Producer cell line engineering for large volume manufacturing of therapeutic Aav

Recombinant adeno-associated viruses (rAAV) remain one of the most promising gene therapy vectors for patients with genetic abnormalities. rAAV can safely deliver long-lasting expression of a therapeutic transgene. To create a rAAV virus the wild-type viral genome is replaced with a functional transgene expression cassette. A constitutive promoter can be utilized to drive strong expression of the transgene once the rAAV vector has infected the target cell. Unlike wtAAV, the recombinant vector avoids the pitfalls of genomic integration by establishing extrachomosomal episomes or concatemers. Multiple studies have shown that rAAV can provide sustained expression of the transgene in cultured cells and pre-clinical models, providing evidence that rAAV could offer a cure for certain diseases. Bioengineering advancements have expanded the viral tropism beyond the constraints of naturally occurring AAV capsids, increasing the types of cells that can be thought of as targets. Taken together, rAAV therapies have attractive qualities to safely address the needs of patients where protein or small molecule therapies would fall short. One challenge with therapeutic rAAV is the ability to generate enough virus for clinical trials and commercial supply. This is particularly true with neuromuscular or hemophilia patients in which doses can exceed 1 x 1014 viral genomes per patient. Typical yields from a rAAV production are around 104 viral genomes per cell, meaning 1010 cells will be needed for a single dose. This amount of therapeutic virus is a challenge to deliver with standard gene therapy production platforms, and will require a production platform that can reliably generate sufficient quantities of therapeutic rAAV to meet patient demand. Biogen has selected the producer cell line (PCL) platform to meet the large demand for therapeutic rAAV. Producer cell lines are generated by stably integrating the AAV viral genes along with the ITR flanked therapeutic gene of interest into a host cell line. rAAV production is then triggered by the addition of a ‘helper virus’ to provide functional genes for AAV replication. Traditional PCL platforms have used HeLa cells as the host and Adenovirus type-5 to deliver helper functions. We have used this platform as a basis for further development. Presented here will be our rationale for selecting the PCL platform, improvements made to the platform for ongoing clinical support, and our vision for the next generation PCL platform

Implementation of An Immediate Postpartum Long-Acting Reversible Contraception Program in a Southern Texas Border Population

Background: Expanding contraceptive access in a Southern Texas border population can reduce the number of unintended and short-interval pregnancies, which have been associated with poor maternal and neonatal health outcomes. Healthcare encounters can be maximized to include immediate postpartum long-acting reversible contraception (IPP LARC) as an additive to the healthcare access many women have during pregnancy. The implementation of the IPP LARC Program allowed for the provision of accessible LARCs to fulfill the contraceptive needs of our patient population. This program was supported by Medicaid reimbursements and a private teaching grant that was awarded to the University of Texas Rio Grande Valley (UTRGV) Ob/Gyn residency program at Doctors Hospital at Renaissance (DHR). Methods: Data was collected retrospectively from participants who received a LARC from 2/2017 to 7/2021 at DHR Women’s Hospital after receiving IRB approval. Patient demographic information, such as age, gravidity, parity, delivery route, primary provider, and funding were assessed, along with LARC distribution and insertion timing. The reimbursement status for Medicaid-funded devices was obtained from the DHR Billing Department. Results: A total of 467 LARCs were inserted under the IPP LARC Program over 4.5 years. Ages ranged from 13-57 years old. A total of 353 (76%) participants received primary care from the UTRGV Ob/Gyn Residency Program. All others were consults who had received care from private physicians at the DHR WH. A total of 314 (67.2%) implants and 153 (32.8%) IUDs were inserted and 313 (75%) devices were placed following a vaginal delivery, while 103 (25%) after a cesarean section. Majority (257 participants (55.2%)) lacked funding, while 193 (41.4%) had Medicaid, and 16 (3.4%) had private insurance. Conclusion: The IPP LARC program provided for 467 participants, with majority of the recipients requesting immediate postpartum contraception. Most lacked adequate funding and many requests came from private providers wanting to provide patients with their desired postpartum contraceptive method. The program helped fulfill the contraceptive needs of this population by expanding access regardless of funding status. Follow-up studies should assess patient satisfaction and LARC continuation rates to help obtain future funding and promote expansion of the program

Pediatric Leukemia: Diagnosis to Treatment–A Review

Leukemia is cancer of the blood and bone marrow, it is the most common cancer found in children and is found to be more than one fourth of pediatric cancers. It causes white blood cells to become abnormal and the body to become weak. This deficiency in the immune system reduces the body\u27s ability to fight infection or simple airborne illnesses, causing extensive treatment of common pathogens and cancer treatment. The present review covers all topics, from diagnosis to treatment of pediatric leukemia, as well as the stages of growth and physiological changes throughout the process. As leukemia has a high mortality rate the chances of survival are low. Discussion on survival rate will be breached and thoroughly deciphered. We also included the topic of treatment and at which point patients may be considered terminal or advanced. By approaching these points, we better understand leukemia (from a scientific standpoint) and its properties throughout the course of treatment

Leaf litter mixtures alter microbial community development: Mechanisms for non-additive effects in litter decomposition

To what extent microbial community composition can explain variability in ecosystem processes remains an open question in ecology. Microbial decomposer communities can change during litter decomposition due to biotic interactions and shifting substrate availability. Though relative abundance of decomposers may change due to mixing leaf litter, linking these shifts to the non-additive patterns often recorded in mixed species litter decomposition rates has been elusive, and links community composition to ecosystem function. We extracted phospholipid fatty acids (PLFAs) from single species and mixed species leaf litterbags after 10 and 27 months of decomposition in a mixed conifer forest. Total PLFA concentrations were 70% higher on litter mixtures than single litter types after 10 months, but were only 20% higher after 27 months. Similarly, fungal-to-bacterial ratios differed between mixed and single litter types after 10 months of decomposition, but equalized over time. Microbial community composition, as indicated by principal components analyses, differed due to both litter mixing and stage of litter decomposition. PLFA biomarkers a15∶0 and cy17∶0, which indicate gram-positive and gram-negative bacteria respectively, in particular drove these shifts. Total PLFA correlated significantly with single litter mass loss early in decomposition but not at later stages. We conclude that litter mixing alters microbial community development, which can contribute to synergisms in litter decomposition. These findings advance our understanding of how changing forest biodiversity can alter microbial communities and the ecosystem processes they mediate

Long-term insect herbivory slows soil development in an arid ecosystem

Although herbivores are well known to alter litter inputs and soil nutrient fluxes, their long-term influences on soil development are largely unknown because of the difficulty of detecting and attributing changes in carbon and nutrient pools against large background levels. The early phase of primary succession reduces this signal-to-noise problem, particularly in arid systems where individual plants can form islands of fertility. We used natural variation in tree-resistance to herbivory, and a 15 year herbivore-removal experiment in an Arizona piñon-juniper woodland that was established on cinder soils following a volcanic eruption, to quantify how herbivory shapes the development of soil carbon (C) and nitrogen (N) over 36–54 years (i.e., the ages of the trees used in our study). In this semi-arid ecosystem, trees are widely spaced on the landscape, which allows direct examination of herbivore impacts on the nutrient-poor cinder soils. Although chronic insect herbivory increased annual litterfall N per unit area by 50% in this woodland, it slowed annual tree-level soil C and N accumulation by 111% and 96%, respectively. Despite the reduction in soil C accumulation, short-term litterfall-C inputs and soil C-efflux rates per unit soil surface were not impacted by herbivory. Our results demonstrate that the effects of herbivores on soil C and N fluxes and soil C and N accumulation are not necessarily congruent: herbivores can increase N in litterfall, but over time their impact on plant growth and development can slow soil development. In sum, because herbivores slow tree growth, they slow soil development on the landscape. http://dx.doi.org/10.1890/ES12-00411.

Evolutionary History and Novel Biotic Interactions Determine Plant Responses to Elevated CO2 and Nitrogen Fertilization

A major frontier in global change research is predicting how multiple agents of global change will alter plant productivity, a critical component of the carbon cycle. Recent research has shown that plant responses to climate change are phylogenetically conserved such that species within some lineages are more productive than those within other lineages in changing environments. However, it remains unclear how phylogenetic patterns in plant responses to changing abiotic conditions may be altered by another agent of global change, the introduction of non-native species. Using a system of 28 native Tasmanian Eucalyptus species belonging to two subgenera, Symphyomyrtus and Eucalyptus, we hypothesized that productivity responses to abiotic agents of global change (elevated CO2 and increased soil N) are unique to lineages, but that novel interactions with a nonnative species mediate these responses. We tested this hypothesis by examining productivity of 1) native species monocultures and 2) mixtures of native species with an introduced hardwood plantation species, Eucalyptus nitens, to experimentally manipulated soil N and atmospheric CO2. Consistent with past research, we found that N limits productivity overall, especially in elevated CO2 conditions. However, monocultures of species within the Symphyomyrtus subgenus showed the strongest response to N (gained 127% more total biomass) in elevated CO2 conditions, whereas those within the Eucalyptus subgenus did not respond to N. Root:shoot ratio (an indicator of resource use) was on average greater in species pairs containing Symphyomyrtus species, suggesting that functional traits important for resource uptake are phylogenetically conserved and explaining the phylogenetic pattern in plant response to changing environmental conditions. Yet, native species mixtures with E. nitens exhibited responses to CO2 and N that differed from those of monocultures, supporting our hypothesis and highlighting that both plant evolutionary history and introduced species will shape community productivity in a changing world

Spatiotemporal characteristics of atrial fibrillation electrograms: a novel marker for arrhythmia stability and termination

Background: Sequentially mapped complex fractionated atrial electrograms (CFAE) and dominant frequency (DF) sites have been targeted during catheter ablation for atrial fibrillation (AF). However, these strategies have yielded variable success and have not been shown to correlate consistently with AF dynamics. Here, we evaluated whether the spatiotemporal stability of CFAE and DF may be a better marker of AF sustenance and termination. Methods: Eighteen sheep with 12 weeks of "one-kidney, one-clip" hypertension underwent open-chest studies. A total of 42 self-terminating (28–100 s) and 6 sustained (>15 min) AF episodes were mapped using a custom epicardial plaque and analyzed in 4-s epochs for CFAE, using the NavX CFE-m algorithm, and DF, using a Fast Fourier Transform. The spatiotemporal stability index (STSI) was calculated using the intraclass correlation coefficient of consecutive AF epochs. Results: A total of 67,733 AF epochs were analyzed. During AF initiation, mean CFE-m and the STSI of CFE-m/DF were similar between sustained and self-terminating episodes, although median DF was higher in sustained AF (p=0.001). During sustained AF, the STSI of CFE-m increased significantly (p=0.02), whereas mean CFE-m (p=0.5), median DF (p=0.07), and the STSI of DF remained unchanged (p=0.5). Prior to AF termination, the STSI of CFE-m was significantly lower (p<0.001), with a physiologically non-significant decrease in median DF (−0.3 Hz, p=0.006) and no significant changes in mean CFE-m (p=0.14) or the STSI of DF (p=0.06). Conclusions: Spatiotemporal stabilization of CFAE favors AF sustenance and its destabilization heralds AF termination. The STSI of CFE-m is more representative of AF dynamics than are the STSI of DF, sequential mean CFE-m, or median DF

The Resolved Stellar Halo of NGC 253

We have obtained Magellan/IMACS and HST/ACS imaging data that resolve red giant branch stars in the stellar halo of the starburst galaxy NGC 253. The HST data cover a small area, and allow us to accurately interpret the ground-based data, which cover 30% of the halo to a distance of 30 kpc, allowing us to make detailed quantitative measurements of the global properties and structure of a stellar halo outside of the Local Group. The geometry of the halo is significantly flattened in the same sense as the disk, with a projected axis ratio of b/a ~ 0.35 +/- 0.1. The total stellar mass of the halo is estimated to be M_halo ~ 2.5 +/- 1.5 x 10^9 M_sun, or 6% of the total stellar mass of the galaxy, and has a projected radial dependence that follows a power law of index -2.8 +/- 0.6, corresponding to a three-dimensional power law index of ~ -4. The total luminosity and profile shape that we measure for NGC 253 are somewhat larger and steeper than the equivalent values for the Milky Way and M31, but are well within the scatter of model predictions for the properties of stellar halos built up in a cosmological context. Structure within the halo is seen at a variety of scales: there is small kpc-scale density variation and a large shelf-like feature near the middle of the field. The techniques that have been developed will be essential for quantitatively comparing our upcoming larger sample of observed stellar halos to models of halo formation.Comment: ApJ, in press. Version with full resolution figures available at http://www.astro.lsa.umich.edu/~jbailin/papers/bailin_n253halo.pd