Probe capsid structure stability and dynamics of adeno-associated virus as an important viral vector for gene therapy by hydrogen-deuterium exchange-mass spectrometry.

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

Role of Microfiltration Membrane Morphology on Nanoparticle Purification to Enhance Downstream Purification of Viral Vectors.

In the rapidly advancing realms of gene therapy and biotechnology, the efficient purification of viral vectors is pivotal for ensuring the safety and efficacy of gene therapies. This study focuses on optimizing membrane selection for viral vector purification by evaluating key properties, including porosity, thickness, pore structure, and hydrophilicity. Notably, we employed adeno-associated virus (AAV)-sized nanoparticles (20 nm), 200 nm particles, and bovine serum albumin (BSA) to model viral vector harvesting. Experimental data from constant pressure normal flow filtration (NFF) at 1 and 2 bar using four commercial flat sheet membranes revealed distinct fouling behaviors. Symmetric membranes predominantly showed internal and external pore blockage, while asymmetric membranes formed a cake layer on the surface. Hydrophilicity exhibited a positive correlation with recovery, demonstrating an enhanced recovery with increased hydrophilicity. Membranes with higher porosity and interpore connectivity showcased superior throughput, reduced operating time, and increased recovery. Asymmetric polyether sulfone (PES) membranes emerged as the optimal choice, achieving ∼100% recovery of AAV-sized particles, an ∼44% reduction in model cell debris (200 nm particles), an ∼35% decrease in BSA, and the fastest operating time of all membranes tested. This systematic investigation into fouling behaviors and membrane properties not only informs optimal conditions for viral vector recovery but also lays the groundwork for advancing membrane-based strategies in bioprocessing.

[DNA assembly by multi-fragment digestion/ligation and homologous recombination].

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Circular single-stranded DNA as a programmable vector for gene regulation in cell-free protein expression systems.

Cell-free protein expression (CFE) systems have emerged as a critical platform for synthetic biology research. The vectors for protein expression in CFE systems mainly rely on double-stranded DNA and single-stranded RNA for transcription and translation processing. Here, we introduce a programmable vector - circular single-stranded DNA (CssDNA), which is shown to be processed by DNA and RNA polymerases for gene expression in a yeast-based CFE system. CssDNA is already widely employed in DNA nanotechnology due to its addressability and programmability. To apply above methods in the context of synthetic biology, CssDNA can not only be engineered for gene regulation via the different pathways of sense CssDNA and antisense CssDNA, but also be constructed into several gene regulatory logic gates in CFE systems. Our findings advance the understanding of how CssDNA can be utilized in gene expression and gene regulation, and thus enrich the synthetic biology toolbox.

Production of VP3-only virus-like particles of Adeno-associated virus 2 in E. coli cells.

Adeno-associated Virus (AAV) is a promising vector for gene therapy. However, few studies have focused on producing virus-like particles (VLPs) of AAV in cells, especially in E. coli. In this study, we describe a method to produce empty VP3-only VLPs of AAV2 in E. coli by co-expressing VP3 and assembly-activating protein (AAP) of AAV2. Although the yields of VLPs produced with our method were low, the VLPs were able to self-assemble in E. coli without the need of in vitro capsid assembly. The produced VLPs were characterized by immunological detection and transmission electron microscopy (TEM). In conclusion, this study demonstrated that capsid assembly of AAV2 is possible in E. coli, and E. coli may be a candidate system for production of VLPs of AAV.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Generation of a human induced pluripotent stem cell line from a patient with GM3 synthase deficiency using self-replicating RNA vector.

GM3 synthase deficiency (GM3SD) is caused by biallelic variants in the ST3GAL5 gene. Early clinical features of GM3SD include infantile onset of severe irritability and feeding difficulties, early intractable seizures, growth failure, hypotonia, sensorineural hearing impairment. We describe the generation and characterization the human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a 13-year-old girl with GM3 synthase deficiency resulted compound heterozygous for two new variants in the ST3GAL5 gene, c.1166A > G (p.His389Arg) and the c.1024G > A (p.Gly342Ser). The generated hiPSC line shows a normal karyotype, expresses pluripotency markers, and is able to differentiate into the three germ layers.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing.

The expansion of the CRISPR-Cas toolbox is highly needed to accelerate the development of therapies for genetic diseases. Here, through the interrogation of a massively expanded repository of metagenome-assembled genomes, mostly from human microbiomes, we uncover a large variety (n = 17,173) of type II CRISPR-Cas loci. Among these we identify CoCas9, a strongly active and high-fidelity nuclease with reduced molecular size (1004 amino acids) isolated from an uncultivated Collinsella species. CoCas9 is efficiently co-delivered with its sgRNA through adeno associated viral (AAV) vectors, obtaining efficient in vivo editing in the mouse retina. With this study we uncover a collection of previously uncharacterized Cas9 nucleases, including CoCas9, which enriches the genome editing toolbox.

Fast, accurate ranking of engineered proteins by target-binding propensity using structure modeling.

Deep-learning-based methods for protein structure prediction have achieved unprecedented accuracy, yet their utility in the engineering of protein-based binders remains constrained due to a gap between the ability to predict the structures of candidate proteins and the ability toprioritize proteins by their potential to bind to a target. To bridge this gap, we introduce Automated Pairwise Peptide-Receptor Analysis for Screening Engineered proteins (APPRAISE), a method for predicting the target-binding propensity of engineered proteins. After generating structural models of engineered proteins competing for binding to a target using an established structure prediction tool such as AlphaFold-Multimer or ESMFold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As proof-of-concept cases, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or programmed death-ligand 1 (PD-L1). APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.

Generation of a TBX20 homozygous knockout stem cell line (WAe009-A-1E) by episomal vector-based CRISPR/Cas9 system.

The T-box family transcription factor gene TBX20 plays a crucial role in cardiac development and function. TBX20 mutations are associated with congenital heart disease, dilated cardiomyopathy, arrhythmias, and heart failure. To further study the role of TBX20 in human heart, here we generated a homozygous TBX20 knockout (TBX20-KO) human embryonic stem cell line using the CRISPR/Cas9 system. This TBX20-KO cell line maintains normal morphology, pluripotency, and karyotype, making it a valuable tool for investigating TBX20's role in cardiac biology.

Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production.

E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins. This study focused on the challenges and engineering approaches for improved expression and solubility in recombinant protein production in E. coli. In this context, solution strategies such as strain and vector selection, codon usage, mRNA stability, expression conditions, translocation to the periplasmic region and addition of fusion tags in E. coli were discussed.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.

Unbiased in vivo selections of diverse capsid libraries can yield engineered capsids that overcome gene therapy delivery challenges like traversing the blood-brain barrier (BBB), but little is known about the parameters of capsid-receptor interactions that govern their improved activity. This hampers broader efforts in precision capsid engineering and is a practical impediment to ensuring the translatability of capsid properties between preclinical animal models and human clinical trials. In this work, we utilize the adeno-associated virus (AAV)-PHP.B-Ly6a model system to better understand the targeted delivery and BBB penetration properties of AAV vectors. This model offers a defined capsid-receptor pair that can be used to systematically define relationships between target receptor affinity and in vivo activity of engineered AAV vectors. Here, we report a high-throughput method for quantifying capsid-receptor affinity and demonstrate that direct binding assays can be used to organize a vector library into families with varied affinity for their target receptor. Our data indicate that efficient central nervous system transduction requires high levels of target receptor expression at the BBB, but it is not a requirement for receptor expression to be limited to the target tissue. We observed that enhanced receptor affinity leads to reduced transduction of off-target tissues but can negatively impact on-target cellular transduction and penetration of endothelial barriers. Together, this work provides a set of tools for defining vector-receptor affinities and demonstrates how receptor expression and affinity interact to impact the performance of engineered AAV vectors in targeting the central nervous system. IMPORTANCE Novel methods for measuring adeno-associated virus (AAV)-receptor affinities, especially in relation to vector performance in vivo, would be useful to capsid engineers as they develop AAV vectors for gene therapy applications and characterize their interactions with native or engineered receptors. Here, we use the AAV-PHP.B-Ly6a model system to assess the impact of receptor affinity on the systemic delivery and endothelial penetration properties of AAV-PHP.B vectors. We discuss how receptor affinity analysis can be used to isolate vectors with optimized properties, improve the interpretation of library selections, and ultimately translate vector activities between preclinical animal models and humans.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Differential interferon-α subtype induced immune signatures are associated with suppression of SARS-CoV-2 infection.

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.

Reprogramming cellular identity in vivo.

Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress.

Gene Transfer of Skeletal Muscle-Type Myosin Light Chain Kinase via Adeno-Associated Virus 6 Improves Muscle Functions in an Amyotrophic Lateral Sclerosis Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients' quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.