Transgene Copy Research Articles

Magnaporthe oryzae pathotype Triticum (MoT) can act as a heterologous expression system for fungal effectors with high transcript abundance in wheat

Plant pathogens deliver effector proteins to reprogramme a host plants circuitry, supporting their own growth and development, whilst thwarting defence responses. A subset of these effectors are termed avirulence factors (Avr) and can be recognised by corresponding host resistance (R) proteins, creating a strong evolutionary pressure on pathogen Avr effectors that favours their modification/deletion to evade the immune response. Hence, identifying Avr effectors and tracking their allele frequencies in a population is critical for understanding the loss of host recognition. However, the current systems available to confirm Avr effector function, particularly for obligate biotrophic fungi, remain limited and challenging. Here, we explored the utility of the genetically tractable wheat blast pathogen Magnaporthe oryzae pathotype Triticum (MoT) as a suitable heterologous expression system in wheat. Using the recently confirmed wheat stem rust pathogen (Puccina graminis f. sp. tritici) avirulence effector AvrSr50 as a proof-of-concept, we found that delivery of AvrSr50 via MoT could elicit a visible Sr50-dependant cell death phenotype. However, activation of Sr50-mediated cell death correlated with a high transgene copy number and transcript abundance in MoT transformants. This illustrates that MoT can act as an effective heterologous delivery system for fungal effectors from distantly related fungal species, but only when enough transgene copies and/or transcript abundance is achieved.

Towards a robust test to detect gene doping for anabolic enhancement in human athletes.

Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone-releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction.

Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology.

Be Cautious to Adopt a Second CAR T-Cell Infusion after Failure of CD19/CD22 Cocktail CAR T-Cell Therapy in Relapsed/Refractory B-NHL

Objective: Chimeric antigen receptor (CAR) T-cell infusion (CTI) has emerged as a breakthrough therapy for relapsed/refractory B-cell non-Hodgkin's lymphoma (R/R B-NHL) patients. Nevertheless, there are still a significant number of patients suffering treatment failure (failed to respond or eventually relapsed after a partial or complete response, PR/CR) post the first time CTI (CTI1). The issue how to salvage this group of patients should be urgently addressed. Our study aimed to provide clues for exploring the potentially effective salvage strategies and finally improving the prognosis of them by analyzing the efficacy and safety especially between the second time CTI (CTI2) and other different therapies (non-CTI2). Methods: Herein,a retrospective analysis was performed on total 61 patients after failure of CD19/22 cocktail CTI1 (ChiCTR-OPN-16008526) between January 2017 and May 2022 in our hospital. Among them, 27 of 61 patients with a prior determination of no antigen loss received CTI2 for compassionate use. The other 34 patients were administrated as non-CTI2 therapies. The clinical characteristics, efficacy, safety, survival analysis and outcome-related risk factors were all evaluated. Results: The CTI2 group achieved a higher overall response rate (ORR) at 30 days assessment (8/27 vs. 2/34, P=0.017). Nevertheless, the non-CTI2 group showed a better event-free survival (EFS) (P=0.007) (Figure1, left panel) and overall survival (OS) (P=0.048) (Figure1, middle panel) than the CTI2 group with a median follow-up of 6.7 months (range, 3-45.5) and 4.7 months (range, 1.1-53). Further analysis on copies of CAR transgene in peripheral blood at 90 days detection revealed that the better CAR T cells expansion was related to a better response in CTI2 group (P=0.012)(data not shown). Additionally, neither of the CTI2 and non-CTI2 was the independent risk factor of survival (HR: 1.665, 95%CI: 0.883-3.141, P=0.115). Either of Ann Arbor stage (HR: 1.970,9 5%CI: 1.944-3.997, P=0.029) or bulky disease (HR: 2.879, 95%CI: 1.321-6.276, P=0.008) at disease onset was the related independent risk factor instead. As shown in Figure1 right panel, the best responses to different salvage therapies suggested that the regimen (Polatuzumab vedotin,-pola, Bendamustine, Rituximab, pola+BR) was likely to be a potentially optimal non-CTI2 salvage therapy, even without statistically significant differences due to the limitation of the sample size. The adverse effects of CTI2 were generally similar to those of non-CTI2. Remarkably the infection-related mortality rate was significantly higher (59% vs. 21%, P=0.025) in CTI2 group. Conclusion: Our study provides novel insights and clues that clinicians should be cautious to adopt a CTI2 after failure of the CD19/22 cocktail CTI1 in R/R B-NHL. It is noteworthy that the choice of pola+BR regimen probably has an advantage in salvage strategies. Large-scale prospective studies are warranted in the future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Utilization of the Tapestri Platform for Quantification of Single Cell Vector Copy Number in CAR T Cell Products

Chimeric antigen receptor (CAR) T cell immunotherapies have been transformative solutions to treat both adult and pediatric patients with a variety of cancers. Given that CAR T cell therapies involve genetic alterations of a patient's T cells by introduction of the CAR into host cells using lentiviral vectors, the quality of CAR T cell therapies are extensively regulated to warrant safety. Key safety and efficacy attributes need to be accurately measured prior to reintroducing modified T cells back into patients, including CAR transgene copy number or viral vector copy number (VCN). We have previously compared digital PCR (dPCR) strategies to the historical standard release assay of quantitative PCR (qPCR) for VCN quantification given that dPCR is known to provide absolute quantification of target nucleic acid sequences with many advantages over qPCR in regards to simplicity, reproducibility, lower limit of detection, and definitive quantification. We were able to demonstrate that dPCR produces accurate, precise, and reproducible CAR T cell VCN measurements when compared to qPCR. However, these conventional PCR methods measuring vector copy number are either population averaged or involve labor-intensive and time-consuming single cell colony picking and outgrowth, with respective drawbacks in resolution and sample representation. Here, we introduce the Tapestri platform, to measure thousands of single cell level VCN with accuracy and precision. The Tapestri platform workflow is enabled by its two droplet steps. First, thousands of cells are each microfluidically encapsulated in a first droplet where lysis of the cells occur, releasing DNA from its heterochromatin state for uniform DNA interrogation. Then, the individual lysates are uniquely barcoded and target sequences amplified via multiplexed PCR inside each droplet. The final products are sequenced on an Illumina sequencing instrument. Single cell VCN Tapestri data was generated and orthogonally compared with dPCR and matches with the bulk measurement on clonal Jurkat cell lines (VCN=2) mixed with control cell lines (VCN=0) at a range of different ratios with accuracy and precision. We demonstrate that the Tapestri platform provides absolute quantification of CAR transgenes at the single cell level and has the potential to provide more precise measurements of CAR T cell therapeutic product attributes prior to release to patients. Future studies will evaluate the utility of this Tapestri platform in measuring both CAR transgene copies and surface expression/density of the CAR at the single cell level, further improving upon current CAR T cell product release assays.

Response, Peak and Persistence of Varnimcabtagene Autoleucel (IMN-003A), First-in-India Industry CD19-Directed CAR-T Cell Therapy, with Fractionated Infusions for Patients with Relapsed and/or Refractory B Cell Malignancies: Early Results (IMAGINE Study)

Background: Varnimcabtagene autoleucel (IMN-003A) is an autologous CD19 directed CAR-T cell product with a 4-1BB co-stimulatory domain and a non-FMC63 murine single chain variable fragment (A3B1 binder), manufactured in India, which is being tested in the IMAGINE study (CTRI/2022/03/041162), a phase-2 clinical trial for patients (pts) with relapsed and/or refractory B cell malignancies (RR BCM). A fractionated infusion of 1 x 106 CAR+ cells (IMN-003A)/kg for B-ALL cohort and 5 x 106 CAR+ cells (IMN-003A)/kg for B-NHL cohort was administered over 3 days (D0, D+1, D+2) as 10%, 30% and 60% fractions after Fludarabine-Cyclophosphamide lymphodepletion regimen (LR). Safety and efficacy results are submitted in a separate abstract (#166297); here we present the early IMN-003A pharmacokinetic data and correlation with disease response. Methods: IMN-003A was manufactured using cGMP compliant closed system (CliniMACS Prodigy®). The manufacturing data were obtained and analyzed, including % CAR transduction in the final product (FP) by flow cytometry (FC). Peripheral blood (PB) samples were obtained after consent at screening and as per patient schedule after infusion. Persistence of IMN-003A was evaluated in PB after infusion by ddPCR. Safety and efficacy data were collected for analysis. Bone marrow (BM) samples for MRD were obtained at screening, D+28 and D+90 for efficacy. Minimal residual disease (MRD) was performed by FC with 10-4 sensitivity. Apheresis, FP, and PB samples were analyzed for T-cell subpopulations and surface markers by FC. Results: Eight apheresis were obtained and transduced to achieve the final product with a median manufacturing time of 11d (range 10-18) with 100% success. The mean % transduction of IMN-003A on autologous T cells was 34.77% (range 15.49 - 58.35) with median transgene copies / genome of 2.25 (range 1.49 - 3.83). The mean CD4/CD8 ratio at apheresis and final product was 0.74 and 0.84 respectively. The T-cell subpopulations at apheresis and FP were analyzed (Figure 1A). The mean proportion of CD4 and CD8 positive naïve cells (CCR7+ RA+) was above 90% in the final product (Figure 1A). Median Product doubling time and Apheresis to Infusion time was 1.04d (range 1.02 - 1.38) and 20d (range 16 - 30) respectively. Post IMN-003A infusion, there was reversal of CD4/CD8 ratio in peripheral blood (Figure 1A). IMN-003A CAR-T cells showed maximum PB expansion (Tmax) at median 10d (range 10 - 14) with median Cmax 180,680 CAR copies / µg gDNA (range 94,675 - 276,412). Among pts with 28-day follow-up, 80% had evidence of CAR+ T cells in PB, with median persistence not reached (range 28 - NR). B-cell aplasia was observed in all pts concurring with CART expansion (Figure 1B). Median loss of B cell aplasia was not reached (range 42 - NR), with no apparent association with response at D+28 and D+90. Hypogammaglobulinemia was noted in 6 of 7 evaluable pts and 2 pts received IVIg. Of evaluable patients with CD4+ T-cell counts below 200/μL, recovery (>200/μL) was seen at median of 10 days (range 2 - 10) after the IMN-003A cell infusion. None of these pts have relapsed. Of 5 MRD-evaluable pts, response at day +28 (n=4) and +90 (n=1) was 100%. Updated results will be presented at the meeting. Conclusion: In this industry-led first-in-India phase-2 study, IMN-003A production in a cGMP closed system was feasible and rapid. The peak of CAR-T expansion occurs at median 10d of infusion. The interim responses obtained are deep. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cre-mediated autoexcision of selectable marker genes in soybean, cotton, canola and maize transgenic plants.

Efficient selectable marker gene autoexcision in transgenic plants of soybean, cotton, canola, and maize is achieved by effective Cre recombinase expression. Selectable marker genes are often required for efficient generation of transgenic plants in plant transformation but are not desired once the transgenic events are obtained. We have developed Cre/loxP autoexcision systems to remove selectable marker genes in soybean, cotton, canola and maize. We tested a set of vectors with diverse promoters and identified promising promoters to drive cre expression for each of the four crops. We evaluated both the efficiency of generating primary transgenic events with low transgene copy numbers, and the frequency of marker-free progeny in the next generation. The best performing vectors gave no obvious decrease in the transformation frequency in each crop and generated homozygous marker-free progeny in the next generation. We found that effective expression of Cre recombinase for marker gene autoexcision can be species dependent. Among the vectors tested, the best autoexcision frequency (41%) in soybean transformation came from using the soybean RSP1 promoter for cre expression. The cre gene expressed by soybean RSP1 promoter with an Arabidopsis AtpE intron delivered the best autoexcision frequency (69%) in cotton transformation. The cre gene expressed by the embryo-specific eUSP88 promoter from Vicia faba conferred the best marker excision frequency (32%) in canola transformation. Finally, the cre gene expressed by the rice CDC45-1 promoter resulted in 44% autoexcision in maize transformation. The Cre/loxP recombinase system enables the generation of selectable marker-free transgenic plants for commercial product development in four agriculturally important crops and provides further improvement opportunities for more specific and better marker excision efficiency.

SIRT6 Mitigates Heart Failure With Preserved Ejection Fraction in Diabetes.

Heart failure with preserved ejection fraction (HFpEF) is a growing health problem without effective therapies. Epidemiological studies indicate that diabetes is a strong risk factor for HFpEF, and about 45% of patients with HFpEF are suffering from diabetes, yet the underlying mechanisms remain elusive. Using a combination of echocardiography, hemodynamics, RNA-sequencing, molecular biology, in vitro and in vivo approaches, we investigated the roles of SIRT6 (sirtuin 6) in regulation of endothelial fatty acid (FA) transport and HFpEF in diabetes. We first observed that endothelial SIRT6 expression was markedly diminished in cardiac tissues from heart failure patients with diabetes. We then established an experimental mouse model of HFpEF in diabetes induced by a combination of the long-term high-fat diet feeding and a low-dose streptozocin challenge. We also generated a unique humanized SIRT6 transgenic mouse model, in which a single copy of human SIRT6 transgene was engineered at mouse Rosa26 locus and conditionally induced with the Cre-loxP technology. We found that genetically restoring endothelial SIRT6 expression in the diabetic mice ameliorated diastolic dysfunction concurrently with decreased cardiac lipid accumulation. SIRT6 gain- or loss-of-function studies showed that SIRT6 downregulated endothelial FA uptake. Mechanistically, SIRT6 suppressed endothelial expression of PPARγ through SIRT6-dependent deacetylation of histone H3 lysine 9 around PPARγ promoter region; and PPARγ reduction mediated SIRT6-dependent inhibition of endothelial FA uptake. Importantly, oral administration of small molecule SIRT6 activator MDL-800 to diabetic mice mitigated cardiac lipid accumulation and diastolic dysfunction. The impairment of endothelial SIRT6 expression links diabetes to HFpEF through the alteration of FA transport across the endothelial barrier. Genetic and pharmacological strategies that restored endothelial SIRT6 function in mice with diabetes alleviated experimental HFpEF by limiting FA uptake and improving cardiac metabolism, thus warranting further clinical evaluation.

Detection of Transgene Location in the CYP2A13/2B6/2F1-transgenic Mouse Model using Optical Genome Mapping Technology.

Most transgenic mouse models are generated through random integration of the transgene. The location of the transgene provides valuable information for assessing potential effects of the transgenesis on the host and for designing genotyping protocols that can amplify across the integration site, but it is challenging to identify. Here, we report the successful utility of optical genome mapping technology to identify the transgene insertion site in a CYP2A13/2B6/2F1-transgenic mouse model, which produces three human cytochrome P450 (P450) enzymes (CYP2A13, CYP2B6, and CYP2F1) that are encoded by neighboring genes on human chromosome 19. These enzymes metabolize many drugs, respiratory toxicants, and chemical carcinogens. Initial efforts to identify candidate insertion sites by whole genome sequencing was unsuccessful, apparently because the transgene is located in a region of the mouse genome that contains highly repetitive sequences. Subsequent utility of the optical genome mapping approach, which compares genome-wide marker distribution between the transgenic mouse genome and a reference mouse (GRCm38) or human (GRCh38) genome, localized the insertion site to mouse chromosome 14, between two marker positions at 4451324 base pair and 4485032 base pair. A transgene-mouse genome junction sequence was further identified through long-polymerase chain reaction amplification and DNA sequencing at GRCm38 Chr.14:4484726. The transgene insertion (∼2.4 megabase pair) contained 5-7 copies of the human transgenes, which replaced a 26.9-33.4 kilobase pair mouse genomic region, including exons 1-4 of Gm3182, a predicted and highly redundant gene. Finally, the sequencing results enabled the design of a new genotyping protocol that can distinguish between hemizygous and homozygous CYP2A13/2B6/2F1-transgenic mice. SIGNIFICANCE STATEMENT: This study characterizes the genomic structure of, and provides a new genotyping method for, a transgenic mouse model that expresses three human P450 enzymes, CYP2A13, CYP2B6, and CYP2F1, that are important in xenobiotic metabolism and toxicity. The demonstrated success in applying the optical genome mapping technology for identification of transgene insertion sites should encourage others to do the same for other transgenic models generated through random integration, including most of the currently available human P450 transgenic mouse models.

Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products

Background aimsVector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. MethodsThe authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer–probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. ResultsddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors’ assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. ConclusionsThe authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test–retest reliability, and that specific assays can be developed for detection of unique constructs.

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

BackgroundGenetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency.ResultsWe have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1 generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes.ConclusionThis new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1–11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

Germline transformation of Artemisia annuaL. plant via in planta transformation technology “Floral dip”

The therapeutic efficacy of Artemisia annua L. is governed by artemisinin (ART), prevalently produced by A. annua extraction. Due to the modest amount of ART (0.01-1 %dw) in this plant, commercialization of ACTs is difficult. In this study, the floral-dip based transformation protocol for A. annua was developed to enhance expression of artemisinin biosynthesis genes and ART content. For dipping, the effective infiltration media components were optimized, and to obtain high transformation (26.9%) partially open bud stage capitulum of floral development was used. Hygromycin phospho-transferase (hptII) selection marker was used to validate the transformed T1 progenies. The copy numbers of the transgene (hptII) in T1 progenies were determined using a sensitive, high-throughput SYBR Green based quantitative RT-PCR. The results of the hptII transgene were compared with those of the low copy number, internal standard (hmgr). Using optimised PCR conditions, one, two and three transgene copies in T1 transformants were achieved.

Genetically Engineered Pigs as Efficient Salivary Gland Bioreactors for Production of Therapeutically Valuable Human Nerve Growth Factor.

Farm animal salivary glands hold great potential as efficient bioreactors for production of human therapeutic proteins. Nerve growth factor (NGF) is naturally expressed in animal salivary glands and has been approved for human clinical treatment. This study aims to employ transgenic (TG) pig salivary gland as bioreactors for efficient synthesis of human NGF (hNGF). hNGF-TG pigs were generated by cloning in combination with piggyBac transposon-mediated gene transfer. These hNGF-TG pigs specifically expressed hNGF protein in their salivary glands and secreted it at high levels into saliva. Surgical and nonsurgical approaches were developed to efficiently collect saliva from hNGF-TG pigs. hNGF protein was successfully purified from collected saliva and was verified to be biologically active. In an additional step, the double-transgenic pigs, where the endogenous porcine NGF (pNGF) gene was replaced by another copy of hNGF transgene, were created by cloning combined with CRISPR/Cas9-mediated homologous recombination. These double-transgenic pigs expressed hNGF but not pNGF, thus avoiding possible “contamination” of hNGF with pNGF protein during purification. In conclusion, TG pig salivary glands can be used as robust bioreactors for a large-scale synthesis of functional hNGF or other valuable proteins. This new animal pharming method will benefit both human health and biomedicine.

A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors.

Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs’ low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

SummaryIn biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue‐types and/or at specific times often precludes co‐expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator‐like effectors (dTALEs) and cognate synthetic TALE‐activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1‐STAP1 and dTALE2‐STAP2—can activate STAP‐driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell‐types, requiring empirical evaluation in each case. dTALE expression leads to off‐target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE‐STAP system thus provides a powerful approach to fine‐tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

CRISPR/Cas9 - mediated knock-in method can improve the expression and effect of transgene in P1 generation of channel catfish (Ictalurus punctatus)

Transgenesis has a wide range of applications in fish breeding and generation of fish models. Previously, it was common to produce transgenic fish by transferring plasmid DNA into early embryos, resulting in random integration, but more precision, targeted integration is possible with CRISPR/Cas9 technology. Channel catfish (Ictalurus punctatus) is an economically important farmed fish in the United States. To make channel catfish an even richer source of nutrients, we produced P1 fish carrying masu salmon (Oncorhynchus masou) elovl2 (OmElovl2) transgene to increase the content of omega-3 (n-3) fatty acids with CRISPR/Cas9-mediated knock-in targeting non-coding region of chromosome 1, and random integration methods. Mosaicism, transgene expression and fatty acids contents were determined. Integration rates of seven-month-old channel catfish generated by CRISPR/Cas9 and random integration methods were 19% and 27.3%, respectively. However, when we tested five tissues including barbel, fin, muscle, liver and kidney of three channel catfish, 13 out of 15 total observations were verified to carry the transgene from three positive P1 fish produced by CRISPR/Cas9 technology. Only five of 15 tissues carrying transgene were detected in three positive P1 fish produced by random integration. Genomic quantitative real-time PCR (qRT-PCR) also suggested that CRISPR/Cas9 transgenic fish had extremely higher average transgene copy numbers than randomly integrated transgenic fish. Additionally, reverse transcription PCR (RT-PCR) and fatty acids analysis revealed that CRISPR/Cas9 P1 fish had strong OmElovl2 transgene expression in most tissues and 20.7% higher DHA than their controls, while randomly integrated P1 fish did not have detectable OmElovl2 expression in any of five tissues detected. There were no significant differences for any fatty acids between transgenic fish produced by random integration and their non-transgenic controls. CRISPR/Cas9 mediated knock-in technology efficiently reduced mosaicism, improved transgene expression and the biological effects of the foreign gene in P1 generation compared to the conventional random integration method. Therefore, transgenesis based on CRISPR/Cas9 technology would shorten breeding programs and improve applications of gene function studies.

Abstract 1138: Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue

Abstract Immune cell therapies derived from induced pluripotent stem cells (iPSC) provide a novel opportunity for the treatment of multiple cancer types. Assessment of the persistence and biodistribution of these product candidates requires specific and sensitive methods to detect engineered cells in both liquid and solid biopsies. Here, we present the development and validation of two complimentary nucleic acid-based detection assays for iPSC-derived natural killer (iNK) cell product candidates containing Fate’s proprietary high-affinity, non-cleavable CD16 transgene (hnCD16). The first assay is a droplet digital PCR (ddPCR) method to detect and quantify hnCD16 transgene copies present in a pool of genomic DNA (gDNA). The primers and probe were designed to recognize the optimized codons of hnCD16. Assay linearity and accuracy were assessed through titration studies using 0.024 to 1 ng of hnCD16-containing DNA spiked into different amounts of hnCD16-negative gDNA. Precision was determined through multiple assay runs by different operators on two instruments. The second assay is an in situ hybridization based method utilizing RNAscope࣪ technology to detect cells expressing hnCD16 in fixed tissue. Probes targeting hnCD16 were used to optimize signal specificity. Cells expressing hnCD16 and tissues from in vivo studies treated with iNK products served as positive controls. For the ddPCR assay, absolute limit of detection (aLoD) was determined to be 4.9 copies of hnCD16 per 20 µL reaction, regardless of total genomic mass input. Absolute limit of quantification (aLoQ) was 12 copies per 20 µL reaction with a %CV ≤30. Relative limit of quantification (rLoQ), assessing transgene to total DNA ratio, is affected by the background gDNA input and is less sensitive with lower input mass. rLoQ for total mass of 70 - 250 ng was 97 - 22 copies/µg gDNA (0.064% - 0.015%) with a %CV ≤30. The sensitivity of this input range allows evaluation of clinical samples with low cellularity. While ddPCR provides robust quantification of the hnCD16 transcript, the RNAscope࣪ assay informs localization of the iNK product. Specificity of the probe was established by confirming its lack of affinity for endogenous CD16 using a variety of human normal and tumor tissues and by staining hnCD16-positive fixed cell pellets and tissues from in vivo studies. In cell pellets, positive RNAscope࣪ signal correlated with the known ratio of transgene positive cells. In murine tissues previously confirmed to contain iNK cell product, the RNAscope࣪ positive staining correlated with NKG2A immunohistochemistry staining, confirming the presence of product NK cells. The combined use of both the ddPCR and RNAscope࣪ assays targeted to hnCD16 allows for detection and quantification of transgene-bearing iNK cells in a wide variety of patient samples including tumor biopsies. Both assays are being utilized for cell detection and quantification in our ongoing clinical trials. Citation Format: Cara E. Bickers, Judy L. Martin, Steven Castro, Jason Zhang, Thomas Dailey, Eric Sung, Suzanna Gasparian, Jason O'Rourke, Moyar Ge, Tom T. Lee, Janel Huffman, Jode Goodridge, Ryan Bjordahl, Bahram Valamehr, Peter M. Szabo, Lilly Wong, Sarah Cooley. Detection of genetically engineered iPSC-derived natural killer cells in blood and tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1138.

Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies.