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The transcription factors (TFs) in the NAC family are involved in regulating multiple biological processes, playing an important role in plant growth, development, and stress adaptation. Our previous studies have demonstrated that TaNAC14, a member of the NAC family in wheat ( Triticum aestivum L.), positively regulates root growth and development and enhances the drought tolerance of wheat seedlings. In this study, we analyzed the physicochemical properties and structure and verified the subcellular localization and transcriptional activation activity of TaNAC14. The prokaryotic expression vector pET21a-HMT-TaNAC14 was constructed and transformed into Escherichia coli BL21 CodonPlus (DE3)-RIPL. The conditions for inducing the expression of the recombinant protein HMT-TaNAC14 were optimized. The solubility of the recombinant protein was analyzed, and the protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The results indicated that TaNAC14 had a conserved domain of the NAM family. It was located in the nucleus and had transcriptional activation activity. The optimal conditions for expression of the recombinant protein in E. coli were induction with 0.2mmol/L IPTG for 4 h. The recombinant protein mainly existed in the soluble form, and the target protein was obtained after purification. This study lays a foundation for the identification of target genes regulated by TaNAC14.

CRISPR-Cas9 editing triggers activation of the TP53-p21 pathway, but the impacts of different editing components and delivery methods have not been fully explored. In this study, we introduce a p21-mNeonGreen reporter iPSC line to monitor TP53-p21 pathway activation. This reporter enables dynamic tracking of p21 expression via flow cytometry, revealing a strong correlation between p21 expression and indel frequencies, and highlighting its utility in guide RNA screening. Our findings show that p21 activation is significantly more pronounced with double-stranded oligodeoxynucleotides (ODNs) or adeno-associated viral vectors (AAVs) compared to their single-stranded counterparts. Lentiviral vectors (LVs) and integrase-defective lentiviral vectors induce notably lower p21 expression than AAVs, suggesting their suitability for gene therapy in sensitive cells such as hematopoietic stem cells or immune cells. Additionally, specific viral promoters like SFFV significantly amplify p21 activation, emphasizing the critical role of promoter selection in vector development. Thus, the p21-mNeonGreen reporter iPSC line is a valuable tool for assessing the potential adverse effects of gene editing methodologies and vectors. Highlights Established a p21-mNeonGreen reporter iPSC line to track activation of the TP53-p21 pathway. Found a direct correlation between p21-mNeonGreen expression and indel frequencies, aiding in gRNA screening. Showed that LVs are preferable over AAVs for certain cells due to lower p21 activation, with viral promoter choice impacting p21 response., (© The Author(s) 2024. Published by Oxford University Press.)

Many types of viruses directly or indirectly target the vascular endothelial growth factor (VEGF) system, which is a central regulator of vasculogenesis and angiogenesis in physiological homeostasis, causing diverse pathologies. Other viruses have been developed into effective therapeutic tools for VEGF modulation in conditions such as cancer and eye diseases. Some viruses may alter the levels of VEGF in the pathogenesis of respiratory syndromes, or they may encode VEGF-like factors, promoting vascular disruption and angiogenesis to enable viruses' systemic spread. Oncogenic viruses may express interactive factors that perturb VEGF's functional levels or downstream signaling, which increases the neovascularization and metastasis of tumors. Furthermore, many viruses are being developed as therapeutic vectors for vascular pathologies in clinical trials. Major examples are those viral vectors that inhibit the role of VEGF in the neovascularization required for cancer progression; this is achieved through the induction of immune responses, by exposing specific peptides that block signaling or by expressing anti-VEGF and anti-VEGF receptor-neutralizing antibodies. Other viruses have been engineered into effective pro- or anti-angiogenesis multitarget vectors for neovascular eye diseases, paving the way for therapies with improved safety and minimal side effects. This article critically reviews the large body of literature on these issues, highlighting those contributions that describe the molecular mechanisms, thus expanding our understanding of the VEGF-virus interactions in disease and therapy. This could facilitate the clinical use of therapeutic virus vectors in precision medicine for the VEGF system.

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in Nicotiana benthamiana leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins.

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism., (© 2024 Wirshing et al.)

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.

Microtubule (MT) consists of α-tubulin and β-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo , we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice ( Oryza sativa ) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7 , OsKCBP , OsCLASP , and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice., Competing Interests: The authors declare that they have no competing interests., (© 2024 Xu et al.)

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis ( B. subtilis ), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, P nprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SP ydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis . These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis .

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD 600 ) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dunaliella salina is an innovative expression system due to its distinct advantages such as high salt tolerance, low susceptibility to contamination, and the absence of the cell wall. While nuclear transformation has been extensively studied, research on D. salina chloroplast transformation remains in the preliminary stages. In this study, we established an efficient chloroplast expression system for D. salina using Golden Gate assembly. We developed a D. salina toolkit comprising essential components such as chloroplast-specific promoters, terminators, homologous fragments, and various vectors. We confirmed its functionality by expressing the EGFP protein. Moreover, we detailed the methodology of the entire construction process. This expression system enables the specific targeting of foreign genes through simple homologous recombination, resulting in stable expression in chloroplasts. The toolkit achieved a relatively high transformation efficiency within a shorter experimental cycle. Consequently, the construction and utilization of this toolkit have the potential to enhance the efficiency of transgenic engineering in D. salina and advance the development of microalgal biofactories., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We constructed a new Aspergillus expression vector (pSENSU2512nid) under the control of the enolase promoter with 12 tandem repeats of cis-acting elements (region III) and the heat shock protein 12 (Hsp12) 5' untranslated region (UTR). Bilirubin oxidase (EC: 1.3.3.5) from Myrothecium verrucaria, which catalyzes the oxidation of bilirubin to biliverdin, was overexpressed in Aspergillus oryzae and A. niger. The productivity was estimated to be approximately 1.2 g/L in the culture broth, which was approximately 6-fold higher than that of recombinant bilirubin oxidase (BOD) expressed in Pichia pastoris (Komagataella phaffii). BOD was purified using hydrophobic interaction chromatography, followed by ion exchange chromatography. The specific activity of the purified BOD against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate was 57.6 U/mg and 66.4 U/mg for A. oryzae and A. niger, respectively. l-Ascorbic acid (4 mM) addition and storage under deoxygenated conditions for 3-7 d increased the specific activity of these Aspergillus-expressed BODs approximately 2.3-fold (154.1 U/mg). The BOD specific activity was enhanced by incubation at higher temperature (30-50 °C). Further characterization of the enzyme catalytic efficiency revealed that the K m value remained unchanged, whereas the k cat value improved 3-fold. In conclusion, this high-level of BOD expression meets the requirements for industrial-level production. Additionally, we identified an effective method to enhance the low specific activity during expression, making it advantageous for industrial applications., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Oxidative Phosphorylation (OXPHOS) defects can cause severe encephalopathies and no effective treatment exists for these disorders. To assess the ability of gene replacement to prevent disease progression, we subjected two different CNS-deficient mouse models (Ndufs3/complex I or Cox10/complex IV conditional knockouts) to gene therapy. We used retro-orbitally injected AAV-PHP.eB to deliver the missing gene to the CNS of these mice. In both cases, we observed survival extension from 5-6 to more than 15 months, with no detectable disease phenotypes. Likewise, molecular and cellular phenotypes were mostly recovered in the treated mice. Surprisingly, these remarkable phenotypic improvements were achieved with only ~30% of neurons expressing the transgene from the AAV-PHP.eB vector in the conditions used. These findings suggest that neurons lacking OXPHOS are protected by the surrounding neuronal environment and that partial compensation for neuronal OXPHOS loss can have disproportionately positive effects., (© 2024. The Author(s).)

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1)., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Protein-protein interactions play critical roles in biological processes. We previously developed the Pil1 co-tethering assay, an imaging-based method to detect protein-protein interactions in living Schizosaccharomyces pombe cells. This assay leverages the distinct localization pattern of the Pil1 protein by fusing a bait protein to Pil1 and examining whether a prey protein co-localize with the Pil1-fused bait. Here, we present an improved protocol of the Pil1 co-tethering assay. In this protocol, modified stable integration vectors (SIVs) with a NotI site as the linearization site are used to express bait and prey proteins. We expect that this protocol will enhance the application of the Pil1 co-tethering assay for studying protein-protein interactions., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%-30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid-filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Gene replacement using adeno-associated virus (AAV) vectors is a promising therapeutic approach for many diseases 1,2 . However, this therapeutic modality is challenged by the packaging capacity of AAVs (approximately 4.7 kilobases) 3 , limiting its application for disorders involving large coding sequences, such as Duchenne muscular dystrophy, with a 14 kilobase messenger RNA. Here we developed a new method for expressing large dystrophins by utilizing the protein trans-splicing mechanism mediated by split inteins. We identified several split intein pairs that efficiently join two or three fragments to generate a large midi-dystrophin or the full-length protein. We show that delivery of two or three AAVs into dystrophic mice results in robust expression of large dystrophins and significant physiological improvements compared with micro-dystrophins. Moreover, using the potent myotropic AAVMYO 4 , we demonstrate that low total doses (2 × 10 13  viral genomes per kg) are sufficient to express large dystrophins in striated muscles body-wide with significant physiological corrections in dystrophic mice. Our data show a clear functional superiority of large dystrophins over micro-dystrophins that are being tested in clinical trials. This method could benefit many patients with Duchenne or Becker muscular dystrophy, regardless of genotype, and could be adapted to numerous other disorders caused by mutations in large genes that exceed the AAV capacity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adeno-associated virus (AAV) has emerged as the most promising vector for in vivo human gene therapy, with several therapeutic approvals in the last few years and countless more under development. Underlying this remarkable success are several attractive features that AAV offers, including lack of pathogenicity, low immunogenicity, long-term gene expression without genomic integration, the ability to infect both dividing and non-dividing cells, etc . However, the commonly used wild-type AAV capsids in therapeutic development present significant challenges, including inadequate tissue specificity and the need for large doses to attain therapeutic effectiveness, raising safety concerns. Additionally, significant preexisting adaptive immunity against most natural capsids, and the development of such anti-capsid immunity after the first treatment, represent major challenges. Strategies to engineer the AAV capsid are critically needed to address these challenges and unlock the full promise of AAV gene therapy. Chemical modification of the AAV capsid has recently emerged as a powerful new approach to engineer its properties. Unlike genetic strategies, which can be more disruptive to the delicate capsid assembly and packaging processes, "late-stage" chemical modification of the assembled capsid-whether at natural amino acid residues or site-specifically installed noncanonical amino acid residues-often enables a versatile approach to introducing new properties to the capsid. This review summarizes the significant recent progress in AAV capsid engineering strategies, with a particular focus on chemical modifications in advancing the next generation of AAV-based gene therapies.

To overcome limitations in the generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by the insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of the human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, which showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to a 5 times higher titer, while AAV-M8 reached a 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. The transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high-throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell-type specificity of virus vectors.

Background: Hemophilia B is an X-linked bleeding disorder caused by a mutation in the gene responsible for encoding coagulation factor IX (FIX). Gene therapy offers promising potential for curing this disease. However, the current method of relatively high dosage of virus injection carries inherent risks. The purpose of this study was to introduce a novel scAAV-DJ/8-LP1-hFIXco vector transduced human umbilical cord blood derived mesenchymal stem cells (HUCMSCs) as an alternative cell-based gene therapy to conventional gene therapy for Hemophilia B., Methods: The LP1-hFIXco gene structure was designed by us through searching the literature from NCBI and the scAAV-DJ/8-LP1-hFIXco vector was constructed by a commercial company. The HUCMSCs were cultivated in routine approach and transduced with scAAV-DJ/8-LP1-hFIXco vector. The human FIX activation system was employed for detection of hFIXco activity. The RNA and protein expression levels of the hFIXco were evaluated using PCR and western blot techniques. In animal studies, both NSG and F9-KO mice were used for the experiment, in which clotting time was utilized as a parameter for bleeding assessment. The immunohistochemical analysis was used to assess the distribution of HUCMSCs in mouse tissue sections. The safety for tumorigenicity of this cell-based gene therapy was evaluated by pathological observation after hematoxylin-eosin staining., Results: The transduction of HUCMSCs with the scAAV-DJ/8-LP1-hFIXco vector results in consistent and sustainable secretion of human FIXco during 5 months period both in vitro and in mouse model. The secretion level (hFIXco activity: 97.1 ± 2.3% at day 7 to 48.8 ± 4.5% at 5 months) was comparable to that observed following intravenous injection with a high dose of the viral vector (hFIXco activity: 95.2 ± 2.2% to 40.8 ± 4.3%). After a 5-month observation period, no clonal expansions of the transduced cells in tissues were observed in any of the mice studied., Conclusions: We have discovered a novel and safer HUCMSCs mediated approach potentially effective for gene therapy in hemophilia B., (© 2024. The Author(s).)

Lentiviral vectors have markedly enhanced gene therapy efficiency in treating congenital diseases, but their long-term safety remains controversial. Most gene therapies for congenital eye diseases need to be carried out at early ages, yet the assessment of related risks to ocular development posed by lentiviral vectors is challenging. Utilizing single-cell transcriptomic profiling on human retinal organoids, this study explored the impact of lentiviral vectors on the retinal development and found that lentiviral vectors can cause retinal precursor cells to shift toward photoreceptor fate through the up-regulation of key fate-determining genes such as PRDM1. Further investigation demonstrated that the intron and intergenic region of PRDM1 was bound by PHLDA1, which was also up-regulated by lentiviral vectors exposure. Importantly, knockdown of PHLDA1 successfully suppressed the lentivirus-induced differentiation bias of photoreceptor cells. The findings also suggest that while lentiviral vectors may disrupt the fate determination of retinal precursor cells, posing risks in early-stage retinal gene therapy, these risks could potentially be reduced by inhibiting the PHLDA1-PRDM1 axis., (© 2024. The Author(s).)

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., Competing Interests: Declarations of completing interest The authors declare no potential conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

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., Competing Interests: Declaration of interests X.J. and X.Z. are co-inventors on in vivo AAV-based Perturb-seq and CRISPR inventions filed by Scripps Research relating to the work in this manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Declaration of interests V.G. is a scientific co-founder and BoD member of Capsida Biotherapeutics. The terms of the arrangements have been reviewed and approved by the California Institute of Technology in accordance with its conflict-of-interest policies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., (© 2024. The Author(s).)

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.

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.

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 (k IN ) of 5.58 × 10 - 2 h-1 and half-life of 12 ± 0 h at 95 °C. K m and V max 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 V max 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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Declaration of interests D.V.S. and J.L. are inventors on patents related to viral vector-directed evolution and engineered AAV variants., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

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., (© 2024. The Author(s).)

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., Competing Interests: The authors declare a conflict of interest. This research was supported by Amicus Therapeutics and Passage Bio. JMW is a paid advisor to and holds equity in iECURE, Scout Bio, Passage Bio, and the Center for Breakthrough Medicines (CBM). He also holds equity in the G2 Bio-associated asset companies. He has sponsored research agreements with Amicus Therapeutics, CBM, Elaaj Bio, FA212, G2 Bio, G2 Bio-associated asset companies, iECURE, Passage Bio, and Scout Bio, which are licensees of Penn technology. JMW and JJS are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments.

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.

To generate stable cell lines that express high levels of recombinant genes often requires screening of a large number of transfected cells using ELISA. The most widely used alternative to ELISA screening is to use an intracellularly expressed GFP reporter construct which allows sorting of recombinant gene expression cells based on GFP fluorescence intensity. The disadvantage of cell sorting, however, is that the resulting population will be polyclonal with the danger of instability and overgrowth of low producers. In addition, GFP or its variants can be toxic to host cells at high concentrations, and thus may reduce growth and robustness of high producer cells or even cause them to become apoptotic. We have developed a new mammalian expression system in which a recombinant protein and a fluorescence protein, AcGFP1, are expressed on the same plasmid separated by an internal ribosome entry site (IRES). A signal peptide was incorporated upstream of AcGFP1 so that the fluorescent protein is secreted from cells, preventing cellular toxicity from intracellular accumulation and enabling convenient and accurate measurement of the protein. Expression tests of Ebola viral envelope GP1 and HIV gp120 proteins using this expression system in 293-H cells showed recombinant protein expression levels were closely correlated with AcGFP1 yield. Therefore, AcGFP1 can serve as an accurate reporter for recombinant protein expression and measuring AcGFP1 concentration provides a convenient, product independent and universal way for efficient clone screening., (Copyright © 2021. Published by Elsevier Inc.)

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC., Competing Interests: Declaration of interests B.D.L., F.S., and Z.X. are inventors on a US provisional patent application no. 63/222723, entitled “Novel COVID vaccine and method for delivery.” All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

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., (© 2022. Published by The Company of Biologists Ltd.)

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 SOD1 G37R 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 SOD1 G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1 G37R mice and the tetanic force of WT and SOD1 G37R 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.

Via the peripheral and autonomic nervous systems, the spinal cord directly or indirectly connects reciprocally with many body systems (muscular, intengumentary, respiratory, immune, digestive, excretory, reproductive, cardiovascular, etc). Accordingly, spinal cord injury (SCI) can result in catastrophe for multiple body systems including muscle paralysis affecting movement and loss of normal sensation, as well as neuropathic pain, spasticity, reduced fertility and autonomic dysreflexia. Treatments and cure for an injured spinal cord will likely require access of therapeutic agents across the blood-CNS (central nervous system) barrier. However, some types of repair within the CNS may be possible by targeting treatment to peripherally located cells or by delivering Adeno-Associated Viral vectors (AAVs) by peripheral routes (e.g., intrathecal, intravenous). This review will consider some future possibilities for SCI repair generated by therapeutic peripheral gene delivery. There are now six gene therapies approved worldwide as safe and effective medicines of which three were created by modification of the apparently nonpathogenic Adeno-Associated Virus. One of these AAVs, Zolgensma, is injected intrathecally for treatment of spinal muscular atrophy in children. One day, delivery of AAVs into peripheral tissues might improve recovery after spinal cord injury in humans; we discuss experiments by us and others delivering transgenes into nerves or muscles for sensorimotor recovery in animal models of SCI or of stroke including human Neurotrophin-3. We also describe ongoing efforts to develop AAVs that are delivered to particular targets within and without the CNS after peripheral administration using capsids with improved tropisms, promoters that are selective for particular cell types, and methods for controlling the dose and duration of expression of a transgene. In conclusion, in the future, minimally invasive administration of AAVs may improve recovery after SCI with minimal side effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has a devastating impact on global populations triggered by a highly infectious viral sickness, produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The third major cause of mortality in the United States, following heart disease and cancer in 2020, was undoubtedly COVID-19. The centers for disease control and prevention (CDC) and the world health organization (WHO) separately developed a categorization system for differentiating new strains of SARS-CoV-2 into variants of concern (VoCs) and variants of interest (VoIs) with the continuing development of various strains SARS-CoV-2. By December 2021, five of the SARS-CoV-2 VoCs were discovered from the onset of the pandemic depending on the latest epidemiologic report by the WHO: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Mutations in the receptor-binding domain (RBD) and n-terminal domain (NTD) have been found throughout all five identified VoCs. All strains other than the delta mutant are often found with the N501Y mutation situated on the RBD, resulting in higher binding between the spike protein and angiotensin-converting enzyme 2 (ACE2) receptors, enhanced viral adhesion, and following the entrance to host cells. The introduction of these new strains of SRAS-CoV-2 is likely to overcome the remarkable achievements gained in restricting this viral disease to the point where it is presented with remarkable vaccine developments against COVID-19 and strong worldwide mass immunization initiatives. Throughout this literature review, the effectiveness of current COVID-19 vaccines for managing and prohibiting SARS-CoV-2 strains is thoroughly described., (Copyright © 2021. Published by Elsevier Masson SAS.)

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections., (© 2022. The Author(s).)

Riboswitches are structured non-coding RNAs often located upstream of essential genes in bacterial messenger RNAs. Such RNAs regulate expression of downstream genes by recognizing a specific cellular effector. Although nearly 50 riboswitch classes are known, only a handful recognize multiple effectors. Here, we report the 2.60-Å resolution co-crystal structure of a class I type I preQ 1 -sensing riboswitch that reveals two effectors stacked atop one another in a single binding pocket. These effectors bind with positive cooperativity in vitro and both molecules are necessary for gene regulation in bacterial cells. Stacked effector recognition appears to be a hallmark of the largest subgroup of preQ 1 riboswitches, including those from pathogens such as Neisseria gonorrhoeae. We postulate that binding to stacked effectors arose in the RNA World to closely position two substrates for RNA-mediated catalysis. These findings expand known effector recognition capabilities of riboswitches and have implications for antimicrobial development., (© 2022. The Author(s).)

Skeletal muscle serves fundamental roles in organismal health. Gene expression fluctuations are critical for muscle homeostasis and the response to environmental insults. Yet, little is known about post-transcriptional mechanisms regulating such fluctuations while impacting muscle proteome. Here we report genome-wide analysis of mRNA methyladenosine (m 6 A) dynamics of skeletal muscle hypertrophic growth following overload-induced stress. We show that increases in METTL3 (the m 6 A enzyme), and concomitantly m 6 A, control skeletal muscle size during hypertrophy; exogenous delivery of METTL3 induces skeletal muscle growth, even without external triggers. We also show that METTL3 represses activin type 2 A receptors (ACVR2A) synthesis, blunting activation of anti-hypertrophic signaling. Notably, myofiber-specific conditional genetic deletion of METTL3 caused spontaneous muscle wasting over time and abrogated overload-induced hypertrophy; a phenotype reverted by co-administration of a myostatin inhibitor. These studies identify a previously unrecognized post-transcriptional mechanism promoting the hypertrophic response of skeletal muscle via control of myostatin signaling., (© 2022. The Author(s).)