202 Combination of transcription activator-like effector nucleases and homology-independent target integration strategy gene editing technologies for knock-in of recombinant human factor IX Under the β-casein native promoter in bovine IVF embryos

Precise DNA modification is a crucial approach for gene function elucidation, biomedical model development, and transgenic bioreactor generation. In livestock, its application was extremely challenging until the development of engineered nucleases such as zinc-finger nucleases, transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9. Still, precise knock-in (KI) techniques remain inefficient. Recently, the homology-independent target integration (HITI) strategy was developed, allowing precise insertion of transgenes in mammalian cells in an easier fashion. The HITI technique allows site-specific gene insertion by means of cleavage of both the target sequence in the genome and the donor plasmid, followed by DNA repair by nonhomologous end joining. Here, we evaluated the use of TALENs to generate precise knockout (KO) alleles of the β-casein gene (CSN2) by creating small insertions or deletions, and precise insertion of recombinant human factor IX (rhFIX) under bovine CSN2 regulatory sequences, using HITI via cytoplasmic injection of bovine IVF zygotes. First, 2 TALEN pairs (Tn1 and Tn2) targeting exon 2 of bovine CSN2 were designed and their activity was confirmed by primary fibroblasts transfection followed by Surveyor assay at Day 3. Then, both TALEN pairs were evaluated for KO embryo generation by zygote cytoplasmic injection of in vitro-transcribed mRNA encoding for Tn1, Tn2, or a mix containing Tn1+Tn2, at 100ng μL−1. A non-injected control (NIC) was also included. Embryos were in vitro cultured until Day 7 and independently analysed by whole-genome amplification followed by PCR and sequencing. Neither the blastocyst rate [28.8% (n=73), 33.8% (n=71), 32.4% (n=74), and 54.3% (n=127) for Tn1, Tn2, Tn1+Tn2, and NIC, respectively] nor the proportion of edited embryos [44% (n=9), 20% (n=10), and 33% (n=9) for Tn1, Tn2, and Tn1+Tn2, respectively] differed between injected groups (Fisher test, P<0.05), demonstrating efficient editing in bovine embryos by TALENs. Finally, to achieve precise CSN2 KI embryos, the rhFIX open reading frame was PCR amplified with a forward primer containing the Tn1 recognition sequence to obtain the HITI donor and bovine IVF zygotes were co-injected with the Tn1 mRNA and the HITI donor. Embryos were in vitro cultured until Day 7 and individually analysed by nested PCR at both the 5′ and 3′ ends of HITI donor. The PCR-based results indicate HITI donor integration in 7% of embryos analysed (n=14). Sanger sequencing analysis is currently in progress to confirm site-specific integration of HITI and possible rearranged DNA integration in other embryos. To our knowledge, this is the first report on the use of TALEN and HITI for gene modification. Our results indicate that TALEN combined with HITI may constitute an easy strategy for precise production of pharmaceuticals in the milk of livestock.

A Highly Productive CHO Cell Line Secreting Human Blood Clotting Factor IX

Hemophilia B patients suffer from an inherited blood-clotting defect and require regular administration of blood-clotting factor IX replacement therapy. Recombinant human factor IX produced in cultured CHO cells is nearly identical to natural, plasma-derived factor IX and is widely used in clinical practice. Development of a biosimilar recombinant human factor IX for medical applications requires the generation of a clonal cell line with the highest specific productivity possible and a high level of specific procoagulant activity of the secreted factor IX. We previously developed plasmid vectors, p1.1 and p1.2, based on the untranslated regions of the translation elongation factor 1 alpha gene from Chinese hamster. These vectors allow one to perform the methotrexate- driven amplification of the genome-integrated target genes and co-transfect auxiliary genes linked to various resistance markers. The natural open reading frame region of the factor IX gene was cloned in the p1.1 vector plasmid and transfected to CHO DG44 cells. Three consecutive amplification rounds and subsequent cell cloning yielded a producer cell line with a specific productivity of 10.7 0.4 pg/cell/day. The procoagulant activity of the secreted factor IX was restored nearly completely by co-transfection of the producer cells by p1.2 plasmids bearing genes of the soluble truncated variant of human PACE/furin signal protease and vitamin K oxidoreductase from Chinese hamster. The resulting clonal cell line 3B12-86 was able to secrete factor IX in a protein-free medium up to a 6 IU/ml titer under plain batch culturing conditions. The copy number of the genome- integrated factor IX gene for the 3B12-86 cell line was only 20 copies/genome; the copy numbers of the genome-integrated genes of PACE/furin and vitamin K oxidoreductase were 3 and 2 copies/genome, respectively. Factor IX protein secreted by the 3B12-86 cell line was purified by three consecutive chromatography rounds to a specific activity of up to 230 IU/mg, with the overall yield 30%. The developed clonal producer cell line and the purification process employed in this work allow for economically sound industrial-scale production of biosimilar factor IX for hemophilia B therapy.

199 Efficient Knock-out of Ovine β-Lactoglobulin (BLG) Gene and Knock-in of Recombinant Human Factor IX (rhFIX) Under BLG Native Regulatory Sequences in Somatic Cells and Zygotes Using TALEN Nuclease

Site-specific genetic engineering is a valuable tool for pharmaceutical research and development of biomedical models. Despite engineered nucleases allow targeted gene edition in a rather simple fashion; few reports are available so far on specific gene knock-in (KI) combined with engineered nucleases in domestic species. Here, we evaluated the possibility of inducing specific KI of cDNAs coding for proteins of pharmaceutical interest under the control of milk native promoter sequences, taking advantage of the TALEN system, both in ovine somatic cells and in zygotes. We designed 2 TALENs, targeting exons 1 and 5 of ovine β-lactoglobulin gene (BLG), respectively, and a homologous recombination vector (pHR), carrying recombinant human factor IX (rhFIX) flanked by homology arms contiguous to the TALEN target sites. In an initial set of experiments, 5 × 105 to 1 × 106 ovine fibroblasts were transfected with 1 μg of each TALEN mRNA, with or without 50 ng μL−1 pHR. The feasibility of inducing knock-out (KO) was confirmed by Cel1 assay. The deletion of the genomic region between TALEN target sites and the occurrence of HR in cell lysates were assessed by PCR. Also, 576 individual colonies were picked up and analyzed by PCR. The deletion of the region between TALEN target sites was achieved with 7.8% efficiency (45/576). The incidence of HR in cells was 0.5% (3/576), as detected by PCR. In order to evaluate the system in zygotes, laparoscopic AI was performed on synchronized and superovulated ewes. Zygotes were recovered 16 h after AI and cytoplasmically injected with (1) 5 ng μL−1 TALEN mix (2.5 ng μL−1 oaBLG T1.1 + 2.5 ng μL−1 oaBLG T5.1) (5TM); (2) 5 ng μL−1 TALEN mix + 25 ng μL−1 pHR-hFIX plasmid (5TM+25pRH); or (3) 15 ng μL−1 TALEN mix (7.5 ng μL−1 oaBLG T1.1 + 7.5 ng μL−1 oaBLG T5.1) + 50 ng μL−1 pHR-hFIX (15TM+50pRH). A non-injected control (NIC) was also included. Embryo analysis was conducted on whole-genome amplified DNA from blastocysts, followed by PCR and sequencing. Non-parametric Fisher test was applied to detect significant differences among treatments. Although blastocyst rates for NIC and 5TM did not statistically differ, 5TM+25pRH and 15TM+50pRH groups resulted in lower blastocysts rates than the NIC [P < 0.05; 13/17 (76%), 6/15 (40%), 4/15 (26%) and 2/14 (14%) for NIC, 5TM, 5TM+25pRH and 15TM+50pRH respectively]. It was possible to detect the PCR product compatible with deletion of the entire region among TALEN target sites in 6/6 blastocysts (100%) from the group 5TM, 3/4 blastocysts (75%) from the group 5TM+25pRH and 2/2 (100%) blastocysts from the group 15TM+50pRH. HR was detected in 1/2 (50%) blastocysts injected with 15TM+50pRH and in 1/4 (25%) blastocysts injected with 5TM+25pRH, by PCR and sequencing of the PCR products. Our results indicate that TALEN combined with homologous recombination constitutes a powerful platform for the production of proteins of pharmaceutical interest under native regulatory sequences in the milk of genetically modified animals.