Site-specific Transgene Integration Research Articles

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.

The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.

Plastid engineering using episomal DNA.

Novel episomal systems have the potential to accelerate plastid genetic engineering for application in plant synthetic biology. Plastids represent valuable subcellular compartments for genetic engineering of plants with intrinsic advantages to engineering the nucleus. The ability to perform site-specific transgene integration by homologous recombination (HR), coordination of transgene expression in operons, and high production of heterologous proteins, all make plastids an attractive target for synthetic biology. Typically, plastid engineering is performed by homologous recombination; however, episomal-replicating vectors have the potential to accelerate the design/build/test cycles for plastid engineering. By accelerating the timeline from design to validation, it will be possible to generate translational breakthroughs in fields ranging from agriculture to biopharmaceuticals. Episomal-based plastid engineering will allow precise single step metabolic engineering in plants enabling the installation of complex synthetic circuits with the ambitious goal of reaching similar efficiency and flexibility of to the state-of-the-art genetic engineering of prokaryotic systems. The prospect to design novel episomal systems for production of transplastomic marker-free plants will also improve biosafety for eventual release in agriculture.

Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human Tcells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) Tcells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR Tcells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Oral booster vaccine antigen-Expression of full-length native SARS-CoV-2 spike protein in lettuce chloroplasts.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), β-casein (Csn2), whey acidic protein (WAP), or β-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a “precise” integration approach, placing the human granulocyte–macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2–11 µg/ml hGMCSF). This was probably caused by the absence of the 3’-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30–40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2–0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals’ health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Generation of a host cell line containing a MAR-rich landing pad for site-specific integration and expression of transgenes.

In recent years, targeted gene integration (TI) has been introduced as a strategy for the generation of recombinant mammalian cell lines for the production of biotherapeutics. Besides reducing the immense heterogeneity within a pool of recombinant transfectants, TI also aims at shortening the duration of the current cell line development process. Here we describe the generation of a host cell line carrying Matrix‐Attachment Region (MAR)‐rich landing pads (LPs), which allow for the simultaneous and site‐specific integration of multiple genes of interest (GOIs). We show that several copies of each chicken lysozyme 5'MAR‐based LP containing either BxB1 wild type or mutated recombination sites, integrated at one random chromosomal locus of the host cell genome. We further demonstrate that these LP‐containing host cell lines can be used for the site‐specific integration of several GOIs and thus, generation of transgene‐expressing stable recombinant clones. Transgene expression was shown by site‐specific integration of heavy and light chain genes coding for a monospecific antibody (msAb) as well as for a bi‐specific antibody (bsAb). The genetic stability of the herein described LP‐based recombinant clones expressing msAb or bsAb was demonstrated by cultivating the recombinant clones and measuring antibody titers over 85 generations. We conclude that the host cell containing multiple copies of MAR‐rich landing pads can be successfully used for stable expression of one or several GOIs.

Abstract 1154: Site-specific gene editing with ABBIE for T-cell therapy

Abstract Therapeutic T-cell engineering using viral vectors has been extensively explored as the most promising therapeutic purpose. However, virus-mediated gene delivery carries many challenges and limitations, such as random insertion of transgene into chromosome, posing risk of oncogenic transformation of the host cells, and complexity in the manufacturing process.We are developing a gene-editing tool, ABBIE (A Binding-Based Integrase Enzyme) which combines the specificity of Cas9 with the non-destructive DNA integration conferred by HIV integrase. Unlike endonucleases, HIV integrase can efficiently insert LTR-flanked DNA without causing dsDNA breaks. In our system, by use of fusing protein of enzymatically dead Cas9 (Cas9DN) to integrase of HIV-1 or its mutants, we were able to perform the gene knock-out (KO) and knock-in (KI) in one step at the target genome loci. For proof of concept, we generated the ABBIE proteins and donor DNA containing fluorescent gene with LTR sequence and tested the ABBIE mediated knock-in with conversion of fluorescent proteins. The integration of fluorescent donor DNA into genome was observed in the 293 T-cells transfected with both WT and Mut ABBIE proteins. Higher integration rate was observed in the cells expressing Mut ABBIE protein. Further, we applied this strategy to deliver the donor fluorescent DNA or chimeric antigen receptors (CARs) via electroporation into the HPRT1 locus for specific insertion using primary human T cells. We observed an average of ~30% and ~16% knock-in efficiency as measured by flow cytometry of GFP+ and CD34+ expression, respectively. The site-specific transgene integration was tested by treating the cells with 6- thioguanine (6-TG). We will further optimize the electroporation condition to maximize editing efficiency. This novel therapeutic toolkit will provide a modular platform for inserting therapeutic genes safely and efficiently into the target genome, further accelerate the offshore immunotherapy development. Citation Format: Jihyun Park, Xiaohong Wang, Leonardo Mirandola, Maurizio Chiriva-Internati. Site-specific gene editing with ABBIE for T-cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1154.

Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

Genome editing in human hematopoietic stem and progenitor cells via CRISPR-Cas9-mediated homology-independent targeted integration

Ex vivo gene correction of hematopoietic stem and progenitor cells (HSPCs) has emerged as a promising therapeutic approach for treatment of inherited human blood disorders. Use of engineered nucleases to target therapeutic transgenes to their endogenous genetic loci addresses many of the limitations associated with viral vector-based gene replacement strategies, such as insertional mutagenesis, variable gene dosage, and ectopic expression. Common methods of nuclease-mediated site-specific integration utilize the homology-directed repair (HDR) pathway. However, these approaches are inefficient in HSPCs, where non-homologous end joining (NHEJ) is the primary DNA repair mechanism. Recently, a novel NHEJ-based approach to CRISPR-Cas9-mediated transgene knockin, known as homology-independent targeted integration (HITI), has demonstrated improved site-specific integration frequencies in non-dividing cells. Here we utilize a HITI-based approach to achieve robust site-specific transgene integration in human mobilized peripheral blood CD34+ HSPCs. As proof of concept, a reporter gene was targeted to a clinically relevant genetic locus using a recombinant adeno-associated virus serotype 6 vector and single guide RNA/Cas9 ribonucleoprotein complexes. We demonstrate high levels of stable HITI-mediated genome editing (∼21%) in repopulating HSPCs after transplantation into immunodeficient mice. Our study demonstrates that HITI-mediated genome editing provides an effective alternative to HDR-based transgene integration in CD34+ HSPCs.

CRISPR-Cas9-Mediated In Vivo Gene Integration at the Albumin Locus Recovers Hemostasis in Neonatal and Adult Hemophilia B Mice

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.

Uniform Expression and Relatively Small Position Effects Characterize Sister Transformants in Maize and Soybean.

Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.

A system for site-specific integration of transgenes in mammalian cells.

Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

Expression of VGRNb-PE immunotoxin in transplastomic lettuce (Lactuca sativa L.).

This research has shown, for the first time, that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins and the transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. Angiogenesis refers to the formation of new blood vessels, which resulted in the growth, invasion and metastasis of cancer. The vascular endothelial growth factor receptor 2 (VEGFR2) plays a major role in angiogenesis and blocking of its signaling inhibits neovascularization and tumor metastasis. Immunotoxins are promising therapeutics for targeted cancer therapy. They consist of an antibody linked to a protein toxin and are designed to specifically kill the tumor cells. In our previous study, VGRNb-PE immunotoxin protein containing anti-VEGFR2 nanobody fused to the truncated form of Pseudomonas exotoxin A has been established. Here, we expressed this immunotoxin in lettuce chloroplasts. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, multigene engineering in a single transformation event and maternal inheritance of the transgenes. Site specific integration of transgene into chloroplast genomes, and homoplasmy were confirmed. Immunotoxin levels reached up to 1.1% of total soluble protein or 33.7µg per 100mg of leaf tissue (fresh weight). We demonstrated that transgenic immunotoxin efficiently causes the inhibition of VEGFR2 overexpression, cell growth and proliferation. These results indicate that plant chloroplasts are a suitable compartment for synthesizing recombinant immunotoxins.

Abstract IA22: Targeting CARs to the TRAC locus enhances T-cell potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T-cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T-cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies. Citation Format: Michel Sadelain, Justin Eyquem. Targeting CARs to the TRAC locus enhances T-cell potency [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA22.

Overexpression of BCL9, a Wnt/β-Catenin Transcriptional Co-Activator, in Transgenic Mice Promotes Spontaneous Development of B-Cell Malignancies

Dysregulation of the Wnt signaling pathway underlies the pathogenesis of a wide range of human cancers, including hematological malignancies such as multiple myeloma (MM). The terminal effector of this signaling pathway is a transcriptional complex formed by β-catenin and BCL9. This complex is of particular interest because the BCL9 locus resides on the frequently recurring 1q21 chromosomal amplification in MM, which has been linked to poor clinical prognosis and outcome. Our previous studies indicate that BCL9-mediated enhancement of β-catenin activity increases cells proliferation, migration, invasion, and the metastatic potential of MM cells. Therefore, in order to: (I) unequivocally determine the oncogenic role of BCL9, (II) better understand its mechanism of action, and (III) develop mouse preclinical model of cancer with dysregulated Wnt/β-catenin/BCL9 activity, we generated transgenic mouse models.To overcome problems inherently related to embryonic lethality, we generated BCL9fl/- conditional transgenic mice using site-specific transgene integration into the mouse ColA1 gene in embryonic stem cells. To remove the stop cassette and activate BCL9 expression in vivo, we generated AID-Cre+/-; BCL9fl/- and ERT2-Cre+/-; BCL9fl/- compound mice. Recombinase activity driven by AID (activation-induced cytidine deaminase) gene promoter or ER receptor after tamoxifen administration, caused removal of the stop cassette and expression of BCL9 in germinal center (GC) B cells or several tissues, respectively, as confirmed by immunoblot, immunohistochemical (IHC) and PCR analysis. Since BCL9 is a β-catenin co-activator, next we generated cohorts of AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- and ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- triple compound transgenic mice carrying the Wnt reporter system that expresses β-galactosidase (β-gal), to determine whether Wnt/β-catenin transcriptional activity is increased as a consequence of BCL9 overexpression in vivo. β-gal stain was increased in frequency and intensity in cells within GCs but not outside them in AID-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- compared to control mice. In ERT2-Cre+/-; BCL9fl/-; TCF/Lef1-lacZ+/- mice β-gal staining was primarily detected in cells outside the GCs, not within them. Overall, these results indicate that Wnt transcriptional activity is increased in B-cells as a consequence of Cre-induced expression of BCL9 and that AID-Cre+/- and ERT2-Cre+/- target expression of BCL9 to GC and non-GC B cells, respectively. Because BCL9 is involved in the pathogenesis of human cancers, we evaluated whether our transgenic mice develop hematological malignancies. Except for mild splenic enlargement, BCL9-transgenic mice were indistinguishable from control mice between 8 and 30 weeks of age as assessed by weight and posture. However, after 40 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-; BCL9fl/- and 70% (28/40) of ERT2-Cre+/-; BCL9fl/- mice but none from control cohorts showed signs of disease. Gross pathologic examination of euthanized animals with BCL9 overexpression revealed enlargement of the spleen and LNs. Two distinct patterns of clonal hematological malignancies were identified after detailed histological, IHC and molecular examination. In AID-Cre+/-; BCL9fl/- mice tumors resembled human plasmacytomas (PCs), whereas in ERT2-Cre+/-; BCL9fl/- mice B-cell acute lymphoblastic leukemia (B-ALL). This later result is of particular interest, since BCL9 was first identified by cloning the t(1;14)(q21;q32) translocation from a patient with B-ALL. These findings indicate that BCL9 overexpression at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated the BCL9 expression in human extramedullary plasmocytomas (EMP) and B-ALL. 32% of EMP cases analyzed by IHC expressed BCL9 at significant levels. Utilizing gene expression data available in the public domain we also showed that BCL9 is significantly overexpressed in ETV6-RUNX1 and TCF3-PBX1 subtypes of human B-ALL when compared to normal bone marrow counterparts, suggesting that BCL9 may play important roles in the pathogenesis of EMP as well as B-ALL in humans. Since BCL9 is highly expressed in tumors but not in the cells of origin and its interaction with β-catenin is specific, these results imply BCL9 as a promising candidate for targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Genetically modified (GM) crops: milestones and new advances in crop improvement.

New advances in crop genetic engineering can significantly pace up the development of genetically improved varieties with enhanced yield, nutrition and tolerance to biotic and abiotic stresses. Genetically modified (GM) crops can act as powerful complement to the crops produced by laborious and time consuming conventional breeding methods to meet the worldwide demand for quality foods. GM crops can help fight malnutrition due to enhanced yield, nutritional quality and increased resistance to various biotic and abiotic stresses. However, several biosafety issues and public concerns are associated with cultivation of GM crops developed by transgenesis, i.e., introduction of genes from distantly related organism. To meet these concerns, researchers have developed alternative concepts of cisgenesis and intragenesis which involve transformation of plants with genetic material derived from the species itself or from closely related species capable of sexual hybridization, respectively. Recombinase technology aimed at site-specific integration of transgene can help to overcome limitations of traditional genetic engineering methods based on random integration of multiple copy of transgene into plant genome leading to gene silencing and unpredictable expression pattern. Besides, recently developed technology of genome editing using engineered nucleases, permit the modification or mutation of genes of interest without involving foreign DNA, and as a result, plants developed with this technology might be considered as non-transgenic genetically altered plants. This would open the doors for the development and commercialization of transgenic plants with superior phenotypes even in countries where GM crops are poorly accepted. This review is an attempt to summarize various past achievements of GM technology in crop improvement, recent progress and new advances in the field to develop improved varieties aimed for better consumer acceptance.

566. Targeted Lentiviral Vector Integration Mediated by the AAV Rep78 Protein

Our goal is to improve the safety of lentiviral vectors by shifting their integration preference to genomic “safe harbor” sites where therapeutic transgenes can integrate and function in a predictable manner. To do this, we have generated integrase-defective LV vectors (IDLVs) capable of inserting transgene sequences at predetermined genomic sites through homologous recombination (HR). To increase the frequency of HR, we employ genome editing approaches involving site-specific nucleases. A major drawback of this approach is safety concerns caused by inadvertent double strand breaks (DSBs) and non-homologous end-joining (NHEJ) events at unintended genomic (off target) sites. To bypass this risk, we are pursuing site-specific nucleases that do not involve DSBs as a potentially safer class of DNA-modifying agents for site-specific integration of transgenes in human cells. The sequence and strand-specific endonucleases Rep78 and Rep68 encoded by adeno-associated virus (AAV) have previously been reported to stimulate HR of plasmids bearing homology arms corresponding to the AAVS1 safe harbor locus on chromosome 19. We tested the integration efficiency of IDLVs containing short homology arms corresponding to the AAVS1 locus mediated by an engineered version of the AAV2-derived Rep78 protein in HEK293 cells. We compared the integration efficiency of IDLVs mediated by the Rep78 protein to that of an AAVS1-specific zinc finger nuclease (ZFN). Furthermore, we explored the potential of IDLV donor vectors for targeted integration mediated by Rep78 in human induced pluripotent stem cells (hiPSCs). The results obtained indicate that in the presence of the Rep78 protein, the integration efficiency of IDLVs was up to 8.3 fold higher compared to cells treated with the donor vector alone. Moreover, the efficiency of site-specific targeting mediated by Rep78 was higher than that of the AAVS1-specific ZFN. We also showed that Rep78 can successfully direct integration of IDLVs at the AAVS1 locus in hiPSCs. We anticipate that the approach used may ultimately contribute to the reduction of risks associated with LV vectors.

In vivo genome editing of the albumin locus as a platform for protein replacement therapy

AbstractSite-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) –mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Transgenic trait deployment using designed nucleases.

The demand for crops requiring increasingly complex combinations of transgenes poses unique challenges for transgenic trait deployment. Future value-adding traits such as those associated with crop performance are expected to involve multiple transgenes. Random integration of transgenes not only results in unpredictable expression and potential unwanted side effects but stacking multiple, randomly integrated, independently segregating transgenes creates breeding challenges during introgression and product development. Designed nucleases enable the creation of targeted DNA double-strand breaks at specified genomic locations whereby repair can result in targeted transgene integration leading to precise alterations in DNA sequences for plant genome editing, including the targeting of a transgene to a genomic locus that supports high-level and stable transgene expression without interfering with resident gene function. In addition, targeted DNA integration via designed nucleases allows for the addition of transgenes into previously integrated transgenic loci to create stacked products. The currently reported frequencies of independently generated transgenic events obtained with site-specific transgene integration without the aid of selection for targeting are very low. A modular, positive selection-based gene targeting strategy has been developed involving cassette exchange of selectable marker genes which allows for targeted events to be preferentially selected, over multiple cycles of sequential transformation. This, combined with the demonstration of intragenomic recombination following crossing of transgenic events that contain stably integrated donor and target DNA constructs with nuclease-expressing plants, points towards the future of trait stacking that is less dependent on high-efficiency transformation.