Efficient and targeted delivery of a DNA payload is vital for developing safe gene therapy. Owing to the recent success of commercial oncolytic vector and multiple COVID-19 vaccines, adenovirus ...vectors are back in the spotlight. Adenovirus vectors can be used in gene therapy by altering the wild-type virus and making it replication-defective; specific viral genes can be removed and replaced with a segment that holds a therapeutic gene, and this vector can be used as delivery vehicle for tissue specific gene delivery. Modified conditionally replicative-oncolytic adenoviruses target tumors exclusively and have been studied in clinical trials extensively. This comprehensive review seeks to offer a summary of adenovirus vectors, exploring their characteristics, genetic enhancements, and diverse applications in clinical and preclinical settings. A significant emphasis is placed on their crucial role in advancing cancer therapy and the latest breakthroughs in vaccine clinical trials for various diseases. Additionally, we tackle current challenges and future avenues for optimizing adenovirus vectors, promising to open new frontiers in the fields of cell and gene therapies.
Pompe disease is a rare, inherited, devastating condition that causes progressive weakness, cardiomyopathy and neuromotor disease due to the accumulation of glycogen in striated and smooth muscle, as ...well as neurons. While enzyme replacement therapy has dramatically changed the outcome of patients with the disease, this strategy has several limitations. Gene therapy in Pompe disease constitutes an attractive approach due to the multisystem aspects of the disease and need to address the central nervous system manifestations. This review highlights the recent work in this field, including methods, progress, shortcomings, and future directions.
Recombinant adeno-associated virus (rAAV) and lentiviral vectors (LV) are well studied platforms for gene therapy in Pompe disease. These products can be further adapted for safe and efficient administration with concomitant immunosuppression, with the modification of specific receptors or codon optimization. rAAV has been studied in multiple clinical trials demonstrating safety and tolerability.
Gene therapy for the treatment of patients with Pompe disease is feasible and offers an opportunity to fully correct the principal pathology leading to cellular glycogen accumulation. Further work is needed to overcome the limitations related to vector production, immunologic reactions and redosing.
Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of ...highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.
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In this work, Clément et al. compared the herpes simplex virus (HSV) infection and polyethyleneimine (PEI) transfection platforms for the production of recombinant AAV vectors. Findings revealed that HSV-produced stocks were of higher quality with respects to AAV full capsids, residual proteins and DNA than PEI-produced stocks, but contained residual HSV DNA.
The increasing demand for adeno-associated virus (AAV) vectors, a result from the surging interest for their potential to cure human genetic diseases by gene transfer, tumbled on low-performing ...production systems. Innovative improvements to increase both yield and quality of the vector produced have become a priority undertaking in the field. In a previous study, we showed that adding a specific concentration of sodium chloride (NaCl) to the production medium resulted in a dramatic increase of AAV vector particle and infectious titers when using the herpes simplex virus (HSV) production system, both in adherent or suspension platforms. In this work, we studied additional salts and their impact on AAV vector production. We found that potassium chloride (KCl), or a combination of KCl and NaCl, resulted in the highest increase in AAV vector production. We determined that the salt-mediated effect was the most impactful when the salt was present between 8 and approximately 16 h post-infection, with the highest rate increase occurring within the first 24 h of the production cycle. We showed that the AAV vector yield increase did not result from an increase in cell growth, size, or viability. Furthermore, we demonstrated that the impact on AAV vector production was specifically mediated by NaCl and KCl independently of their impact on the osmolality of the production media. Our findings convincingly showed that NaCl and KCl were uniquely efficacious to promote up to a 10-fold increase in the production of highly infectious AAV vectors when produced in the presence of HSV. We think that this study will provide unique and important new insights in AAV biology toward the establishment of more successful production protocols.
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In this work, Clément and colleagues demonstrated that the addition of potassium or sodium chloride during the production cycle of adeno-associated virus (AAV) vector led to up to a 10-fold increase in vector genome and infectious particle titers. This novel finding provides a robust, yet simple, process to improve AAV vector production.
To demonstrate the correlation of proinflammatory cytokines (PCs), intercellular adhesion molecule (sICAM-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) in CSF of ...tuberculous meningitis (TBM) patients with magnetic resonance imaging (MRI) including diffusion tensor imaging (DTI) and also to look for the changes in imaging parameters after antitubercular treatment (ATT) in these patients.
Forty patients with TBM (median age, 27.7 years) and 30 age-/sex-matched controls were included in this study. PCs were quantified from the CSF of TBM patients at the time of hospital admission (baseline). MRI including DTI was performed at the time of baseline study and 6 months after ATT.
Significant positive correlation of PCs with fractional anisotropy (FA) values and post-contrast signal intensity (PCSI) collected from cerebral cortical regions was observed in TBM patients. A significant positive correlation of FA values with PCSI was also observed at both time points in patient groups. At baseline study significantly high FA values were observed in patients compared to controls. Significantly decreased FA values and PCSI were observed in the patients after 6 months of ATT compared to the baseline study.
Results of this study suggest that the DTI-derived anisotropy have the potential to delineate meningeal inflammation and it may be used in assessment of therapeutic response in TBM patients as an additional method to conventional imaging.
Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout ...screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types.
Chromothripsis, the shattering and imperfect reassembly of one (or a few) chromosome(s)
, is an ubiquitous
mutational process generating localized and complex chromosomal rearrangements that drive ...genome evolution in cancer. Chromothripsis can be initiated by mis-segregation errors in mitosis
or DNA metabolism
that lead to entrapment of chromosomes within micronuclei and their subsequent fragmentation in the next interphase or following mitotic entry
. Here we use inducible degrons to demonstrate that chromothriptically produced pieces of a micronucleated chromosome are tethered together in mitosis by a protein complex consisting of mediator of DNA damage checkpoint 1 (MDC1), DNA topoisomerase II-binding protein 1 (TOPBP1) and cellular inhibitor of PP2A (CIP2A), thereby enabling en masse segregation to the same daughter cell. Such tethering is shown to be crucial for the viability of cells undergoing chromosome mis-segregation and shattering after transient inactivation of the spindle assembly checkpoint. Transient, degron-induced reduction in CIP2A following chromosome micronucleation-dependent chromosome shattering is shown to drive acquisition of segmental deletions and inversions. Analyses of pancancer tumour genomes showed that expression of CIP2A and TOPBP1 was increased overall in cancers with genomic rearrangements, including copy number-neutral chromothripsis with minimal deletions, but comparatively reduced in cancers with canonical chromothripsis in which deletions were frequent. Thus, chromatin-bound tethers maintain the proximity of fragments of a shattered chromosome enabling their re-encapsulation into, and religation within, a daughter cell nucleus to form heritable, chromothriptically rearranged chromosomes found in the majority of human cancers.
The Plasmodium FIKK kinases are diverged from human kinases structurally. They harbour conserved ATP-binding domains that are non-homologous to other existing kinases. FIKK9.1 kinase is considered as ...an essential protein for parasite survival. It is localized in major organelles present in parasite and trafficked throughout the infected RBC. It is speculated that FIKK9.1 may phosphorylate several substrates in the parasite’s proteome and contribute to parasite survival. Therefore, FIKK9.1 is an attractive target that may lead to a novel class of antimalarials. To identify specific FIKK9.1 kinase inhibitors, we virtually screened organic structural scaffolds from a library of 623 entries. The top hits were identified based on conformations and molecular interactions with the ATP biophore. The hits were also validated under in vitro conditions. In this study, we identified seven top hit organic compounds that may arrest the growth of parasites by inhibiting FIKK9.1 kinase. Evaluation of top hit compounds in antimalarial activity assay identifies that the highly substituted 1,3-selenazolidin-2-imine 1 and thiophene 2 are inhibiting parasite growth with an IC
50
of 3.2 ± 0.27 μg/ml and 3.13 ± 0.16 μg/ml, respectively. These functionalized heterocyclic compounds 1 and 2 kills the malaria parasite with an IC
50
of 2.68 ± 0.02 μg/ml and 3.08 ± 0.14 μg/ml, respectively. Isothermal titration calorimetry analysis indicate that ATP is binding to the FIKK9.1 kinase. The dissociation constant (
K
d
) is measured to be 27.8 ± 2.07 μM with a stoichiometry of
n
= 1. The heterocyclic scaffolds 1 and 2 were abolishing the binding of ATP into the binding pocket. They in-turn reduce the ability of FIKK9.1 kinase to phosphorylate its substrate. Our study found that compounds 1 and 2 are potent inhibitor of FIKK9.1 kinase and the inhibition of FIKK9.1 kinase using small molecules disturbs the parasite life cycle and leads to the death of parasites. This provides new insight in development of novel antimalarials.
Bronchiectasis is a common but neglected chronic lung disease. Most epidemiological data are limited to cohorts from Europe and the USA, with few data from low-income and middle-income countries. We ...therefore aimed to describe the characteristics, severity of disease, microbiology, and treatment of patients with bronchiectasis in India.
The Indian bronchiectasis registry is a multicentre, prospective, observational cohort study. Adult patients (≥18 years) with CT-confirmed bronchiectasis were enrolled from 31 centres across India. Patients with bronchiectasis due to cystic fibrosis or traction bronchiectasis associated with another respiratory disorder were excluded. Data were collected at baseline (recruitment) with follow-up visits taking place once per year. Comprehensive clinical data were collected through the European Multicentre Bronchiectasis Audit and Research Collaboration registry platform. Underlying aetiology of bronchiectasis, as well as treatment and risk factors for bronchiectasis were analysed in the Indian bronchiectasis registry. Comparisons of demographics were made with published European and US registries, and quality of care was benchmarked against the 2017 European Respiratory Society guidelines.
From June 1, 2015, to Sept 1, 2017, 2195 patients were enrolled. Marked differences were observed between India, Europe, and the USA. Patients in India were younger (median age 56 years IQR 41–66 vs the European and US registries; p<0·0001) and more likely to be men (1249 56·9% of 2195). Previous tuberculosis (780 35·5% of 2195) was the most frequent underlying cause of bronchiectasis and Pseudomonas aeruginosa was the most common organism in sputum culture (301 13·7%) in India. Risk factors for exacerbations included being of the male sex (adjusted incidence rate ratio 1·17, 95% CI 1·03–1·32; p=0·015), P aeruginosa infection (1·29, 1·10–1·50; p=0·001), a history of pulmonary tuberculosis (1·20, 1·07–1·34; p=0·002), modified Medical Research Council Dyspnoea score (1·32, 1·25–1·39; p<0·0001), daily sputum production (1·16, 1·03–1·30; p=0·013), and radiological severity of disease (1·03, 1·01–1·04; p<0·0001). Low adherence to guideline-recommended care was observed; only 388 patients were tested for allergic bronchopulmonary aspergillosis and 82 patients had been tested for immunoglobulins.
Patients with bronchiectasis in India have more severe disease and have distinct characteristics from those reported in other countries. This study provides a benchmark to improve quality of care for patients with bronchiectasis in India.
EU/European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis Consortium, European Respiratory Society, and the British Lung Foundation.
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