Vector Integration Research Articles

Non-clinical safety assessment of novel drug modalities: Genome safety perspectives on viral-, nuclease- and nucleotide-based gene therapies

Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.

Innovations in Time Related Expression Recognition Using LSTM Networks

The proposed architecture leverages the strengths of both Convolutional Neural Network (CNN) and Bidirectional Long Short-Term (BLSTM) to create a robust model for temporal expression recognition in clinical texts. The CNN component effectively captures morphological and orthographic features at the character level, which enriches the semantic understanding of complex medical terminologies that are often abbreviated or have unique suffixes and prefixes. The BLSTM component excels in capturing long-range dependencies in text, which is crucial for understanding the context in which temporal expressions occur. By integrating these models with a CRF layer, the system not only predicts discrete labels but also ensures that the sequence of predicted labels is coherent and contextually appropriate, addressing the limitations of models that predict labels independently. The integration of pre-trained biomedical word vectors provides significant contextual grounding tailored to the medical domain, enhancing the model's ability to discern and interpret the nuances of medical language. This is crucial in clinical contexts where accurate interpretation of temporal phrases can be critical for patient management and treatment timelines. Further, experiments conducted on the dataset validate the effectiveness of the proposed model, demonstrating a notable improvement over traditional methods that rely heavily on hand-crafted features and rule-based approaches. Future work could explore the adaptability of this model to other subdomains of the medical field and its efficacy in processing multilingual texts, potentially increasing its applicability in global healthcare settings, with further refinement of the neural architecture and optimization of training strategies potentially yielding even better performance and faster processing times essential for real-time clinical decision support systems.

ANALYSIS OF TOLERANCE AS A SYSTEMIC CHARACTERISTIC OF A STUDENT'S PERSONALITY

In the article, it is noted that in the modern world, in the context of global socio-economic and political changes, the problem of tolerance is attracting increasingly more attention, and this, in turn, engages more researchers to its solution. Intolerance has become one of the biggest global problems of the modern world. In these conditions, it is important for science and practice to resolve the issue of identifying the conditions for the development of tolerance, tools with which to determine the level and ways of its development, based on individual, age, and other personality characteristics. It can best be achieved in the conditions of higher education. In the article, the key and root issues of intolerance among student youth are outlined, and vectors of systemic integration of shaping tolerance into higher education curricula are suggested.

A new teaching-learning sequence to promote secondary school students’ learning of quantum physics using Dirac notation

This paper describes the design of a new teaching-learning sequence on quantum physics aimed at upper secondary school students. In this teaching-learning sequence, GeoGebra simulations and interactive screen experiments are used to investigate the behaviour of a single photon at beam splitter and single photon interference in a Michelson interferometer. We propose a minimal formalism using Dirac notation, which avoids complex numbers and elaborate vector calculus, to make a quantitative description of the quantum optics experiments accessible to secondary school students. With this new educational pathway, we take into account findings from physics education research, which suggest that the introduction of a mathematical formalism tailored to students’ abilities might help them to overcome naive-realist views of quanta or space-time descriptions of quantum phenomena, while at the same time facilitating a transition to a functional understanding of quantum models.

Vectorial Analysis of Deep Plane Face and Neck Lift.

The vector of aging and consequently the vector of lift in rhytidectomy has aided surgeons in improving movement of tissues during facial rejuvenation procedures. The goal was to analyze the vector of lift in patients undergoing primary and revisional facelift to achieve proper vectorial lifting. Patients undergoing deep-plane facelift surgery were included for analysis. Intraoperative photographs and measurements were taken of the skin, superficial musculoaponeurotic system (SMAS), and platysmal suture suspension with mastoid crevasse inset. Measurements were compared between patients who were undergoing primary vs secondary surgery, site of lift, age, and gender. Seventy-one patients (90% female, mean age 57.8) with a total of 142 hemifaces were analyzed, 57 (73%) of which were primary and 14 (27%) secondary facelifts. The average vector of SMAS lifting was 70.8°. Females had a more vertical vector than males (71.3° vs 65.4°; P < .01). The average vectors of platysmal and skin lift were 87.0° and 58.2°, respectively. There was intrapatient difference between hemifaces. Despite there being more intersuture disparity in secondary cases than primary cases (16.9° vs 4.5°; P < .05), the mean vector of lifting was similar between them. Proper release of the deep plane helps determine the appropriate vectors of lift, without relying on guidelines based on population averages. Each patient presents with a unique vector required to correct their descent. This technique provides an optimal result by directly suspending against the vectors of greatest descent.

What should we understand by the four-momentum of physical system?

It is shown that, in general, in curved spacetime, none of the known definitions of four-momentum correspond to the definition, in which all the system’s particles and fields, including fields outside matter, make an explicit contribution to the four-momentum. This drawback can be eliminated under the assumption that the primary representation of four-momentum is the sum of two nonlocal four-vectors of the integral type with covariant indices. The first of these four-vectors is the generalized four-momentum, found with the help of Lagrangian density. The time component of the generalized four-momentum, in theory of vector fields, is proportional to the particles’ energy in scalar field potentials, and the space component is related to vector field potentials. The second four-vector is the four-momentum of fields themselves, and its time component is related to the energy given by tensor invariants. As a result, the system’s four-momentum is defined as a four-vector with a covariant index. The standard approach makes it possible to find the four-momentum in covariant form only for a free point particle. In contrast, the obtained formulas for calculating the four-momentum components are applied to a stationary and moving relativistic uniform system, consisting of many particles. In this case, the main fields of the system under consideration are taken into account, including the electromagnetic and gravitational fields, the acceleration field and the pressure field. All these fields are considered vector fields, which makes it possible to unambiguously determine the equations of motion of the fields themselves and the equations of motion of matter in these fields. The formalism used includes the principle of least action, charged and neutral four-currents, corresponding four-potentials and field tensors, which ensures unification and the possibility of combining fields into a single interaction. Within the framework of the special theory of relativity, it is shown that due to motion, the four-momentum of the system increases in proportion to the Lorentz factor of the system’s center of momentum, while in the matter of the system the sum of the energies of all fields is equal to zero. The calculation of the integral vector’s components in the relativistic uniform system shows that the so-called integral vector is not equal to four-momentum and is not a four-vector at all, although it is conserved in a closed system. Thus, in the theory of relativistic vector fields, the four-momentum cannot be found with the help of an integral vector and components of the system’s stress-energy tensor, in contrast to how it is assumed in the general theory of relativity.

Asymptotic distribution for pairs of linear and quadratic forms at integral vectors

Asymptotic distribution for pairs of linear and quadratic forms at integral vectors

Integrable 2D and 3D piecewise smooth vector fields with chaotic behavior and preserving energy or not

In this paper we exhibit 2D and 3D piecewise smooth vector fields that have, at the same time, first integrals and chaotic behavior. The fact of existing first integrals means that the trajectories are tangent to foliations. Also, in some of these examples the trajectories live in an unique level of the first integrals, and so the system preserves energy, and in other cases the trajectories visit distinct levels of the first integrals and the system does not preserves the energy.

A historical sequence deletion in a commonly used Bacillus subtilis chromosome integration vector generates undetected loss-of-function mutations.

Since the 1980s, chromosome-integration vectors have been used as a core method of engineering Bacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into the amyE locus. Here, we report a gap in the homology region inherited from the original amyE integration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3' portion of amyE and the downstream gene ldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation of ldh, which encodes lactate dehydrogenase. Although both types of colonies test positive for amyE disruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

ABSTRACT The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.

ПУБЛІЧНІ ФІНАНСИ ТА ПУБЛІЧНІ ІНВЕСТИЦІЇ В КОНТЕКСТІ ІНТЕНСИФІКАЦІЇ ЕКОНОМІЧНОГО РОЗВИТКУ

The purpose of this article is to study the interdependence of the state, local governments, institutional investors and the level of foreign direct investment in Ukraine's economy in conditions of instability. The article examines the role of institutional investors and clarifies the main trends in attracting investment resources into the economy of Ukraine, taking into account the European integration vector, the state investment policy of Ukraine should include the main aspects of the main goals and objectives of the new generation investment policy, which takes into account the latest trends and needs of the world economy. In this context, it is especially important to establish the optimal ratio of foreign direct and domestic investment. In the context of European integration processes and ensuring investment security in Ukraine, it is necessary to protect domestic markets from monopolization by foreign investors by setting a limit on foreign direct investment in each sector.

First integrals, conserved vectors of nonlinear partial difference equations

We perform a symmetry analysis of some nonlinear partial difference equations (nP △ Es), where the discrete version is obtained using some discretization approach. The discrete versions of the wave, diffusion, Fisher and Huxley equations are the subject of this research. At first, the initial invariance approach is the Lie symmetry approach. The first integrals technique that Hydon introduced to be used with discrete ordinary difference equations (O △ Es) serves as our inspiration in this situation. We develop a similar technique for generating the first integral vectors of the nP △ Es without recourse to symmetry generators.

Clinical outcomes of a toric continuous range of vision presbyopia-correcting intraocular lens.

To evaluate the clinical outcomes of the toric version of a presbyopia-correcting intraocular lens (IOL) based on the combination of a diffractive-based extended depth-of-focus (EDOF) pattern and a diffractive multifocal platform. Miranza COI Bilbao, Bilbao, Spain. Prospective case series. 35 patients (51 to 84 years) with corneal astigmatism ranging from 0.75 to 2.19 diopters (D) undergoing bilateral cataract surgery with implantation of the Synergy Toric II IOL were evaluated during a 3-month follow-up. Visual acuity, refraction, defocus curve, and patient-reported outcomes with the Catquest-9SF questionnaire were analyzed. A vectorial analysis was used to analyze the accuracy of astigmatic correction. Mean 3-month monocular postoperative uncorrected distance, intermediate (80 cm) and near (40 cm) visual acuities were 0.06 ± 0.11 logMAR, 0.13 ± 0.12 logMAR, and 0.13 ± 0.09 logMAR, respectively. Mean monocular distance-corrected intermediate (80 cm) and near visual acuity (40 cm) were 0.11 ± 0.12 logMAR and 0.10 ± 0.10 logMAR, respectively. Mean binocular defocus curve showed visual acuities of 0.10 logMAR or better for defocus levels from +0.50 to -2.50 D. Residual cylinder was within ±0.50 D in 97.0% of eyes. The surgically induced astigmatism prediction error ranged between -0.49 D and 0.50 D, with a mean value of 0.04 ± 0.16 D. Mean absolute IOL rotation was 3.79 ± 2.94 degrees. Significant improvements were found in all Rasch-calibrated scores obtained with Catquest-9SF ( P < .001). The implantation of the toric presbyopia-correcting IOL evaluated provides an efficacious astigmatic correction while providing a fully restoration of the visual function across different distances.

Predicting the Risk of Morbidity by GLIM-Based Nutritional Assessment and Body Composition Analysis in Oncologic Abdominal Surgery in the Context of Enhanced Recovery Programs : The PHase Angle Value in Abdominal Surgery (PHAVAS) Study.

Preoperative nutritional status and body structure affect short-term prognosis in patients undergoing major oncologic surgery. Bioimpedance vectorial analysis (BIVA) is a reliable tool to assess body composition. Low BIVA-derived phase angle (PA) indicates a decline of cell membrane integrity and function. The aim was to study the association between perioperative PA variations and postoperative morbidity following major oncologic upper-GI surgery. Between 2019 and 2022 we prospectively performed BIVA in patients undergoing surgical resection for pancreatic, hepatic, and gastric malignancies on the day before surgery and on postoperative day (POD) 1. Malnutrition was defined as per the Global Leadership Initiative on Malnutrition criteria. The PA variation (ΔPA) between POD1 and preoperatively was considered as a marker for morbidity. Uni and multivariable logistic regression models were applied. Overall, 542 patients with a mean age of 64.6 years were analyzed, 279 (51.5%) underwent pancreatic, 201 (37.1%) underwent hepatobiliary, and 62 (11.4%) underwent gastric resections. The prevalence of preoperative malnutrition was 16.6%. The overall morbidity rate was 53.3%, 59% in those with ΔPA<-0.5 versus 46% when ΔPA≥-0.5. Age [odds ratio (OR) 1.11; 95% confidence interval (CI) (1.00; 1.22)], pancreatic resections [OR 2.27; 95% CI (1.24; 4.18)], estimated blood loss (OR 1.20; 95% CI (1.03; 1.39)], malnutrition [OR 1.77; 95% CI (1.27; 2.45)], and ΔPA [OR 1.59; 95% CI (1.54; 1.65)] were independently associated with postoperative complications in the multivariate analysis. Patients with preoperative malnutrition were significantly more likely to develop postoperative morbidity. Moreover, a decrease in PA on POD1 was independently associated with a 13% increase in the absolute risk of complications. Whether proactive interventions may reduce the downward shift of PA and the complication rate need further investigation.

Abstract 7250: Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors

Abstract GEN2 is a non-replicating retroviral gene transfer vector encoding a sequence-optimized herpes simplex thymidine kinase (HSV-TK), which activates the antiherpetic pro-drug ganciclovir (GCV), resulting in chain termination during DNA replication, triggering cancer cell apoptosis and release of neoantigens (NA). GEN2 also encodes hGM-CSF, which may augment antitumor immunity by enhancing NA presentation to immune effector cells. GEN2 in combination with valganciclovir (an oral formulation of GCV) was evaluated in a dose-escalating Phase 1 clinical trial in 61 adult patients (pts) with an advanced solid tumor (NCT04313868), with serial assessment of PBMCs to detect any GEN2 integration and replication competent retrovirus (RCR). Methods: PBMCs were obtained in Cycle 1 (following 1st/3rd dose), prior to and 2 hours after Cycle 2, and thereafter at 6-month intervals for 15 years, or until death or withdrawal of consent. Digital droplet PCR (ddPCR) for psi and envelope gene sequences in extracted genomic DNA from PBMCs was used to detect vector genome integration and replication competent retrovirus (RCR), respectively. Results: All 82 samples obtained from the intravenous cohorts 1-12 were below LOD (Limit of Detection) for RCR. All results for integration were BLOQ (Below Limit of Quantitation), whereas 4 samples collected at two hours following the end of infusion were above LOD, likely resulting from non-integrated cDNA. GEN2 continues to be studied in clinical trials in adult patients with solid tumors. Per the 2020 FDA Guidance for Industry (Long Term Follow-Up After Administration of Human Gene Therapy Products), serial assessment of vector integration is mandated throughout a pt’s participation in a gene therapy clinical trial and for 15 years following the last dose. These guidance documents were originally conceived when gene therapy was developed for treatment of rare hereditary diseases in pediatric pts; however, adult pts enrolled in early-stage clinical trials for cancer typically do not have a prolonged life expectancy (BMC Cancer 2011 11:426). In the context of proper and warranted usage of pts’ blood for testing, attention should be drawn to the limited expectation for a 2nd malignancy within the lifespan of this pt population, and the absence of detectable RCR in cancer patients receiving retroviral vectors over decades. A recent review noted no evidence of RCR in 1,595 post-treatment PBMCs obtained from 60 clinical trials (Mol Ther 2023 31(3):801). Our results confirm that GEN2 also shows negligible genotoxic risk for gene therapy of cancer at doses tested to date. Citation Format: Alison L. Hannah, Noriyuki Kasahara, Li Liu, Allen Wang, Joe McNulty, Robert G. Johnson. Serial assessment of GEN2, a retroviral vector for gene transfer of an optimized thymidine kinase and GM-CSF, for genomic integration into peripheral blood mononuclear cells (PBMCs) in a Phase 1 clinical trial in adult patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7250.

Abstract 5335: Immune cell population variations in the livers of commonly used mouse strains

Abstract Previous studies have shown that the distribution of major immune cell populations differ across mouse strains in peripheral blood and hematopoietic organs at baseline. Additionally, variations in immune responses exist between mouse strains in Leishmania spp. and Melanoma models, but these studies in the context of liver diseases including Non Alcoholic Fatty Liver Disease (NAFLD) and Hepatocellular Carcinoma (HCC) have not been carried out. With the effort to state if these variations exist within the liver, we performed comprehensive spectral flow cytometry analysis on the liver and spleens of three female mouse strains commonly used in cancer research (BALB/c, C57BL/6, and FVBN). NAFLD was induced by providing mice with a methionine choline deficient diet. To promote HCC, a single genotype (myc/p53+/-) was induced by administering transposon-based vectors to overexpress Myc-Luc and CRISPR/Cas-9 vectors to knock down p53 via hydrodynamic tail-vein injection. To confirm vector integration and monitor tumor growth (luciferase activity), in vivo imaging was performed at different time points. As a result, we confirm throughout this study that differences exist within immune cell populations of the liver and spleen at baseline. Moreover, we depict differences in the shift of immune cell populations across mouse strains in the pathologic states included. In NAFLD, there is an increase of dendritic cells (DCs), macrophages, NK cells, ILC1s, T regulatory cells (Tregs), TH1, and TH2, and a decrease of CD4+ T cells, B cells, γδ-T cells, and NKT cells in the liver across all mouse strains, when compared to controls. However, the population of neutrophils in BALB/c decreases, and the population of CD8+ T cells increases in C57BL/6 mice, which differs from the patterns of change seen in the other mouse strains. Moreover, in spleens, NAFLD promotes an increase of TH1 and NK cells, and a decrease of TH2 and NKT Cells across the mouse strains included in the study, whereas the patterns of change differ between mouse strains among the remaining immune cell types. When evaluating liver immune cell population shifts on our HCC model, BALB/c mice showed a decrease in macrophages and ILC1s, C57BL/6 mice showed a decrease in γδ-T cells, prominent shifts across Neutrophils, TH1, and NKT cells, and null changes in TH2 and CD4+ T cell shifts, and FVBN mice had a less prominent decrease of B cells, which differed from the shifts observed in the mouse strains used for comparison. Immune cell populations in the spleens of our HCC mouse model also presented with differences across the mouse strains studied. In conclusion, we highlight that key differences in immune profiles exist across commonly used mouse strains for cancer research in the presence of liver pathology including HCC, suggesting that mouse strains could be possible confounders in the study of immune oncology of HCC. Citation Format: Francisco J. Rodriguez-Matos, Patrick Huang, Rajiv Trehan, Benjamin Ruf, Chi Ma, Tim F. Greten. Immune cell population variations in the livers of commonly used mouse strains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5335.

Reduction procedure for obtaining solutions of the scalar additive Jump problem and Riemann boundary value problem in vectorial Clifford analysis

In this paper, we study existence of solutions to the scalar additive Jump problem and the Riemann boundary value problems in the context of vectorial Clifford analysis on domains with fractal boundaries. A reduction procedure is applied with great effectiveness to find the solution of the problems.

Dynamic typhoon visualization based on the integration of vector and scalar fields

Dynamic typhoon visualization based on the integration of vector and scalar fields

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer

AbstractGene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e−4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

A research-informed graphical tool to visually approach Gauss’ and Stokes’ theorems in vector calculus

Gauss’ and Stokes’ theorems are fundamental results in vector calculus and important tools in physics and engineering. When students are asked to describe the meaning of Gauss’ divergence theorem, they often use statements like this: ‘The sum of all sources of a vector field in a region gives the net flux out of the region’. In order to raise this description to a mathematically sound level of understanding, we present an educational approach based on the visual interpretation of the vector differential operators, i.e. divergence and curl. As a starting point, we use simple vector field diagrams for a qualitative approach to connect both sides of the integral theorems, and present an interactive graphical tool to support this connection. The tool allows to visualise two-dimensional vector fields, to specify vector decomposition, to evaluate divergence and curl point wise, and to draw rectangles to determine surface and line integrals. From a meta-perspective, we situate this educational approach into learning with (multiple) representations. Based on prior research, the graphical tool addresses various learning difficulties of vector fields that are connected to divergence and curl. The tool was incorporated into the weekly lecture-based recitations of Physics II (electromagnetism) in 2022 and 2023, and we assessed various educational outcome measures. The students overall reported the tool to be intuitive and user-friendly (level of agreement 76%, N=125 ), considered it helpful for understanding and recommended its use for introductory physics courses (level of agreement 65%, N=65 ).