Review

Authors: Alan Cabrera, Hailey I Edelstein, Fokion Glykofrydis...

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

Topics: Animals; Transgenes; Genetic Engineering; Gene Regulatory Networks; Cell Communication; Mammals

PubMed: 36549273
DOI: 10.1016/j.cels.2022.11.005

Review

Authors: Sara Kathleen Powell, Ricardo Rivera-Soto, Steven James Gray...

Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy.

Topics: Dependovirus; Genetic Therapy; Humans; Transgenes

PubMed: 25636961
DOI: No ID Found

Authors: Lei Yang, Frank Machin, Shuangfeng Wang...

Generation of stable gene-edited plant lines using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) requires a lengthy process of outcrossing to eliminate CRISPR-Cas9-associated sequences and produce transgene-free lines. We have addressed this issue by designing fusions of Cas9 and guide RNA transcripts to tRNA-like sequence motifs that move RNAs from transgenic rootstocks to grafted wild-type shoots (scions) and achieve heritable gene editing, as demonstrated in wild-type Arabidopsis thaliana and Brassica rapa. The graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors. We anticipate that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants.

Topics: Gene Editing; CRISPR-Cas Systems; Plant Breeding; Plants, Genetically Modified; Transgenes

PubMed: 36593415
DOI: 10.1038/s41587-022-01585-8

Authors: Angela Lek, Brenda Wong, Allison Keeler...

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dCas9 (i.e., "dead" Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×10 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Topics: Adult; Humans; Antibodies; Dystrophin; Genetic Therapy; Muscular Dystrophy, Duchenne; Respiratory Distress Syndrome; Transgenes; Fatal Outcome; Immunity, Innate

PubMed: 37754285
DOI: 10.1056/NEJMoa2307798

Authors: Tanya L Daigle, Linda Madisen, Travis A Hage...

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Topics: Animals; Brain; Calcium; Cell Line; Gene Knockout Techniques; Genes, Reporter; In Situ Hybridization, Fluorescence; Light; Mice; Mice, Transgenic; Microscopy, Fluorescence; Neurons; Optogenetics; RNA, Untranslated; Transgenes

PubMed: 30007418
DOI: 10.1016/j.cell.2018.06.035

Review

Authors: Valeria V Kolesnik, Ruslan F Nurtdinov, Ezekiel Sola Oloruntimehin...

Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.

Topics: Dependovirus; Transgenes; Genetic Therapy; Genetic Vectors

PubMed: 38488469
DOI: 10.1002/ctm2.1607

Review

Authors: Nikolas Zeh, Moritz Schmidt, Patrick Schulz...

Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.

Topics: CHO Cells; Animals; Cricetulus; Transgenes; Recombinant Proteins

PubMed: 38950872
DOI: 10.1016/j.biotechadv.2024.108402

Authors: Leslie O Goodwin, Erik Splinter, Tiffany L Davis...

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

Topics: Animals; Cells, Cultured; Genomic Structural Variation; Genotyping Techniques; Mice; Mice, Transgenic; Mutagenesis, Insertional; Nucleic Acid Amplification Techniques; Phenotype; Recombination, Genetic; Transgenes

PubMed: 30659012
DOI: 10.1101/gr.233866.117

Authors: Jin-Lei Liu, Meng-Meng Chen, Wen-Qiang Chen...

An integrated transgene-free multiplex gene-editing toolkit based on the Transgene Killer CRISPR technology greatly saves labor, time, and cost.

Topics: CRISPR-Cas Systems; Gene Editing; Transgenes

PubMed: 34893900
DOI: 10.1093/plphys/kiab573

Review

Authors: Reinhard Fässler

Green light for efficient production of transgenic farm animals

Topics: Animals; Gene Transfer Techniques; Genetic Vectors; Lentivirus; Microinjections; Swine; Transgenes

PubMed: 14710182
DOI: 10.1038/sj.embor.7400053