Multivariate data analysis on multisensor measurement for inline process monitoring of adenovirus production in HEK293 cells.

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.

Serotonin transporter knockdown relieves depression-like behavior and ethanol-induced CPP in mice after chronic social defeat stress.

Patients with stress-triggered major depression disorders (MDD) can often seek comfort or temporary relief through alcohol consumption, as they may turn to it as a means of self-medication or coping with overwhelming emotions. The use of alcohol as a coping mechanism for stressful events can escalate, fostering a cycle where the temporary relief it provides from depression can deepen into alcohol dependence, exacerbating both conditions. Although, the specific mechanisms involved in stress-triggered alcohol dependence and MDD comorbidities are not well understood, a large body of literature suggests that the serotonin transporter (SERT) plays a critical role in these abnormalities. To further investigate this hypothesis, we used a lentiviral-mediated knockdown approach to examine the role of hippocampal SERT knockdown in social defeat stress-elicited depression like behavior and ethanol-induced place preference (CPP). The results showed that social defeat stress-pro depressant effects were reversed following SERT knockdown demonstrated by increased sucrose preference, shorter latency to feed in the novelty suppressed feeding test, and decreased immobility time in the tail suspension and forced swim tests. Moreover, and most importantly, social stress-induced ethanol-CPP acquisition and reinstatement were significantly reduced following hippocampal SERT knockdown using short hairpin RNA shRNA-expressing lentiviral vectors. Finally, we confirmed that SERT hippocampal mRNA expression correlated with measures of depression- and ethanol-related behaviors by Pearson's correlation analysis. Taken together, our data suggest that hippocampal serotoninergic system is involved in social stress-triggered mood disorders as well as in the acquisition and retrieval of ethanol contextual memory and that blockade of this transporter can decrease ethanol rewarding properties.

High temperatures and low humidity promote the occurrence of microsporidians (Microsporidia) in mosquitoes (Culicidae).

BACKGROUND: In the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae). METHODS: In our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively. RESULTS: DNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months. CONCLUSIONS: These results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.

Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

Proceedings of the 2023 Viral Clearance Symposium, Session 2: Viral Clearance Strategy and Case Studies.

This session deals with the rational design of viral clearance studies for biopharmaceuticals including recombinant proteins such as monoclonal antibodies and, as new in scope of the symposium, also viral clearance for adeno-associated viral (AAV) vectors. For recombinant proteins, large datasets were accumulated over the last decades and are intended to be used for accelerated product process development and streamlining of viral clearance studies. How to utilize prior knowledge in viral clearance validation and how it can be used in a risk assessment tool to decide whether additional virus clearance studies are necessary during product development is being addressed by three of the presentations of this session. This also includes an a priori intended design and generation of validation data for a new kind of detergent such as CG-110, to build up a platform dataset to be used as prior knowledge in future marketing application. Another presentation investigates the virus removal mechanism of a newly developed hydrophobic interaction chromatography (HIC) resin and demonstrates for highly hydrophobic antibodies appropriate reduction for a retrovirus and impurities in a defined process range in contrast to the moderate to poor virus reduction of recent HIC resins. The last two presentations deal with virus clearance approaches for AAV, which will become mandatory with approval of the ICH Q5A revision. Appropriate virus removal and virus inactivation procedures can be implemented into the manufacturing processes of AAV vectors including viral filtration, viral inactivation (e.g., heat inactivation), affinity chromatography, and anion-exchange chromatography with which it seems possible to achieve a good clearance for helper and also adventitious viruses. The heat treatment step can be even a robust step for adenovirus helper inactivation for AAV products when product characteristics and process conditions are understood.

The Kotake-Narasimhan theorem in general ultradifferentiable classes.

We prove a Kotake-Narasimhan type theorem in general ultradifferentiable classes given by weight matrices. In doing so we simultaneously recover and generalize significantly the known results for classes given by weight sequences and weight functions. In particular, we obtain a sharp Kotake-Narasimhan theorem for Beurling classes.

Combining CRISPR-Cas-mediated terminal resolution with a novel genetic workflow to achieve high-diversity adenoviral libraries.

While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 104 unique rAds per cm2 of transfected cell culture.

Prediction of protein N-terminal acetylation modification sites based on CNN-BiLSTM-attention model.

N-terminal acetylation is one of the most common and important post-translational modifications (PTM) of eukaryotic proteins. PTM plays a crucial role in various cellular processes and disease pathogenesis. Thus, the accurate identification of N-terminal acetylation modifications is important to gain insight into cellular processes and other possible functional mechanisms. Although some algorithmic models have been proposed, most have been developed based on traditional machine learning algorithms and small training datasets. Their practical applications are limited. Nevertheless, deep learning algorithmic models are better at handling high-throughput and complex data. In this study, DeepCBA, a model based on the hybrid framework of convolutional neural network (CNN), bidirectional long short-term memory network (BiLSTM), and attention mechanism deep learning, was constructed to detect the N-terminal acetylation sites. The DeepCBA was built as follows: First, a benchmark dataset was generated by selecting low-redundant protein sequences from the Uniport database and further reducing the redundancy of the protein sequences using the CD-HIT tool. Subsequently, based on the skip-gram model in the word2vec algorithm, tripeptide word vector features were generated on the benchmark dataset. Finally, the CNN, BiLSTM, and attention mechanism were combined, and the tripeptide word vector features were fed into the stacked model for multiple rounds of training. The model performed excellently on independent dataset test, with accuracy and area under the curve of 80.51% and 87.36%, respectively. Altogether, DeepCBA achieved superior performance compared with the baseline model, and significantly outperformed most existing predictors. Additionally, our model can be used to identify disease loci and drug targets.

Prime and pull of T cell responses against cancer-exogenous antigens is effective against CPI-resistant tumors.

Neoantigen (neoAg)-based cancer vaccines expand preexisting antitumor immunity and elicit novel cancer-specific T cells. However, at odds with prophylactic vaccines, therapeutic antitumor immunity must be induced when the tumor is present and has already established an immunosuppressive environment capable of rapidly impairing the function of anticancer neoAg T cells, thereby leading to lack of efficacy. To overcome tumor-induced immunosuppression, we first vaccinated mice bearing immune checkpoint inhibitor (CPI)-resistant tumors with an adenovirus vector encoding a set of potent cancer-exogenous CD8 and CD4 T cell epitopes (Ad-CAP1), and then "taught" cancer cells to express the same epitopes by using a tumor-retargeted herpesvirus vector (THV-CAP1). Potent CD8 effector T lymphocytes were elicited by Ad-CAP1, and subsequent THV-CAP1 delivery led to a significant delay in tumor growth and even cure.

Gene editing as a therapeutic strategy for spinocerebellar ataxia type-3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a neurodegenerative disease caused by expanded polyglutamine repeats in exon 10 of the ataxin-3 gene, ATXN3. The accumulation of mutant ATXN3 protein leads to severe clinical manifestations and premature death. Clinically, SCA3 pathology is characterized by progressive ataxia leading to motor incoordination that may affect balance, gait and speech, and neuropathologically by a progressive degeneration of the spinal cord and cerebellum, as well as the cerebral cortex and basal ganglia. Although SCA3 is a rare disease, it is the most common autosomal dominant spinocerebellar ataxia worldwide. Its geographical distribution varies worldwide, with peak prevalence in certain regions of Brazil, Portugal and China. In 1994, the identification of the polyglutamine expansion in the ATXN3 gene made it possible not only to diagnose this pathology but also to dissect the mechanisms leading to cellular degeneration. As a monogenic disease for which only symptomatic treatment is available, the ATXN3 gene represents an attractive therapeutic target for gene editing strategies.

A pilot community science program in Mexico for passive triatomine surveillance and increase Chagas disease awareness / Programa piloto de ciencia ciudadana en México para vigilancia pasiva de triatominos y concientizar sobre la enfermedad de Chagas

Objective. To generate data about Chagas disease vectors through passive surveillance and inform the public using social media and community science. Materials and methods. We used social media to inform, raise awareness and to promote the public to report their triatomine encounters. We received pictures and specimens collected to be tested for Trypanosoma cruzi and to identify recent bloodmeal source through PCR. Results. Community scientists reported 44 triatomines from 15 states in Mexico and one triatomine from Nicaragua, including 9 species with Triatoma dimidiata sensu lato and T. gerstaeckeri being the most common. We received 12 collected specimens and T. cruzi was detected in 8 (67%) of the discrete typing unit TcI. We identified recent bloodmeal source in 6 triatomines including: human (Homo sapiens), dog (Canis lupus familiaris), wood rat (Neotoma sp.), dove (Columbidae) and amphibius (Bufonidae). Conclusion. The use of community science can be a complementary method to generate information about the ecology and epidemiology of Chagas disease vectors.

Objetivo. Generar datos sobre vectores de la enfermedad de Chagas (EC) mediante vigilancia pasiva e informar a la población mediante redes sociales y ciencia ciudadana. Material y métodos. Utilizando redes sociales informamos, concientizamos y alentamos al público a reportarnos sus encuentros con triatominos. Recibimos reportes fotográficos y especímenes colectados a los que analizamos para detectar infección por Trypanosoma cruzi e identificar la fuente reciente de alimentación mediante PCR. Resultados. Nos reportaron 44 triatominos de 15 estados en México y uno de Nicaragua, incluyendo 9 especies siendo Triatoma dimidiata sensu lato y T. gerstaeckeri las más comunes. Recibimos 12 especímenes colectados y encontramos T. cruzi en 8 (67%) de la unidad taxonómica discreta TcI. Identificamos fuente reciente de alimentación en 6 triatominos incluyendo: humano (Homo sapiens), perro (Canis lupus familiaris), rata de campo (Neotoma sp.), paloma (Columbidae) y anfibio (Bufonidae). Conclusión. Ciencia ciudadana puede ser un método complementario para generar información sobre ecología y epidemiología de EC.

A new era of targeting cystic fibrosis with non-viral delivery of genomic medicines.

Cystic fibrosis (CF) is a complex genetic respiratory disorder that necessitates innovative gene delivery strategies to address the mutations in the gene. This review delves into the promises and challenges of non-viral gene delivery for CF therapy and explores strategies to overcome these hurdles. Several emerging technologies and nucleic acid cargos for CF gene therapy are discussed. Novel formulation approaches including lipid and polymeric nanoparticles promise enhanced delivery through the CF mucus barrier, augmenting the potential of non-viral strategies. Additionally, safety considerations and regulatory perspectives play a crucial role in navigating the path toward clinical translation of gene therapy.

Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Combined 100 keV Cryo-Electron Microscopy and Image Analysis Methods to Characterize the Wider Adeno-Associated Viral Products.

Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products.

Surveillance of mosquitoes harnessing their buzzing sound.

Mosquito surveillance for vector-borne disease management relies on traditional morphological and molecular techniques, which are tedious, time-consuming, and costly. The present study describes a simple and efficient recording device that analyzes mosquito sound to estimate species composition, male-female ratio, fed-unfed status, and harmonic convergence interaction using fundamental frequency (F0) bandwidth, harmonics, amplitude, and combinations of these parameters. The study examined a total of 19 mosquito species, including 3 species of Aedes, 7 species of Anopheles, 1 species of Armigeres, 5 species of Culex, 1 species of Hulecoetomyia, and 2 species of Mansonia. Among them, the F0 ranges between 269.09 ± 2.96 Hz (Anopheles culicifacies) to 567.51 ± 3.82 Hz (Aedes vittatus) and the harmonic band (hb) number ranges from 5 (An. culicifacies) to 12 (Ae. albopictus). In terms of species identification, the success rate was 95.32% with F0, 84.79% with F0-bandwidth, 84.79% with harmonic band (hb) diversity, and 49.7% with amplitude (dB). The species identification rate has gone up to 96.50% and 97.66% with the ratio and multiplication of F0 and hb, respectively. This is because of the matrices that combine multiple sound attributes. Comparatively, combinations of the amplitude of the F0 and the higher harmonic frequency band were non-significant for species identification (60.82%). The fed females have shown a considerable increase in F0 in comparison to the unfed. The males of all the species possessed significantly higher frequencies with respect to the females. Interestingly, the presence of male-female of Ae. vittatus together showed harmonic convergence between the 2nd and 3rd harmonic bands. In conclusion, the sound-based technology is simple, precise, and cost-effective and provides better resolution for species, sex, and fed-unfed status detection in comparison to conventional methods. Real-time surveillance of mosquitoes could potentially utilize this technology.

One-Component Cationic Lipids for Systemic mRNA Delivery to Splenic T Cells.

Unlocking the full potential of mRNA immunotherapy necessitates targeted delivery to specific cell subsets in the spleen. Four-component lipid nanoparticles (LNPs) utilized in numerous clinical trials are primarily limited in hepatocyte and muscular targeting, highlighting the imperative demand for targeted and simplified non-liver mRNA delivery systems. Herein, we report the rational design of one-component ionizable cationic lipids to selectively deliver mRNA to the spleen and T cells with high efficacy. Unlike the tertiary amine-based ionizable lipids involved in LNPs, the proposed cationic lipids rich in secondary amines can efficiently deliver mRNA both in vitro and in vivo as the standalone carriers. Furthermore, these vectors facilitate efficacious mRNA delivery to the T cell subsets following intravenous administration, demonstrating substantial potential for advancing immunotherapy applications. This straightforward strategy extends the utility of lipid family for extrahepatic mRNA delivery, offering new insights into vector development beyond LNPs to further the field of precise mRNA therapy.

Bois noir management in vineyard: a review on effective and promising control strategies.

Among grapevine yellows, Bois noir (BN), associated with 'Candidatus Phytoplasma solani', represents the biggest threat in the main wine-growing areas worldwide, causing significant losses in berry quality and yields. BN epidemiology involves multiple plant hosts and several insect vectors, making considerably complex the development of effective management strategies. Since application of insecticides on the grapevine canopy is not effective to manage vectors, BN management includes an integrated approach based on treatments to the canopy to make the plant more resistant to the pathogen and/or inhibit the vector feeding, and actions on reservoir plants to reduce possibilities that the vector reaches the grapevine and transmit the phytoplasma. Innovative sustainable strategies developed in the last twenty years to improve the BN management are reviewed and discussed.

Development and characterization of a non-human primate model of disseminated synucleinopathy.

Introduction: The presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery. Methods: Here we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location. Results: Intraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular). Discussion: Obtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein.

Stabilization of an infectious enveloped virus by spray-drying and lyophilization.

Enveloped viruses are attractive candidates for use as gene- and immunotherapeutic agents due to their efficacy at infecting host cells and delivering genetic information. They have also been used in vaccines as potent antigens to generate strong immune responses, often requiring fewer doses than other vaccine platforms as well as eliminating the need for adjuvants. However, virus instability in liquid formulations may limit their shelf life and require that these products be transported and stored under stringently controlled temperature conditions, contributing to high cost and limiting patient access. In this work, spray-drying and lyophilization were used to embed an infectious enveloped virus within dry, glassy polysaccharide matrices. No loss of viral titer was observed following either spray-drying (at multiple drying gas temperatures) or lyophilization. Furthermore, viruses embedded in the glassy formulations showed enhanced thermal stability, retaining infectivity after exposure to elevated temperatures as high as 85°C for up to one hour, and for up to 10 weeks at temperatures as high as 30°C. In comparison, viruses in liquid formulations lost infectivity within an hour at temperatures above 40°C, or after incubation at 25°C for longer periods of time.