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PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗

PubMed2025

Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.

Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.

Populus↗

PubMed2025

Effect of transgene on salt tolerance of tobacco.

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

Plants, Genetically Modified↗

PubMed2024

Identifying transgene insertions in Caenorhabditis elegans genomes with Oxford Nanopore sequencing.

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Caenorhabditis elegans↗

PubMed2026

Inducing fixation of transgenic alleles in open-pollinated populations.

The progeny of chlorsulfuron-resistant forage rape (Brassica napus L. subsp. napus, cv. Giant) and potato (Solanum tuberosum L. cv. Iwa) plants hemizygous for a single transgenic locus were screened for transgene segregation following an application of either water or chlorsulfuron. The transgenic locus contained three transgenes conferring kanamycin resistance (NOS-NPTII-NOS), β-glucuronidase (GUS) activity (35S-GUS-OCS), and chlorsulfuron resistance (a complete acetohydroxyacid synthase gene with a proline197 to serine substitution). In the absence of the herbicide application, plants segregated for GUS activity as expected for single locus inheritance. However, the progeny of plants sprayed with chlorsulfuron exhibited a highly distorted segregation for GUS activity, with a significant excess of transgenic progeny. Inducing such biased segregation provides a simple treatment to rapidly drive the fixation of transgenic alleles to homozygosity in open pollinated populations during seed increases of new cultivars.

Alleles↗

PubMed2026

Transgene sequence codon optimization and composition determines replication competence of self-amplifying RNA.

Self-amplifying RNA (saRNA) is an emerging RNA therapeutic modality that can facilitate higher magnitude and more durable protein expression at substantially lower doses than nonreplicating mRNA. Unlike conventional messenger RNA (mRNA), alphavirus-derived saRNA must support a replicase-driven RNA amplification step in addition to translation, raising the possibility that transgene coding sequences impose sequence-level constraints on replication. Here, saRNA replication was found to be dependent on the codon composition of the transgene; multiple therapeutic transgenes were replication defective despite an intact Venezuelan Equine Encephalitis Virus (VEEV)-derived saRNA backbone. Replication defects were rescued by synonymous codon re-optimization of the same transgenes, indicating that nucleotide-level features of the coding sequence, rather than the encoded protein, govern replication competence. Comparative compositional analyses identified a distinct signature associated with productive replication, characterized by elevated GC (>53%) and GC3 (>63%) content, higher codon adaptation to human (>0.75), and reduced UpA (<43/kb) and UpU (<41/kb) dinucleotide density. Moreover, deliberate compositional perturbation of an otherwise replication-competent transgene shifted these features and abolished replication, supporting a causal and combinatorial role for sequence composition in defining saRNA replication outcome. These findings define an underappreciated constraint in saRNA therapeutics and motivate saRNA-specific payload design frameworks that incorporate alphavirus-associated compositional biases during transgene sequence optimization.

Codon↗

PubMed2026

Expression dynamics of mCry3A and eCry3.1Ab transgenes in Bt corn hybrids across growth and environments.

In 2017 and 2018, studies were conducted in Iowa, US across three environments with a history of greater than expected corn rootworm injury i.e., greater than one node of injury to (US EPA 2009) and one environment without rootworm injury, and at different growth stages, to determine the expression levels of mCry3A and eCry3.1Ab transgenes in the roots of different Bt corn hybrids namely the molecular stack (MZIR098), breeding stack (MIR604 × 5307), 5307 and MIR604. ELISA results showed that expressions of both mCry3A and eCry3.1Ab transgenes, were higher in the MZIR098 molecular stack and breeding stack (MIR604 × 5307), than the MIR604 and 5307 at V3 and VT growth stages over both years. To protect the roots from feeding damage by the corn rootworm larvae, expression of the transgenes must be high at the V3 growth stage. The expression of the transgene was significantly impacted by the stage of plant growth while the environments with greater than expected corn rootworm injury did not impact expression of the transgenes. It was found that the expression of mCry3A and eCry3.1Ab transgenes were high at the V3 plant growth stage compared to the VT growth stage. Stacking two or more genes together in the same plant such as the molecular and breeding stacks have the potential to protect roots in environments with higher-than-expected damage and slow down the evolution of resistance in field populations of rootworms.

Zea mays↗

PubMed2013

Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.

Base Pairing↗

PubMed2026

Pathogen Species-Specific Differences in Induction of the Maize Polyubiquitin Gene Promoter in Transgenic Wheat.

The maize polyubiquitin promoter (ZmUbi) is a mainstay in molecular biology for transgene expression and is used for constitutive expression of defense-related gene products. Transgenic wheat lines were produced expressing a ZmUbi-RUBY reporter gene that produces the red pigment betalain. Some lines showed transgene silencing with reduced RUBY transcript accumulation and chimeric sectors of betalain. Infection of these plants with Blumeria graminis, Puccinia graminis f. sp. tritici (Pgt), or P. triticina (Pt) each resulted in localized betalain accumulation at infection sites and increased RUBY transcript accumulation. In contrast, two isolates of P. striiformis f. sp. tritici (Pst) caused no detectable RUBY transcript accumulation and no visible betalain accumulation at infection sites, although a modest betalain increase was detected in infected tissue extracts. Compared with Pst, Pgt more strongly induced host genes involved in transcriptional and post-transcriptional regulatory processes, although no obvious pathogen-induced changes in ZmUbi promoter methylation were observed. ZmUbi-GUS transgenic wheat plants were also pathogen challenged, and, unlike Pst, both Pgt and Pt induced localized GUS staining at infection sites. Database mining showed that the endogenous maize polyubiquitin gene from which ZmUbi is derived was pathogen inducible, albeit in a species-specific fashion. These pathogen differences in ZmUbi induction have implications when using this regulatory element to express defense-related transgenes in wheat. Comparing the resistance efficacy of transgenes against different pathogens using this promoter is potentially influenced by significant, localized expression differences occurring at infection sites of different pathogen species. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Promoter Regions, Genetic↗

PubMed2025

Development of a plant-based vaccine against brucellosis: stable expression of Brucella abortus OMP25 in transgenic tobacco.

Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.

Plants, Genetically Modified↗

PubMed2026

An All-in-One Photothermal Nanocomposite Hydrogel for Controlling Inducible Transgene Expression.

We have developed a remotely near-infrared (NIR)-activated, implantable fibrin hydrogel for the controlled induction of transgene expression, designed to decouple the therapeutic efficacy of rapamycin from its systemic toxicity. Rapamycin, a drug widely used in clinical practice as an immunosuppressant and antiproliferative agent, is a potent transcriptional inducer that enables tightly regulated temporal transgene expression through chemically induced dimerization. However, its utility as a dimerizer is hindered by the unintended systemic immunosuppression and off-target effects inherent to its conventional administration. To address this, we developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles to encapsulate rapamycin, aiming to facilitate localized delivery and enhance drug stability. Engineered cells harboring a dual heat- and dimerizer-responsive gene switch exhibited robust reporter transgene expression following nanoparticle treatment and thermal activation. Nanoencapsulation preserved rapamycin activity against thermal and hydrolytic degradation, enabling superior, long-term dimerizer function compared to the free drug. To create a remotely actuated platform, we developed photothermal hydrogels by incorporating hollow gold nanoparticles and rapamycin-loaded PLGA nanoparticles within a fibrin matrix hosting the reporter cells. In mice, NIR irradiation of subcutaneously implanted constructs achieved transgene induction levels comparable to systemic administration of rapamycin. Notably, nanoparticle-mediated delivery resulted in negligible circulating rapamycin concentrations. Furthermore, localized rapamycin release initially promoted a pro-healing M2 macrophage phenotype, followed by a late-stage transition toward an M1-dominant profile that likely facilitated the clearance of scaffold degradation products. In hydrogels incorporating cells harboring a gene switch to control human VEGF165 production, NIR irradiation triggered a robust angiogenic cascade characterized by transient erythema followed by an increase in CD31+ microvascular density. Collectively, these data demonstrate the potential of this light-triggered and rapamycin-dependent platform as a customizable and safe tool for achieving the control required to advance the next-generation of site-specific, transgenic protein therapies.

Animals↗

PubMed2025

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. Here, we develop a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression that is extensible to additional transcription factors. Lentiviral delivery of DIAL generates multiple setpoints in primary cells and induced pluripotent stem cells. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotype and fate in direct conversion to induced motor neurons. The DIAL framework opens opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications.

Promoter Regions, Genetic↗

PubMed2025

Abiotic conditions can modify the penetrance of transgene-based lethality systems for insect population control.

Modern genetic biocontrol techniques for insect pest management, when compared to chemical insecticide spraying, offer high species specificity and reduced environmental impact, and some of these methods require the environmental release of genetically modified (GM) insects. Because organisms exposed to different environments often show variability in phenotype and gene expression, it is likely that GM insects will also experience environmentally mediated variation, potentially compromising pest control efficiency. This study examines the impact of temperature and nutrition on the early embryonic Tet-off conditional lethality system in Drosophila melanogaster. By independently manipulating parental and offspring environments, we assessed how exposure to variable environments influenced the probability of larval hatching and the transcript abundance of the transgenic system. Our findings revealed that: (i) transgene performance distinctly responds to temperature and nutrition; (ii) thermal stress has a greater impact when embryos, rather than parents, are exposed; and (iii) extreme nutritional conditions can markedly reduce the penetrance of transgenic lethality. Although changes in transgene transcript abundance were observed across environments, these changes did not fully explain the phenotypic variation, suggesting that factors downstream of transcription probably drive variation in transgenic lethality.

Animals↗

PubMed2026

A CHO-Derived Matrix Attachment Region Enhances Transgene Dosage, SATB1 Recruitment, and Monoclonal Antibody Expression in a Dual-Promoter Vector System.

The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.

Animals↗

PubMed2026

Epigenetic regulation of transgenes.

Gene therapy holds significant potential for treating genetic disorders, but the use of viral vectors is limited by factors such as immunogenicity, payload capacity, and high manufacturing costs. Nonviral gene delivery (NVGD) using plasmid DNA presents an attractive alternative; however, it typically provides a limited magnitude or duration of transgene expression. One potential reason for these shortcomings is the host cell's epigenetic regulation mechanisms, which can silence both viral and nonviral transgenes. Specifically, when foreign DNA enters the nucleus, it is detected by nuclear DNA sensors, such as IFI16, which initiate the assembly of a "restrictosome" or nuclear domain 10 (ND10) body. This multiprotein complex contains several components, such as PML, Speckled Proteins (e.g., SP100), DAXX, and ATRX that act as a scaffold for recruiting various epigenetic modifiers that subsequently deposit repressive histone modifications like H3K9me3 and H3K27me3 on the transgene chromatin. These marks induce DNA methylation and the subsequent condensation of plasmids or episomes into heterochromatin, which represses transgene expression. Alternatively, unmethylated CpG motifs in bacterial plasmid DNA can trigger innate immune responses in the cytosol, but this review will specifically focus on the detailed mechanisms of epigenetic regulation responsible for silencing plasmid DNA within the host cell nucleus. Addressing these nuclear defense mechanisms, potentially through strategies that manipulate DNA methylation or inhibit restrictosome activity, is crucial for advancing the development of safe, effective, and long-lasting plasmid viral and non-viral gene therapies.

Epigenesis, Genetic↗

PubMed2024

Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo.

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to 1/2 binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (1) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (2) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (3) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (4) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo; and (5) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE 1/2 binding site, and insertion of a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y AAV vectors, which are serotype, transgene, or promoter agnostic, should be useful in human gene therapy.

AAV vectors↗

PubMed2026

Conserved innate immunity components limit transgene expression in adult planarians.

The planarian flatworm Schmidtea mediterranea has become a powerful model for studying whole-body regeneration, tissue patterning, and stem cell regulation. Yet the absence of reliable tools for transgene expression still limits the elucidation of molecular mechanisms in in this system. Here, we establish a proof-of-principle system for plasmid-based expression of NanoLuciferase (NanoLuc) in S. mediterranea, employing commercially available transfection reagents and a panel of endogenous promoter sequences. Despite successful delivery, reporter expression remained low and transient. To identify biological barriers to robust transgene expression, we investigated the role of innate immune pathways. Candidate gene searches and biochemical pull-down of cytoplasmic DNA coupled to mass spectrometry identified several planarian homologs of conserved immune regulators and putative DNA sensors. Through RNA interference screening of conserved innate immune components, we uncover roles for S. mediterranea homologs of Tank-binding kinase 1 (TBK1) and macrophage mannose receptor 1 (MRC1) as potent repressors of transgene expression. Transcriptomic and functional analyses further implicate TBK1 in regulating broad innate immune and stress-response programs, akin to its vertebrate function. Together, our findings demonstrate that innate immune signaling limits transgene expression in S. mediterranea and suggest that modulating these pathways may be key to enabling stable and efficient genetic manipulation in planarians.

Animals↗

PubMed2024

Adeno-Associated Virus Engineering and Load Strategy for Tropism Modification, Immune Evasion and Enhanced Transgene Expression.

Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.

Dependovirus↗