heart failure models, and discuss the advantages and disadvantages of these strategies in vector-mediated cardiac gene delivery, with the main focus on the high efficiency approach of a molecular cardiac surgery delivery system.

The Cardiovascular Intensive Care Unit-An Evolving Model for Health Care Delivery.

Prior to the advent of the coronary care unit (CCU), patients having an acute myocardial infarction (AMI) were managed on the general medicine wards with reported mortality rates of greater than 30%. The first CCUs are believed to be responsible for reducing mortality attributed to AMI by as much as 40%. This drastic improvement can be attributed to both advances in medical technology and in the process of health care delivery. Evolving considerably since the 1960s, the CCU is now more appropriately labeled as a cardiac intensive care unit (CICU) and represents a comprehensive system designed for the care of patients with an array of advanced cardiovascular disease, an entity that reaches far beyond its early association with AMI. Grouping of patients by diagnosis to a common physical space, dedicated teams of health care providers, as well as the development and implementation of evidence-based treatment algorithms have resulted in the delivery of safer, more efficient care, and most importantly better patient outcomes. The CICU serves as a platform for an integrated, team-based patient care delivery system that addresses a broad spectrum of patient needs. Lessons learned from this model can be broadly applied to address the urgent need to improve outcomes and efficiency in a variety of health care settings.

Continuous release of oregano oil effectively and safely controls Varroa destructor infestations in honey bee colonies in a northern climate.

The ectoparasitic mite Varroa destructor is responsible for the death of millions of honey bee (Apis mellifera) colonies worldwide. Testing potential miticide compounds with different delivery methods that effectively control V. destructor and have low toxicity for honey bees is crucial to manage this parasite in hives. We determined the varroacide efficacy of three natural compounds delivered to hives with three application methods over a 4-week period. Oxalic acid in a sucrose solution was applied impregnated in cardboard (T1). A mixture of oregano and clove oils in an ethanol-gelatin solution was applied impregnated in absorbent pads (T2). Oregano oil alone was delivered using electric vaporizers (T3) to test the hypothesis that continuous release of miticides increases the varroacidal efficacy of essential oils. The varroa mite control rates for treatments T1-T3 were 76.5 ± 7.11, 57.8 ± 12.79 and 97.4 ± 0.68%, respectively, and there were no differences for bee mortality between control and treatments 1 and 3. Additionally, most mites were killed in the first 2 weeks in T3 colonies compared to the last 2 weeks in colonies of the other treatments. These results demonstrate the importance of continuously releasing natural miticides to achieve safe and high rates of mite control in hives. They also show that oregano oil may be an effective miticide against V. destructor infestations in colonies.

CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods.

While human gene therapy has gained significant attention for its therapeutic promise, CRISPR/Cas9 technology has made a breakthrough as an efficient genome editing tool by emulating prokaryotic immune defense mechanisms. Although many studies have found that CRISPR/Cas9 technology is more efficient, specific and manipulable than previous generations of gene editing tools, it can be further improved by elevating its overall efficiency in a higher frequency of genome modifications and reducing its off-target effects. Here, we review the development of CRISPR/Cas9 technology, focusing on enhancement of its sequence specificity, reduction of off-target effects and delivery systems. Moreover, we describe recent successful applications of CRISPR/Cas9 technology in laboratory and clinical studies.

Digital delivery methods of parenting training interventions: a systematic review.

BACKGROUND: Alternative delivery methods to implement evidence-based parent training programs are emerging to address barriers related to parent attendance in face-to-face administration. The purpose of this systematic review is to summarize and critically evaluate the research on the use of technology and digital delivery methods for parenting training and offer recommendations for advancing the science and practice of parent training using digital delivery methods. METHODS: A systematic review was conducted using PubMed, PsychInfo, CINAHL, Scopus database, and ERIC to identify articles published between 2000 and 2012 reporting studies using digital methods to deliver parent training. Eleven studies were included in the review and were analyzed related to the digital delivery methods used, participant rates of intervention completion (dose), and the efficacy for improving parent and child outcomes. FINDINGS: The final sample of papers (n = 11) represent seven parent training interventions and nine digital delivery methods. Six of the nine used the Internet as the primary delivery method. The proportion of digitally delivered content completion ranged from 41.7% to 99.2%. Of the studies (n = 4) that reported behavioral outcomes of the interventions, the average effect size (Cohen's d) for child outcomes was .61 and for parent outcomes .46. LINKING EVIDENCE TO ACTION: Findings from this review indicate that the use of technology and digital delivery is a growing and emerging method of delivering parent training interventions with high potential for increasing reach and sustainability as we implement interventions in real world settings. Gaps in the studies reviewed highlight the need for consistency of dose calculations using digital methods, more research related to efficacy and comparative effectiveness studies of delivery methods.

The Dilemma of Insulin Delivery into the Brain: A Comprehensive Review.

Insulin is a peptide hormone that is essential for regulating body homeostasis. Furthermore, it is involved in various neurological functions such as memory, behaviors, and cognition. The ubiquitous distribution of insulin receptors on various brain cells, such as neurons, microglia, astrocytes, and oligodendrocytes, and their differential localization across various brain regions, including the hippocampus, hypothalamus, and olfactory bulb, collectively underscore the crucial involvement of insulin in the modulation of cerebral functions. Along with ageing, in some pathological conditions such as diabetes and brain insulin resistance, the need for exogenous insulin is felt to compensate for insulin deficiency. In these cases, the biggest obstacle to the delivery of insulin to the brain is the blood-brain barrier (a physical barrier consisting of endothelial cells with tight junctions), which prevents the direct entry of most substances possessing high molecular weight, like insulin, into the brain. Therefore, different delivery methods have been proposed by researchers for insulin delivery that directly or indirectly cause the transfer of insulin to the brain. Some of these methods lack high efficiency and cause many side effects for the patient. In this regard, many new technologies have come to the aid of researchers and have introduced more effective delivery strategies, including the use of nanocarriers. Despite the promising outcomes demonstrated in the experimental models, the utilization of these techniques in human studies remains at a nascent stage and necessitates further comprehensive investigation. This review article aims to examine the diverse methods of insulin administration to the brain by gathering extensive information on insulin and its obstacles to brain delivery.

Gene therapy for heart failure: A novel treatment for the age old disease.

Across the globe, cardiovascular disease (CVD) is the leading cause of mortality. According to reports, around 6.2 million people in the United states have heart failure. Current standards of care for heart failure can delay but not prevent progression of disease. Gene therapy is one of the novel treatment modalities that promises to fill this limitation in the current standard of care for Heart Failure. In this paper we performed an extensive search of the literature on various advances made in gene therapy for heart failure till date. We review the delivery methods, targets, current applications, trials, limitations and feasibility of gene therapy for heart failure. Various methods have been employed till date for administering gene therapies including but not limited to arterial and venous infusion, direct myocardial injection and pericardial injection. Various strategies such as AC6 expression, S100A1 protein upregulation, VEGF-B and SDF-1 gene therapy have shown promise in recent preclinical trials. Furthermore, few studies even show that stimulation of cardiomyocyte proliferation such as through cyclin A2 overexpression is a realistic avenue. However, a considerable number of obstacles need to be overcome for gene therapy to be part of standard treatment of care such as definitive choice of gene, gene delivery systems and a suitable method for preclinical trials and clinical trials on patients. Considering the challenges and taking into account the recent advances in gene therapy research, there are encouraging signs to indicate gene therapy for heart failure to be a promising treatment modality for the future. However, the time and feasibility of this option remains in a situation of balance.

Exploring preference for delivery methods for a psychosocial intervention for prenatal anxiety: A qualitative study from a tertiary care hospital in Pakistan.

Objective: This qualitative study explores therapists' and participants' preferences for delivery methods (face-to-face and phone sessions) of a cognitive behavioral therapy-based psychosocial intervention for prenatal anxiety delivered in a tertiary care hospital. Setting: The research was conducted in a randomized controlled trial in Pakistan, where a shift from face-to-face to phone-based therapy occurred during the coronavirus disease-2019 (COVID-19) pandemic. Participants: Twenty in-depth interviews and a focus group discussion were conducted with participants and therapists, respectively. Transcripts were analyzed using thematic analysis. Results: Participants generally preferred face-to-face sessions for rapport building, communication, and comprehension. However, barriers like venue accessibility, childcare, and lack of family support hindered engagement. Telephone sessions were favored for easy scheduling and the comfort of receiving the session at home, but there were challenges associated with phone use, distractions at home, and family members' limited mental health awareness. A mix of face-to-face and telephone sessions was preferred, with rapport from in-person sessions carrying over to telephone interactions. Conclusion: This study underscores the need for adaptable intervention delivery strategies that consider cultural norms, logistical challenges, and individual family dynamics. By combining the benefits of both delivery methods, mental health interventions can be optimized to effectively address prenatal anxiety and promote well-being in resource-constrained settings like Pakistan.

Oncolytic viral therapy for gliomas: Advances in the mechanisms and approaches to delivery.

Glioma is a diverse category of tumors originating from glial cells encompasses various subtypes, based on the specific type of glial cells involved. The most aggressive is glioblastoma multiforme (GBM), which stands as the predominant primary malignant tumor within the central nervous system in adults. Despite the application of treatment strategy, the median survival rate for GBM patients still hovers around 15 months. Oncolytic viruses (OVs) are artificially engineered viruses designed to selectively target and induce apoptosis in cancer cells. While clinical trials have demonstrated encouraging results with intratumoral OV injections for some cancers, applying this approach to GBM presents unique challenges. Here we elaborate on current trends in oncolytic viral therapy and their delivery methods. We delve into the various methods of delivering OVs for therapy, exploring their respective advantages and disadvantages and discussing how selecting the optimal delivery method can enhance the efficacy of this innovative treatment approach.

The Future of Gene Therapy: A Review of In Vivo and Ex Vivo Delivery Methods for Genome Editing-Based Therapies.

The development of gene therapy based on genome editing has opened up new possibilities for the treatment of human genetic disorders. This field has developed rapidly over the past few decades, some genome editing-based therapies are already in phase 3 clinical trials. However, there are several challenges to be addressed before widespread adoption of gene editing therapy becomes possible. The main obstacles in the development of such therapy are safety and efficiency, so one of the biggest issues is the delivery of genetic constructs to patient cells. Approaches in genetic cargo delivery divide into ex vivo and in vivo, which are suitable for different cases. The ex vivo approach is mainly used to edit blood cells, improve cancer therapy, and treat infectious diseases. To edit cells in organs researches choose in vivo approach. For each approach, there is a fairly large set of methods, but, unfortunately, these methods are not universal in their effectiveness and safety. The focus of this article is to discuss the current status of in vivo and ex vivo delivery methods used in genome editing-based therapy. We will discuss the main methods employed in these approaches and their applications in current gene editing treatments under development.

In vivo liver targeted genome editing as therapeutic approach: progresses and challenges.

The liver is an essential organ of the body that performs several vital functions, including the metabolism of biomolecules, foreign substances, and toxins, and the production of plasma proteins, such as coagulation factors. There are hundreds of genetic disorders affecting liver functions and, for many of them, the only curative option is orthotopic liver transplantation, which nevertheless entails many risks and long-term complications. Some peculiar features of the liver, such as its large blood flow supply and the tolerogenic immune environment, make it an attractive target for in vivo gene therapy approaches. In recent years, several genome-editing tools mainly based on the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system have been successfully exploited in the context of liver-directed preclinical or clinical therapeutic applications. These include gene knock-out, knock-in, activation, interference, or base and prime editing approaches. Despite many achievements, important challenges still need to be addressed to broaden clinical applications, such as the optimization of the delivery methods, the improvement of the editing efficiency, and the risk of on-target or off-target unwanted effects and chromosomal rearrangements. In this review, we highlight the latest progress in the development of in vivo liver-targeted genome editing approaches for the treatment of genetic disorders. We describe the technological advancements that are currently under investigation, the challenges to overcome for clinical applicability, and the future perspectives of this technology.