scholarly journals Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells

2021 ◽  
Author(s):  
Matias Ilmari Autio ◽  
Efthymios Motakis ◽  
Arnaud Perrin ◽  
Talal Bin Amin ◽  
Zenia Tiang ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Viral Vectors ◽  
Integration Site ◽  
Embryonic Stem ◽  
Transgene Integration ◽  
Safe Harbour ◽  
Random Fashion ◽  
Endogenous Genes ◽  
Bona Fide

Stable expression of transgenes is essential in both therapeutic and research applications. Traditionally, transgene integration has been accomplished via viral vectors in a semi-random fashion, but with inherent integration site biases linked to the type of virus used. The randomly integrated transgenes may undergo silencing and more concerningly, can also lead to dysregulation of endogenous genes. Gene dysregulation can lead to malignant transformation of cells and has unfortunately given rise to cases of leukaemia in gene therapy trials. Genomic safe harbour (GSH) loci have been proposed as safe sites for transgene integration. To date, a number of sites in the human genome have been used for directed integration; however none of these pass scrutiny as bona fide GSH. Here, we conducted a computational analysis to identify 25 putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

scholarly journals Regulated hAAT Expression from a Novel rAAV Vector and Its Application in the Prevention of Type 1 Diabetes

2019 ◽  
Vol 8 (9) ◽  
pp. 1321 ◽  
Author(s):  
Hongxia Ma ◽  
Yuanqing Lu ◽  
Keith Lowe ◽  
Lonneke van der Meijden-Erkelens ◽  
Clive Wasserfall ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Fine Tuning ◽  
Vector System ◽  
Regulation System ◽  
Raav Vector

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

Improved Production of Genetically Modified Fetuses with Homogeneous Transgene Expression After Transgene Integration Site Analysis and Recloning in Cattle

2011 ◽  
Vol 13 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Fabiana Fernandes Bressan ◽  
Moyses dos Santos Miranda ◽  
Felipe Perecin ◽  
Tiago Henrique De Bem ◽  
Flavia Thomaz Verechia Pereira ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Integration Site ◽  
Genetically Modified ◽  
Site Analysis ◽  
Transgene Integration ◽  
Integration Site Analysis

scholarly journals Modeling motor neuron resilience in ALS using stem cells

2018 ◽  
Author(s):  
Ilary Allodi ◽  
Jik Nijssen ◽  
Julio Aguila Benitez ◽  
Christoph Schweingruber ◽  
Andrea Fuchs ◽  
...  
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Embryonic Stem ◽  
Spinal Motor Neurons ◽  
Neuronal Progenitors ◽  
Oculomotor Neurons ◽  
Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

scholarly journals In Vitro- and In Vivo-Induced Transgene Expression in Human Embryonic Stem Cells and Derivatives

Stem Cells ◽  
2008 ◽  
Vol 26 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Xiaofeng Xia ◽  
Melvin Ayala ◽  
Benjamin R. Thiede ◽  
Su-Chun Zhang
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Transgene Expression ◽  
Embryonic Stem

scholarly journals The human FLT1 regulatory element directs vascular expression and modulates angiogenesis pathways in vitro and in vivo

2021 ◽  
Author(s):  
Julian Stolper ◽  
Holly K. Voges ◽  
Michael See ◽  
Neda Rahmani Mehdiabadi ◽  
Gulrez Chahal ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Regulatory Element ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Homeodomain Protein ◽  
Cardiovascular Development ◽  
Vascular Expression

AbstractThere is growing evidence that mutations in non-coding cis-regulatory elements (CREs) disrupt proper development. However, little is known about human CREs that are crucial for cardiovascular development. To address this, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined a highly conserved CRE within the FLT1 locus termed enFLT1. We show that the human enFLT1 is an enhancer capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka. Deletion of the human enFLT1 enhancer (ΔenFLT1) triggered molecular perturbations in extracellular matrix organisation and blood vessel morphogenesis in vitro in endothelial cells derived from human embryonic stem cells and vascular defects in vivo in medaka. These findings highlight the crucial role of the human FLT1 enhancer and its function as a regulator and buffer of transcriptional regulation in cardiovascular development.

scholarly journals Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 1911 ◽  
Author(s):  
Jamie R. Bhagwan ◽  
Emma Collins ◽  
Diogo Mosqueira ◽  
Mine Bakar ◽  
Benjamin B. Johnson ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Single Cell Analysis ◽  
Induced Pluripotent Stem Cell ◽  
Transgene Silencing ◽  
Variable Expression ◽  
Safe Harbour ◽  
Calcium Indicator ◽  
Models Of Disease ◽  
Aavs1 Locus

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

scholarly journals Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1911 ◽  
Author(s):  
Jamie R. Bhagwan ◽  
Emma Collins ◽  
Diogo Mosqueira ◽  
Mine Bakar ◽  
Benjamin B. Johnson ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Single Cell Analysis ◽  
Induced Pluripotent Stem Cell ◽  
Transgene Silencing ◽  
Variable Expression ◽  
Safe Harbour ◽  
Calcium Indicator ◽  
Models Of Disease ◽  
Aavs1 Locus

Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.

scholarly journals Identifying a novel role for the master regulator Tal1 in the Endothelial to Hematopoietic Transition

2021 ◽  
Author(s):  
Yasmin Natalia Serina Secanechia ◽  
Isabelle Bergiers ◽  
Matt Rogon ◽  
Christian Arnold ◽  
Nicolas Descostes ◽  
...  
Keyword(s):  
Developmental Process ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Hematopoietic Stem ◽  
Hematopoietic Development ◽  
Stem And Progenitor Cells ◽  
Bona Fide ◽  
Endothelial Precursors

ABSTRACTRecent progress in the generation of bona-fide Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro and ex vivo has been built on the knowledge of developmental hematopoiesis, underscoring the importance of understanding in detail this developmental process. Here, we sought to elucidate the function of the hematopoietic regulators Tal1, Lmo2 and Lyl1 in the Endothelial to Hematopoietic Transition (EHT), the process through which HSPCs are generated from endothelial precursors during embryogenesis. We used a mouse embryonic-stem cell (mESC)-based differentiation system to model hematopoietic development, and combined gain-of-function experiments in sorted vascular smooth muscle cells (VSM) with multi-omics to obtain mechanistic insights into the mode of action of Tal1, Lmo2 and Lyl1. We found that these factors promote the silencing of the VSM transcriptional program and the activation of the hematopoietic one. Through this approach and the use of a Tet-on system to control the expression of Tal1 during hematopoietic specification from mESCs, we discovered that its expression in endothelial cells is crucial for the EHT to occur.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

Challenges and progress in the production of transgenic cattle

1994 ◽  
Vol 6 (5) ◽  
pp. 647 ◽  
Author(s):  
WH Eyestone
Keyword(s):  
Embryonic Stem ◽  
Developmental Potential ◽  
High Frequencies ◽  
Transgene Integration ◽  
Transgenic Cattle ◽  
Genetic Manipulations ◽  
Near Future ◽  
Positive Results

The production of transgenic cattle presents a number of unique challenges not encountered in other species. First, the survival of microinjected zygotes is low; only 15% in vivo-derived develop into morulae and blastocysts and, of these, only about 18% yield live calves. Second, transgene integration frequency is relatively low, around 3%. Thus, more than 1000 zygotes must be injected to produce a single transgenic calf. Obtaining sufficient zygotes from donor cattle to sustain a transgenic cattle programme is logistically and financially prohibitive, since the average superovulated donor yields only about four microinjectable zygotes per collection attempt. In vitro oocyte maturation and fertilization techniques may be used to alleviate this problem, although initially the developmental potential of in vitro-derived microinjected zygotes is lower than their in vivo-produced counterparts (8% v. 15%, respectively, yield morulae and blastocysts). Since only 3-5% of calves born from microinjected zygotes produced in either fashion yield transgenics, at least 20-30 pregnancies must be carried to term for every transgenic calf born. These conditions require that large herds of donor and recipient cattle be maintained. Recipient requirements could be reduced if transgene integration frequency could be increased, but improvements in the near future are unlikely since the mechanism of integration after pronuclear microinjection is poorly understood. Alternatively, embryos could be screened for integrated transgenes before transfer; however, efforts in this area have been complicated by high frequencies of false positive results. Although yet to be developed, bovine embryonic stem cells would alleviate many of these problems and permit a wider range of genetic manipulations.

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