Search

The field of gene therapy using Adeno-associated viral (AAV) vector delivery is rapidly advancing in the biotherapeutics industry. Despite its successes, AAV manufacturing remains a challenge due to limited production yields. The triple plasmid transfection of HEK293 cells represents the most extensively utilized system for AAV production. The regulatory factors and mechanisms underlying viral production in HEK293 cells are largely unknown. In this study, we isolated high-titer AAV production clones from a parental HEK293 population using a single limiting dilution step, and subsequently elucidating their underlying molecular mechanisms through whole transcriptome analysis. LncRNA TCONS_00160397 was upregulated in clones and shown to promoted HEK293 cells proliferation and improved the titer of AAV production. Mechanistically, results from proteomics and metabolomics indicated that TCONS_00160397 regulated the ABC transporters pathway. These findings furnish a rich repository of knowledge and actionable targets for the rational optimization of HEK293-based producer lines, thereby paving the way for tangible improvements in AAV vector output and expediting the broad implementation of gene therapies., 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.)

The development of gene therapy based on genome editing has opened up new possibilities for the treatment of human genetic disorders. This field has developed rapidly over the past few decades, some genome editing-based therapies are already in phase 3 clinical trials. However, there are several challenges to be addressed before widespread adoption of gene editing therapy becomes possible. The main obstacles in the development of such therapy are safety and efficiency, so one of the biggest issues is the delivery of genetic constructs to patient cells. Approaches in genetic cargo delivery divide into ex vivo and in vivo, which are suitable for different cases. The ex vivo approach is mainly used to edit blood cells, improve cancer therapy, and treat infectious diseases. To edit cells in organs researches choose in vivo approach. For each approach, there is a fairly large set of methods, but, unfortunately, these methods are not universal in their effectiveness and safety. The focus of this article is to discuss the current status of in vivo and ex vivo delivery methods used in genome editing-based therapy. We will discuss the main methods employed in these approaches and their applications in current gene editing treatments under development., Competing Interests: Declarations. Competing interests: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This study investigates the crucial role of transfection methods in the manufacturability and potency of recombinant adeno-associated virus (rAAV) gene therapies. By employing a novel analytical approach, multiplex digital PCR (dPCR), we evaluated the impact of different transfection reagents and conditions on the scalability and quality of rAAV. Our research demonstrates that the selection of transfection approach significantly influences not only the yield and ease of scale-up but also the potency of the final product. Importantly, later changes to transfection parameters established in the early stages of development can be challenging, potentially compromising product quality and leading to comparability issues. Leveraging multiplex dPCR has proven instrumental in guiding these early-stage decisions, ensuring a reliable manufacturing process that consistently delivers high-quality therapeutic products. Our findings highlight the importance of optimizing transfection strategies early in development to guarantee the successful production of potent and cost-effective gene therapies, ultimately impacting patient accessibility., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owners of stock options. Other than that, no conflict of interest is declared., (Copyright © 2025 Baxalta Innovations Gmbh (part of Takeda). Published by Elsevier B.V. All rights reserved.)

Recombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 10 6 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV-1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV-1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer- probe set targeting the packaging signal sequence common to murine leukemia virus- and murine stem cell virus- based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are employees of Takeda Pharmaceutical Company Limited, and have no commercial, proprietary, or financial interests in the products or companies described in this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Efficient recombinant protein production requires mammalian stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. In our study, we aim to develop a high expression cassette to increase yields of baculoviral transduction. The establishment follows a sequential approach by first identifying the strongest promoter, followed by intron and WPRE sequences as enhancer elements for transcription and translation. The resulting REMBAC-cassette was compared to conventional transfection in suspension and adherent cells. Irrespective of the cell line, transduction reached nearly 100 % efficiency and led to almost 10-fold increases of gene expression levels. We confirmed these results in larger scale with batch and fed-batch cultivations. Finally, expression of different soluble proteins with high degrees of complexity confirmed the versatility of our established cassette. Overall, the REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes., 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 © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Cytokines play pivotal roles in anticancer immune response. We previously reported that adenovirus armed with an IL18 variant (DR18) that overcomes IL18BP neutralizing effect displayed powerful therapeutic effects in local and distant tumors when delivered intratumorally. Here, we tested a combined delivery of IL12 and DR18 in tumor models since IL12 and IL18 are known to act synergistically in potentiating IFNγ production and antitumor immunity. To minimize adverse effects associated with systemic delivery, we constructed oncolytic adenoviruses (oAd) harboring DR18 and IL12 (oAd.DR18/IL12). IL12 was expressed as a single chain IL12 (scIL12) peptide composed of the IL12/p40 and IL12/p35 subunits. Intratumoral administration of oAd.DR18/IL12, oAd-expressing DR18 (oAd.DR18), or oAd-expressing IL12 (oAd.IL12) showed antitumor effect in syngeneic colorectal tumor models. Compared to oAd.DR18 or oAd.IL12, administration of oAd.DR18/IL12 improved the antitumor effects as well as increased survival rate in these models. We detected enhanced tumor infiltrating T lymphocytes and NK cells in oAd.DR18/IL12-treated mice than those from mock-treated or individually treated groups. Moreover, mice received oAd.DR18/IL12 had more robust tumor-specific cytotoxicity. Importantly, mice that had tumor regression after oAd.DR18/IL12 treatment established anti-tumor specific immune memory. These results show that adenovirus armed with engineered cytokines boosts tumor specific immunity and antitumor effect., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethic statement: The use and care of laboratory animals was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines., (© 2025. The Author(s).)

Adeno-associated virus (AAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a synthetic ITR (SynITR), harboring substitutions in putative p53 binding sites was generated and evaluated for vector production and gene delivery. Replication of SynITR flanked transgenic genome was similar compared to wild type (wt) ITR, with a modest increase in vector titers. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cells without toxicity. Following AAV2-wtITR vector infection of hESCs, rapid apoptosis was observed as reported. In contrast, infection by AAV2 vectors packaged with SynITRs attenuated the wtITR-induced hESC toxicity. While hESC particle entry and the abundance of double stranded circular episomes was similar for the ITR contexts, reporter expression was inhibited from transduced SynITR genomes. Mechanistically, infection of hESCs induced γH2AX in an ITR-independent manner, however, canonical activation of p53α was uncoupled using AAV-SynITR. Further investigations in hESCs revealed additional novel findings: (i) p53β is uniquely and constitutively active and (ii) AAV vector infection, independent of the ITR sequence, induces activation of p53ψ. The data herein reveal an ITR-dependent AAV vector transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Muscular dystrophies are a group of heterogenic disorders characterized by progressive muscle weakness, the most common of them being Duchenne muscular dystrophy (DMD). Muscular dystrophies are caused by mutations in over 50 distinct genes, and many of them are caused by different genetic mechanisms. Currently, none of these diseases have a cure. However, in recent years, significant progress has been made to correct the underlying genetic cause. The clinical development of adeno-associated viral vector-based therapies has simultaneously produced excitement and disappointment in the research community due to the moderate effect, making it clear that new methods of muscle delivery have to be created. Herein, we review the main characteristics of major muscular dystrophies and outline various muscle-targeted delivery methods being explored for genetic medicines., Competing Interests: Declaration of interests D.P. receives licensing fees (to patents on which he was an inventor) from, invested in, consults (or on scientific advisory boards or boards of directors), or is a founder of, or conducts sponsored research at TAU for the following entities: ART Bioscience; BioNTech SE; Earli, Inc.; Kernal Biologics; Geneditor Biologics, Inc.; New Phase Ltd.; NeoVac Ltd.; RiboX Therapeutics; Roche; SirTLab Corporation; and Teva Pharmaceuticals, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Gene therapy has emerged as a promising frontier in the management of diabetes, offering innovative approaches to address both type 1 and type 2 diabetes. This narrative review examines the advancements in gene therapy applications, focusing on both animal and human studies, and includes a total of 11 studies in adherence to PRISMA guidelines. These studies utilize various viral vectors, such as adeno-associated virus (AAV) and lentivirus, to deliver genes that regulate insulin production and enhance angiogenesis. This review aims to synthesize recent advancements in gene therapy for both type 1 and type 2 diabetes and its complications, and to explore the evolving role of pharmacists in this emerging field. Methods: A comprehensive search was conducted to identify relevant studies on gene therapy for diabetes. Databases such as PubMed, the Cochrane Database of Systematic Reviews, the Cochrane Central Register of Controlled Trials, and Google Scholar were queried using keywords such as "Diabetes", "gene therapy", "Type 1 diabetes", and "Type 2 diabetes". Both animal and human studies were included to provide a broad perspective on the advancements in this field. Results: Animal model studies have shown promising results, including sustained insulin production, improved glucose homeostasis, and enhanced wound healing. Human studies, though fewer in number, have reported significant advancements. Patients with diabetic neuropathy treated with plasmid VEGF and recombinant adeno-associated virus (rAAV) showed improvements in neuropathic symptoms and glycemic control. Other studies involving intramuscular injections of VM202 and bicistronic VEGF165/HGF plasmid have reported pain reduction, improved healing of ischemic lesions, and increased angiogenesis. Conclusions: Despite these encouraging results, limitations such as small sample sizes, short follow-up periods, and the necessity for more extensive clinical trials persist. Diabetes is a metabolic syndrome that requires the collaboration of a multidisciplinary team to assist in several aspects of implementing successful gene therapy. Several healthcare providers and policy makers may play a crucial role in patient education, counseling, and the management of gene therapy treatments.

Semaphorin 3A (Sema3A) is an axon guidance molecule, which is also abundant in the adult central nervous system (CNS), particularly in perineuronal nets (PNNs). PNNs are extracellular matrix structures that restrict plasticity. The cellular sources of Sema3A in PNNs are unknown. Most Sema3A-bearing neurons do not express Sema3A mRNA, suggesting that Sema3A may be released from other neurons. Another potential source of Sema3A is the choroid plexus. To identify sources of PNN-associated Sema3A, we focused on the cerebellar nuclei, which contain Sema3A + PNNs. Cerebellar nuclei neurons receive prominent input from Purkinje cells (PCs), which express high levels of Sema3A mRNA. By using a non-invasive viral vector approach, we overexpressed Cre in PCs, the choroid plexus, or throughout the CNS of Sema3A fl/fl mice. Knocking out Sema3A in PCs or the choroid plexus was not sufficient to decrease the amount of PNN-associated Sema3A. Alternatively, knocking out Sema3A throughout the CNS induced a decrease in PNN-associated Sema3A. However, motor deficits, microgliosis, and neurodegeneration were observed, which were due to Cre toxicity. Our study represents the first attempt to unravel cellular sources of PNN-associated Sema3A and shows that non-invasive viral-mediated Cre expression throughout the CNS could lead to toxicity, complicating the interpretation of Cre-mediated Sema3A knock-out.

Adeno-associated viruses (AAVs) are non-pathogenic, replication-deficient viruses that have gained widespread attention for their application as gene therapy vectors. While these vectors offer high transduction efficiency and long-term gene expression, the host immune response poses a significant challenge to their clinical success. This review focuses on the obstacles to evaluating the humoral response to AAVs. We discuss the problems with the validation of in vitro tests and the possible approaches to overcome them. Using published data on neutralizing titers of AAV serotypes, we built the first antigenic maps of AAVs in order to visualize the antigenic relationships between varying serotypes.

Genetic functions have evolved over long timescales and can be encoded by multiple genes dispersed in different locations in genomes, and although contemporary molecular biology enables control over single genes, more complex genetic functions remain challenging. Here, we study the restructuring and mobilization of a complex genetic function encoded by 10 genes, originally expressed from four operons and two loci on the Escherichia coli genome. We observe subtle phenotypic differences and reduced fitness when expressed from episomal DNA and demonstrate that mutations in the transcriptional machinery are necessary for successful implementation in different bacteria. The work provides new approaches for advanced genome editing and constitutes a first step toward modularization and genome-level engineering of complex genetic functions.

Glioblastoma multiforme (GBM) is a devastating, aggressive primary brain tumor with poor patient outcomes and a five-year survival of less than 10%. Significant limitations to effective GBM treatment include poor drug delivery across the blood-brain barrier, drug resistance, and complex genetic tumor alterations. Gene therapy uses a mechanism different from other GBM therapies to reduce tumor growth and enhance antitumor immunity. This review article will provide an update on various viral and nonviral vectors, their DNA and RNA cargoes, and how they genetically modify tumor cells and evoke therapeutic responses to GBM. The article explores the oncolytic and immunogenic effects of gene therapy agents. It reviews promising DNA transgenes, RNA inhibitors, and vectors for anti-GBM therapy. The possible benefits of combining gene therapy with standard GBM treatments will also be covered.

Recombinant adeno-associated virus (rAAV) has emerged as the vector of choice for in vivo gene delivery, with numerous clinical trials underway for the treatment of various human diseases. Utilizing rAAV in gene therapy requires a highly precise quantification method to determine the viral genome titer and further establish the optimal therapeutic dosage for a rAAV product. The conventional single-channel droplet digital PCR (1D ddPCR) method offers only partial information regarding the viral vector genome titer, lacking insights into its integrity. In our pursuit of further advancing rAAV analysis, we have developed a novel 3D ddPCR assay with advanced 3D linkage analysis. We have designed the three amplicon sites targeting both ends of the viral genome, as well as the center of key therapeutic gene of interest (GOI). This study aims to offer a more comprehensive and insightful assessment of rAAV products which includes not only quantity of viral genome titer but also the quality, distinguishing between partial ones and intact full-length viral genomes with the right GOI. Importantly, due to the random partitioning property of a digital PCR system, the 3D linkage analysis of rAAV viral genome requires a proper mathematical model to identify the true linked DNA molecules (full-length/intact DNA) from the population of false/unlinked DNA molecules (fragmented/partial DNA). We therefore have developed an AAV 3D linkage analysis workflow to characterize genomic integrity and intact titer for rAAV gene therapy products. In this study, we focus on evaluating our 3D linkage mathematical model by performing DNA mixing experiments and a case study using multiple rAAV samples. Particularly, we rigorously tested our algorithms by conducting experiments involving the mixing of seven DNA fragments to represent various AAV viral genome populations, including 3 single partials, 3 double partials, and 1 full-length genomes. Across all 37 tested scenarios, we validated the accuracy of our workflow's output for the percentages of 3D linkage by comparing to the known percentages of input DNA. Consequently, our comprehensive AAV analytical package not only offers insights into viral genome titer but also provides valuable information on its integrity and identity. This cost-effective approach, akin to the setup of traditional 1D or 2D dPCR, holds the potential to advance the application of rAAV in cell and gene therapy for the treatment of human diseases., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. Lonza Houston Inc., 2024.)

Most rare diseases (RDs) encompass a diverse group of inherited disorders that affect millions of people worldwide. A significant proportion of these diseases are driven by functional haploinsufficiency, which is caused by pathogenic genetic variants. Currently, most treatments for RDs are limited to symptom management, emphasizing the need for therapies that directly address genetic deficiencies. Recent advancements in gene therapy, particularly with adeno-associated viruses (AAVs) and lipid nanoparticle-encapsulated messenger RNA (mRNA), have introduced promising therapeutic approaches. AAV vectors offer durable gene expression, extensive tissue tropism, and a safety profile that makes them a leading choice for gene delivery; however, limitations remain, including packaging size and immune response. In contrast, mRNA therapeutics, formulated in LNPs, facilitate transient protein expression without the risk of genomic integration, supporting repeated dosing and pharmacokinetic control, though with less long-term expression than AAVs. This review analyzes the latest developments in AAV and mRNA technologies for rare monogenic disorders, focusing on preclinical and clinical outcomes, vector design, and delivery challenges. We also address key regulatory and immunological considerations impacting therapeutic success. Together, these advancements in AAV and mRNA technology underscore a new era in RD treatment, providing innovative tools to target the genetic root of these diseases and expanding therapeutic approaches for patients who currently face limited medical options.

Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite's biology at scale., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Current plasmid propagation in E. coli compromises large inverted repeats, such as inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying strains and culture conditions revealed ITR instability caused by a slipped misalignment mechanism, although other mechanism probably contribute. ITRs stabilized in absence of SbcC, which is part of the SbcCD nuclease complex, a human Mre11-Rad50 homolog, or at elevated growth temperatures (e.g. 42°C), with a combination being optimal. Resulting full ITR transgene plasmids improved rAAV yield and purity in HEK-293 productions. The findings advance plasmid biology, cloneable sequences and therapeutic AAV manufacturing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recombinant Adeno-associated virus (rAAV) is a popular vector for treating genetic diseases caused by absent or defective genes. rAAVs can be produced that contain a therapeutic transgene, i.e., a correct copy of the affected gene, which is then delivered into target cells. A further application of rAAV is to deliver pro-apoptotic genes such as TNF-related apoptosis-inducing ligand ( TRAIL ) into cancer cells, leading to tumor regression. However, rAAV production is expensive and insufficient yields may hinder wide-spread adoption especially in systemic conditions. During rAAV production, the therapeutic transgene may be expressed in the producer cell line, and in the case of an oncolytic gene, this would likely lead to cell death thus reducing rAAV yields. Here we demonstrate that expression of TRAIL during rAAV production in HEK293F cells negatively impacts rAAV yield. A shRNA-based strategy was developed to suppress the expression of TRAIL in rAAV-producing cells specifically during the production process. Incorporating a TRAIL -targeting shRNA expression cassette within the backbone of the rAAV genome-encoding plasmid during triple-transfection of HEK293F cells reduced transgene expression and led to a 60% increase in the yield of rAAV- TRAIL compared to controls.

Objective: Adeno-associated viruses (AAVs) are widely used as gene therapy vectors due to their safety, stability, and long-term expression characteristics. The objective of this work is to develop an aqueous two-phase system (ATPS) as a universal platform for the separation and purification of AAVs., Results: This study utilized polyethylene glycol (PEG)/salt ATPSs to separate and purify various AAV serotypes, including AAV5, AAV8, and AAV9, which focusing on serotype-specific performance and partial empty capsid removal. The results showed that all the AAV serotypes were mainly enriched in the interphase of ATPS, with achieving high recovery (> 95%) and impurity removal (> 95%). The PEG/sodium citrate ATPS was serotype-independent, but the process optimization of component concentrations for each serotype was necessary to attain the best performance. Notably, a single-step aqueous two-phase extraction also demonstrated the ability to remove some amount of empty capsids from the crude cell lysate, with removal rate ranging from 4 to 25%., Conclusions: The results demonstrated the practical applicability of PEG/sodium citrate ATPS in separating and purifying different AAV serotypes, which addressing key challenges in gene therapy vector production., Competing Interests: Declarations. Conflict of interests: 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. Data availability: Data will be made available on request. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice., Competing Interests: Declaration of interests B.P.K. is an inventor of patents or patent applications filed by Mass General Brigham (MGB) that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital and Novartis Venture Fund and is on the scientific advisory boards of Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. B.P.K. has a financial interest in Prime Medicine, Inc., a company developing therapeutic CRISPR-Cas technologies for gene editing. B.P.K.'s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. C.A.M. has financial interests in Chameleon Biosciences, Skylark Bio, and Sphere Gene Therapeutics, companies developing AAV vector technologies for gene therapy applications. C.A.M. performs paid consulting work for all three companies. C.A.M.’s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenylketonuria (PKU) is a liver metabolic disorder mainly caused by a deficiency of the hepatic phenylalanine hydroxylase (PAH) enzyme activity, often leading to severe brain function impairment in patients if untreated or if treatment is delayed. In this study, we utilized dual-AAV8 vectors to deliver a near PAM-less adenine base editor variant, known as ABE8e-SpRY, to treat the Pah R408W PKU mouse model carrying a frequent R408W mutation in the Pah gene. Our findings revealed that a single intravenous injection in adult mice and a single intraperitoneal injection in neonatal mice resulted in 19.1%-34.6% A-to-G editing efficiency at the pathogenic mutation site with minimal bystander edits. Furthermore, the dual-AAV8-treated mice exhibited reduced blood Phe levels to below the therapeutic threshold of 360 μmol L -1 and restored weight and fur color to normal levels. Importantly, the brain function of the mice was restored after the treatment, particularly when administered during the neonatal stage, as levels of monoamine neurotransmitters and metabolites in the brain returned to normal and near-normal levels. Our study demonstrated that ABE8e-SpRY-based base editing could effectively correct the point mutation in the Pah R408W PKU mouse model, indicating potential clinical applications for PKU and other genetic diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

We conducted a genome-wide CRISPR/Cas9 screen in suspension 293 F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screening included the previously reported KIAA0319L , TM9SF2 , and RNF121 , along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization, and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 ( SLC35A1 ), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, was only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3, which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (∆C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting that the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.IMPORTANCErAAV is an essential tool for gene delivery in the treatment of genetic disorders; however, the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293 F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV., Competing Interests: R.M. is an employee of GeneGoCell Inc. The remaining authors have no competing financial interests.

Dorsal root ganglion (DRG) toxicity has been consistently reported as a potential safety concern after delivery of adeno-associated viruses (AAVs) containing gene-replacement vectors but has yet to be reported for RNAi-based vectors. Here, we report DRG toxicity after AAV intra-CSF delivery of an RNAi expression construct-artificial microRNA targeting superoxide dismutase 1 (SOD1)-in non-human primates (NHPs) and provide evidence that this can be recapitulated within mice. Histopathology evaluation showed that NHPs and mice develop DRG toxicity after AAV delivery, including DRG neuron degeneration and necrosis and nerve-fiber degeneration that were associated with increases in cerebrospinal fluid (CSF) and serum phosphorylated neurofilament heavy chain (pNF-H). RNA-sequencing analysis of DRGs showed that dysregulated pathways were preserved between NHPs and mice, including increases in innate/adaptive immune responses and decreases in mitochondrial- and neuronal-related genes, following AAV treatment. Finally, endogenous miR-21-5p was upregulated in DRGs of AAV-treated NHPs and mice. Increases in miR-21-5p were also identified within the CSF of NHPs, which significantly correlated with pNF-H, implicating miR-21-5p as a potential biomarker of DRG toxicity in conjunction with other molecular analytes. This work highlights the importance of assessing safety concerns related to DRG toxicity when developing RNAi-based AAV vectors for therapeutic purposes., Competing Interests: Declaration of interests M.L.H. is a paid advisor from Charter Preclinical Services. All other authors are or were paid employees at Biogen., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

CD19 CAR-T therapy has achieved remarkable responses in relapsed/refractory non-Hodgkin lymphoma (NHL). However, challenges persist, with refractory responses or relapses after CAR-T administration linked to CD19 loss or downregulation. Given the co-expression of CD19 and BCMA in NHL, we hypothesized that dual targeting could enhance long-term efficacy. We optimized different dual-targeting approaches, including co-transduction of two lentiviral vectors, bicistronic, tandem, and loop and pool strategies, based on our academic anti-CD19 (ARI0001) and anti-BCMA (ARI0002h) CAR-T cells. Comparison with anti-CD19/CD20 or anti-CD19/CD22 dual targeting was also performed. We demonstrate that anti-CD19/BCMA CAR-T cells can be effectively generated through the co-transduction of two lentiviral vectors after optimization to minimize competition for cellular resources. Co-transduced T cells, called ARI0003, effectively targeted NHL tumor cells with high avidity, outperforming anti-CD19 CAR-T cells and other dual-targeting approaches both in vitro and in vivo, particularly in low CD19 antigen density models. ARI0003 maintained effectiveness post-CD19 CAR-T treatment in xenograft models and in spheroids from relapsed CART-treated patients. ARI0003 CAR-T cells were effectively manufactured under Good Manufacturing Practice conditions, with a reduced risk of genotoxicity compared to other dual-targeting approaches. A first-in-human phase 1 clinical trial (CARTD-BG-01; this study was registered at ClinicalTrials.gov [NCT06097455]) has been initiated to evaluate the safety and efficacy of ARI0003 in NHL., Competing Interests: Declaration of interests S.G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity Therapeutics, and has received commercial research funding from Gilead. A.R.-G. is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania. M.P., M.J., J.D., and A.U.-I. are inventors of a patent describing ARI0001. B.M.-A., A.U.-I., and M.J. are inventors of a patent describing ARI0002h., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to develop a reliable and efficient genetic transformation method for the ornamental Indian Lotus (Nelumbo nucifera Gaertn.) using the sonication-assisted Rhizobium radiobacter-mediated transformation technique. To conduct the transformation, shoot apical meristem explants were infected with Rhizobium radiobacter (synonym Agrobacterium tumefaciens) strain LBA 4404 containing a binary vector pBI121 that harbours the GUS reporter gene (uidA) and kanamycin resistance gene nptII for plant selection. To improve the transformation efficiency, we optimized parameters such as bacterial cell density, sonication duration, infection time, co-cultivation duration, acetosyringone concentration, cefotaxime, and kanamycin concentrations. Sonication treatment at 42 kHz for 90 s recorded the highest transformation efficiency. The selection of regenerated plantlets was performed on a kanamycin-supplemented selection medium. The putative transformants showed GUS expression in the leaves and petioles. The presence of the GUS gene was also confirmed in the putative transformants through PCR, with the appearance of the expected amplicon size of 520 bp. The presence of nptII was confirmed by PCR in the putatively transformed plants with an amplicon size of 530 bp. The maximum regeneration frequency obtained was 72.66%, and the highest transformation efficiency achieved was 9.0% in the Indian Lotus., Competing Interests: Declarations. Conflict of interests: The authors declare no competing interests. Human or animal rights: The authors declare the research did not involve human participants and animals. Informed consent: Authors consent to publish this manuscript., (© 2025. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Key Message: Viral vector-mediated gene editing is enhanced for cultivated tomato under low temperature conditions, enabling higher mutation rates, heritable, and virus-free gene editing for efficient breeding. The CRISPR/Cas system, a versatile gene-editing tool, has revolutionized plant breeding by enabling precise genetic modifications. The development of robust and efficient genome-editing tools for crops is crucial for their application in plant breeding. In this study, we highly improved virus-induced genome-editing (VIGE) system for cultivated tomato. Vectors of tobacco rattle virus (TRV) and potato virus X (PVX) were used to deliver sgRNA targeting phytoene desaturase (SlPDS), along with mobile RNA sequences of tFT or tRNA Ileu , into Cas9-overexpressing cultivated tomato (S. lycopersicum cv. Moneymaker). Our results demonstrate that low temperature significantly enhanced viral vector-mediated gene editing efficiency in both cotyledons and systemic upper leaves. However, no mutant progeny was obtained from TRV- and PVX301-infected MM-Cas9 plants. To address this challenge, we employed tissue culture techniques and found that low-temperature incubations at the initiation stage of tissue culture lead to enhanced editing efficiency in both vectors, resulting in a higher mutation rate (> 70%) of SlPDS in regenerated plants. Heritable gene-edited and virus-free progenies were successfully identified. This study presents a straightforward approach to enhance VIGE efficiency and the expeditious production of gene-edited lines in tomato breeding., Competing Interests: Declarations. Conflict of interest: The authors declare no competing financial interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Expression of recombinant genes can be controlled using inducible promoters. However, the most commonly used IPTG- and arabinose-inducible promoters result in an 'all-or-nothing' response, leading to fully induced and uninduced bacterial subpopulations. Here, we investigate whether appropriate modifications to these promoter systems can be combined into a single vector system, enabling homogenous expression of two genes of interest that can be precisely tuned using inducer concentration. We show that modifications of positive feedback loops related to inducer uptake result in homogeneous gene expression in both the T7 lactose and pBAD arabinose systems. Furthermore, these two modified systems were combined into a single vector, pRAT-sfGFP that provides the desired tunable expression of two genes of interest. Finally, we test this single-vector system as a tool for studying two-component genetic circuits, using toxin-antitoxin modules as model systems. This novel low-copy single vector expression system opens up new possibilities for investigating the function of two-component bacterial genetic circuits., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Gene therapy using hematopoietic stem and progenitor cells is altering the therapeutic landscape for patients with hematologic, immunologic, and metabolic disorders but has not yet been successfully developed for individuals with the bone marrow failure syndrome Diamond-Blackfan anemia (DBA). More than 30 mutations cause DBA through impaired ribosome function and lead to inefficient translation of the erythroid master regulator GATA1, providing a potential avenue for therapeutic intervention applicable to all patients with DBA, irrespective of the underlying genotype. Here, we report the development of a clinical-grade lentiviral gene therapy that achieves erythroid lineage-restricted expression of GATA1. We show that this vector is capable of augmenting erythropoiesis in DBA models and diverse patient samples without impacting hematopoietic stem cell function or demonstrating any signs of premalignant clonal expansion. These preclinical safety and efficacy data provide strong support for the first-in-human universal gene therapy trial for DBA through regulated GATA1 expression., Competing Interests: Declaration of interests V.G.S. serves as an advisor to Ensoma, unrelated to the present work. R.A.V. and V.G.S. are named as inventors on patent PCT/US2020/036600 filed by Boston Children’s Hospital related to this approach., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA., (© 2025 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Delandistrogene moxeparvovec is an rAAVrh74 vector-based gene transfer therapy that delivers a transgene encoding delandistrogene moxeparvovec micro-dystrophin, an engineered, functional form of dystrophin shown to stabilize or slow disease progression in DMD. It is approved in the US and in other select countries. Two serious adverse event cases of immune-mediated myositis (IMM) were reported in the phase Ib ENDEAVOR trial (NCT04626674). We hypothesized that immune responses to the micro-dystrophin transgene product may have mediated these IMM events. An interferon-gamma ELISpot assay was used to detect T cell responses to delandistrogene moxeparvovec micro-dystrophin peptide pools. ELISpot analysis suggested that IMM resulted from T cell-mediated responses directed against specific micro-dystrophin peptides corresponding to exons 8 and 9 (Case 1) and exon 8 (Case 2) of the DMD gene. In silico epitope mapping based on the patients' HLA-I alleles indicated greater probability for peptides derived from exons 8 and/or 9 to bind HLA-I, providing further evidence that peptides derived from corresponding micro-dystrophin regions may have higher immunogenic potential. Collectively, these data suggest that patients with DMD gene deletions involving exons 8 and/or 9 may be at increased risk of IMM following delandistrogene moxeparvovec micro-dystrophin gene therapy infusion., Competing Interests: Declarations. Competing interests: R.A.P., I.H.M., S.K., D.R.A., E.P., D.A.G., and S.M. are employees of Sarepta Therapeutics and may have stock options. H.H., A.H., G.S., and C.W. are employees of F. Hoffmann-La Roche Ltd and may have stock options. A.P.M. is an employee of Roche Products Ltd and may have stock options. S.T.I. receives research support from industry (Elpida, Novartis, Biogen, Sarepta Therapeutics, PTC Therapeutics, Scholar Rock, FibroGen, RegenxBio, and ReveraGen) and the Department of Defense W81XWH2010293, Parent Project Muscular Dystrophy, and Cure SMA. She has served on medical advisory boards for Novartis, Biogen, Genentech, and Sarepta Therapeutics. She receives partial salary support from the following grants: National Institutes of Health Wellstone Muscular Dystrophy Center P50HD087351, NeuroNEXT U24NS107176, and the Muscular Dystrophy Association. C.M.Z. receives research support from Biogen and Novartis and has served on an advisory board for Sarepta Therapeutics. L.R.R.K. is an employee of Sarepta Therapeutics and may have stock options and is a co-inventor of AAVrh74.MHCK7.SRP-9001-dys technology., (© 2024. The Author(s).)

Background: Fabry disease is an X-linked lysosomal storage disorder due to a deficiency of α-galactosidase A (α-gal A) activity. Our goal was to correct the enzyme deficiency in Fabry patients by transferring the cDNA for α-gal A into their CD34+ hematopoietic stem/progenitor cells (HSPCs). Overexpression of α-gal A leads to secretion of the hydrolase; which can be taken up and used by uncorrected bystander cells. Gene-augmented HSPCs can circulate and thus provide sustained systemic correction. Interim results from this 'first-in-the-world' Canadian FACTs (Fabry Disease Clinical Research and Therapeutics) trial were published in 2021. Herein we report 5-year 'End-of-Study' results., Methods: Five males with classical Fabry disease were treated. Their HSPCs were mobilized, enriched, and transduced with a recombinant lentivirus engineering expression of α-gal A. Autologous transduced cells were infused after conditioning with a nonmyeloablative, reduced dose, melphalan regimen. Safety monitoring was performed. α-Gal A activity was measured in plasma and peripheral blood (PB) leucocytes. Globotriaosylceramide (Gb3) and lyso-Gb3 levels in urine and plasma were assessed by mass spectrometry. qPCR assays measured vector copy number in PB leucocytes. Antibody titers were measured by ELISA. Body weight, blood pressure, urinary protein levels, eGFR, troponin levels, and LVMI were tracked., Results: Four out of 5 patients went home the same day as their infusions; one was kept overnight for observation. Circulating α-gal A activity was observed at Day 6-8 in each patient following infusion and has remained durable for 5+ years. LV marking of peripheral blood cells has remained durable and polyclonal. All 5 patients were eligible to come off biweekly enzyme therapy; 3 patients did so. Plasma lyso-Gb3 was significantly lower in 4 of 5 patients. There was no sustained elevation of anti-α-gal A antibodies. Patient weight was stable in 4 of the 5 patients. All blood pressures were in the normal range. Kidney symptoms were stabilized in all patients., Conclusions: This treatment was well tolerated as only two SAEs occurred (during the treatment phase) and only two AEs were reported since 2021. We demonstrate that this therapeutic approach has merit, is durable, and should be explored in a larger clinical trial., Highlights: This was the first gene therapy clinical trial to be completed for Fabry disease. There were no adverse events of any grade attributable to the cellular gene therapy intervention or host conditioning throughout the follow-up interval of 5 years. After reduced-intensity melphalan treatment, all patients engrafted their autologous modified α-gal A expressing cells. All patients synthesized and secreted α-gal A throughout the course of the study. Expression of α-gal A resulted in a decrease in plasma lyso-Gb3 in four of five patients and stabilization of kidney symptoms in all patients., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae., (© 2025 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Increasing demand for adeno-associated virus (AAV) used in gene therapy highlights the need to enhance AAV production. When intracellular AAV2 and extracellular AAV9 were produced in HEK293T cells using the triple transfection method, apoptosis occurred during the AAV production. To mitigate apoptosis induced by AAV production, the pro-apoptotic BAX/BAK1 genes were knocked out in HEK293T cells. BAX/BAK1 knockout (BBKO) in HEK293T cells significantly increased the production of both AAV2 and AAV9. For AAV2, BBKO increased the genome titer of AAV2 by 55% without negatively affecting the proportion of unwanted empty capsids generated during AAV production. Empty capsid ratios were determined based on viral genome and capsid titers and confirmed via transmission electron microscopy (TEM). Likewise, for AAV9, BBKO increased the genome titer of AAV9 by 66% without negatively affecting the proportion of empty capsids. Additionally, as assessed using a transduction assay, BBKO increased the functional titers of AAV2 and AAV9 by 30% and 46%, respectively. Therefore, BBKO increased AAV production, while maintaining full capsid ratio and infectivity. Taken together, BBKO proved to be an efficient method for enhancing AAV production in HEK293T cells for both AAV2 and AAV9., (© 2025 Wiley‐VCH GmbH.)

Adeno-associated virus (AAV) vectors have become a leading platform for gene delivery. A major portion of gene therapy currently in clinical trials are AAV-based for a wide range of diseases. A commonly used method for AAV production is by mammalian or insect cell culture, with or without added viruses to introduce needed genetic elements for AAV production. There are potential risks of virus contamination from the production cell line, process residuals, or adventitious contamination in the production of biotherapeutics, including AAV-based gene therapy products; therefore, it is imperative to demonstrate that the drug substance manufacturing process has sufficient capability to clear process-related or adventitious viruses. In the AAV-based gene therapy manufacturing process, cell lysis, affinity chromatography, and ion exchange chromatography steps are often effective to inactivate or remove viruses. To increase the viral clearance robustness, virus filtration (VF) is increasingly recommended by regulatory agencies for gene therapy products as a dedicated viral clearance step in the downstream purification process. In the current study, two commercially available virus filters were evaluated in the context of AAV manufacturing. The filter throughput and process yield were assessed under different operational modes. Virus clearance performance was evaluated by spiking in Adenovirus type 5 (Adv-5) and Simian virus 40 (SV-40). The viral filters assessed in this study demonstrated manufacturable throughputs, acceptable process yields, and robust virus clearance capabilities for viruses greater than 40 nm., (© 2025 Wiley‐VCH GmbH.)

Species A rotaviruses (RVs), which belong to the family Sedoreoviridae and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.

Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here, we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors, we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody, with Chinese hamster ovary cells expressing 20-50 mg l -1 after 5 days when cells were grown to a density of ~1×10 6  cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo .

Genetic transformation is a powerful tool in plant biotechnology. However, its application is limited to species that are well-studied and easy to transform. There is a critical need to establish transformation protocols for non-model species. A stable transformation method using Agrobacterium rhizogenes for hairy root transformation and regeneration of transgenic Linum grandiflorum was established. This protocol shows the successful co-transformation of different T-DNA fragments from both the native Ri plasmid and the binary vector with the reporter gene. Hairy roots were produced after inoculation with Agrobacterium rhizogenes from which later shoots were formed from the callus, and subsequently, whole plants were regenerated. This protocol significantly facilitates genomic studies in Linum grandiflorum, particularly in investigating genes at the S-locus supergene, which are crucial for understanding self-incompatibility. Moreover, the established transformation method enables the production of hairy root lines, which can be utilized for the biosynthesis of medically useful and commercially valuable plant metabolites., (© 2025 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Treatment with autologous chimeric antigen receptor (CAR)-modified T cells can achieve outstanding clinical response rates in heavily pretreated patients with B and plasma cell malignancies. However, relapses occur, and they limit the efficacy of this promising treatment approach. The complex GMP-compliant production and high treatment costs cause that CAR T cells cannot yet be used in a broad population. Among others, CAR T cell therapy has evolved regarding vector design and manufacturing process. Optimal production of CAR T cells is not yet defined, far from being standardized. Quality, cellular composition and immunophenotype of the administered CAR T cells are influenced by the manufacturing protocol and therefore play a crucial role for therapeutic success. For the gene transfer, viral and non-viral strategies are available. Retrovirus-based protocols for CAR T cell production offer advantages in terms of stable gene integration, sufficient transduction efficiency, proven clinical success, and scalability. Here, we detail a retrovirus-based generation protocol of human CAR-modified T cells for experimental immunotherapeutic treatment of cancer cells. For the CAR generation, HEK-293-based packaging cell lines, CD3 + selection, CD3/CD28-coated bead-based activation and IL-2/IL-15-mediated expansion were used. This protocol can be applied for every possible CAR construct after being successfully transfected in HEK-293-based packaging cell lines., Competing Interests: Conflict of interest SS, LF, VDM and TCS have no relevant financial or non-financial interests to disclose relevant for this work. MC is a co-founder and a shareholder of BioVec Pharma. SK has received honoraria from TCR2 Inc., Novartis, Galapagos, Cymab, Miltenyi, BMS and GSK. SK is an inventor of several patents in the field of immuno-oncology. SK received license fees from TCR2 Inc. and Carina Biotech. SK received research support from TCR2 Inc., Tabby Therapeutics, Plectonic GmBH, Catalym GmBH and Arcus Bioscience for work unrelated to the manuscript., (Copyright © 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Recombinant adeno-associated virus (rAAV) has emerged as one of the best gene delivery vectors for human gene therapy in vivo. However, the clinical efficacy of rAAV gene therapy is often hindered by the host immune response against its transgene products. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is specialised to process peptides presented by class I molecules of major histocompatibility complex. Therefore, we hypothesise that modulation of the ERAP1 activity in rAAV transduced cells may be favoured to evade immune response against transgene products. In this study, we incorporated either miRNA-UL112-5p or ERAP1 shRNA into rAAV vectors expressing full-length ovalbumin (OVA) as a model antigen, and evaluated their effects for antigen presentation, cellular and humour immune response induced by OVA expression. The results indicated that silencing ERAP1 using miR-UL112-5p or ERAP1 shRNA did not affect the expression of OVA in cells, but inhibited the processing and presentation of OVA antigen peptide SIINFEKL in antigen presenting cells (APCs). Moreover, the rAAV vector co-expressing ERAP1 shRNA maintains stable and high expression of OVA in vivo, while simultaneously suppressing the humoral immunity of OVA. In addition, experimental results demonstrated that rAAV vectors incorporated ERAP1 shRNA efficiently repress costimulatory signals in dendritic cells (DCs), significantly attenuated the cytotoxic T-cell response, allowed for sustained transgene expression and reduced clearance of transduced muscle cells in mice. Moreover, our study suggested that the incorporation of miRNA-UL112-5p or ERAP1 shRNA into rAAV vectors effectively reduced transgene products induced immune response. The proposed method may potentially be applied in clinics to deliver therapeutic proteins safely and efficiently., (© 2025 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Rationale: Adenovirus-based therapies have encountered significant challenges due to host immunity, particularly from pre-existing antibodies. Many trials have struggled to evade antibody response; however, the efficiency of these efforts was limited by the diversity of antibody Fv-region recognizing multiple amino acid sequences. Methods: In this study, we developed an antibody-evading adenovirus vector by encoding a plasma-rich protein transferrin-binding domain. The coding sequence was employed from Neisseria Meningitides and inserted in the experimentally-optimized site within the adenovirus capsid protein. Result: This engineered antibody-evading oncolytic adenovirus overcame the reduction in productivity and infectivity typically caused by the insertion of a foreign domain. We observed decreased immune recognition and compromised formation of anti-adenovirus antibodies. Furthermore, the anti-tumor efficacy was demonstrated both in vitro and in vivo , with increased recruitment of CD8 + T cells. Conclusion: This novel antibody-evading strategy effectively evades neutralizing antibodies and innate immunity while boosting cytotoxic immunity by recruiting CD8 + T cells at the tumor site. Additionally, this strategy holds potential for application in other gene therapies and adenovirus vectors., Competing Interests: Competing Interests: D. H. L., Y. K., K. H. U., J. K. Y., W. H., K. K., and M. J. K. partially received research grants from Curigin. Ltd. J. C., H. C., K. S. P., and J. W. C. declares no competing interests associated with this manuscript., (© The author(s).)

The bimolecular fluorescence complementation (BiFC) technique is a powerful tool for visualizing protein-protein interactions in vivo. It involves genetically fused nonfluorescent fragments of green fluorescent protein (GFP) or its variants to the target proteins of interest. When these proteins interact, the GFP fragments come together, resulting in the reconstitution of a functional fluorescent protein complex that can be observed using fluorescence microscopy. In this chapter, we provide a detailed overview of the BiFC method and its application in studying protein-protein interactions in mouse hippocampal neurons. We discuss experimental procedures, including virus construct design, neuronal transduction, and imaging optimization. Additionally, we explore complementary assays for result validation and address potential challenges associated with BiFC experiments in the neuronal system. Overall, the BiFC offers researchers a valuable approach for investigating the spatial and temporal dynamics of protein interactions in living neuronal cells., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The overproduction of certain protein components of the Spindle Assembly Checkpoint (SAC) such as human BUB3, CDH1, and CDC20 either in isolation or associated with other proteins in prokaryotic heterologue gene expression systems remains a technical challenge. Recent advances in virus engineering methods and the development of genetically modified insect cells have been exploited to overproduce these SAC proteins as natively folded, functional molecules. One widely used baculovirus-based expression system is flashBac, which exhibits several advantages over other insect cells-based expression systems such as the straightforward production of recombinant baculoviruses through homologous recombination and the positive selection of recombinant clones., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor. We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level -1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)