(19) TZZ ZZ__T (11) EP 2 500 417 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 19.09.2012 Bulletin 2012/38 C12N 5/0735 (2010.01) A01K 67/027 (2006.01) (21) Application number: 12167371.9 (22) Date of filing: 09.08.2007 (84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • Valarche, Isabelle HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE 44000 Nantes (FR) SI SK TR • Batard, Luc Designated Extension States: 44000 Nantes (FR) AL BA HR MK RS • Mehtali, Majid 44220 Coueron (FR) (30) Priority: 09.08.2006 US 836378 P (74) Representative: Flesselles, Bruno F.G. et al (62) Document number(s) of the earlier application(s) in BF IP accordance with Art. 76 EPC: 4, rue Ribera 07802553.3 / 2 054 507 75016 Paris (FR) (71) Applicant: Vivalis Remarks: 49450 Roussay (FR) This application was filed on 09-05-2012 as a divisional application to the application mentioned under INID code 62. (54) Method of production of transgenic avian using embryonic stem cells (57) The present invention concerns a method of cul- inhibitory factor (LIF), interleukin 11 (II-11), oncostatin turing embryonic stem (ES) cells of avian, comprising the and cardiotrophin; b) seeding the suspension of ES cells steps of: a) suspending ES cells originating from the blas- obtained in step a) on a layer of feeder cells and further toderm disk of fertilized un-incubated avian egg(s) in a culturing the ES cells for at least between 2 to 10 pas- basal culture medium supplemented with: insulin-like sages; c) optionally removing at least one growth factors growth factor-1 (IGF-1) and ciliary neurotrophic factor selected SCF, FGF, II-6, II-6R, LIF, oncostatin and car- (CNTF); and animal serum; and optionally, at least one diotrophin and II-11 from the culture medium; d) further growth factors selected in the group comprising inter- culturing the ES cells in the medium of step c) on a layer leukin 6 (II-6), interleukin 6 receptor (II-6R), stem cell of feeder cells. factor (SCF), fibroblast growth factor (FGF), leukaemia EP 2 500 417 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 500 417 A1 Description [0001] The present invention relates to avian biotechnology and in particular to a method of producing transgenic birds. The invention is particularly useful to generate transgenic birds expressing high amount of a recombinant protein 5 of interest in egg. [0002] With over 400 biomolecules in clinical development and a market of over 30 billions dollars, the therapeutic field has witnessed an accelerated growth in the last fifteen years. Most recombinant proteins require specific post- translational modifications for full biological activities and must thus be produced in mammalian cells grown in large- scale bio-reactors. The cost associated with such production system, combined with important quantitative needs is 10 responsible for increasing delays in the overall process of product development. In this context, transgenic animals could represent an economically attractive alternative, especially if the modification could be transmitted to the progeny through the germ-line. [0003] While rabbits, goats and cows have elicited plenty of interest, the hen has long been considered as the most appealing biological production system for fast and cost-effective production of recombinant therapeutic proteins in 15 chicken eggs. The chicken egg-laying capabilities are indeed remarkable since commercial hen lays about 250 eggs a year, each egg containing nearly 4 grams of egg white proteins. If only 100 mg of recombinant proteins were to be produced in an egg, a small flock of 1000 hens would thus be able to produce 25 kg of raw recombinant proteins material a year. In addition, the commercial egg industry is already highly automated and regulatory agencies are comfortable with egg-derived products since many human vaccines are produced in the chicken eggs since decades. 20 [0004] Likewise, the poultry industry may also have interest in using transgenesis for accelerated selection of genetic traits of commercial interest (i.e "meta-cloning technology"). The idea would be to bypass the classical selection scheme: [Pedigree -> GGP -> GP -> PS] to get in a shorter time the desired chick. The most likely use of transgenic technology in poultry production could be to impart disease resistance which is a common practise in transgenic plants or to improve food assimilation . In addition, transgenic technology could be a strategy to conserve avian genetic resources. So far, 25 the splitting into different places of pedigree animals, the most valuable animals in poultry industry, is the only way to prevent the loss of years of selection programs in case of troubles (eg. viral infections) in a breeding facility. Such an approach is expensive and does not guarantee against national sanitary decisions enforcing the preventive elimination of all local poultry to check viral infection, as in the Netherlands in 2003. [0005] The engineering of a modified animal implies first the stable introduction of the transgene into the genome of 30 the animal. The different technologies to introduce DNA investigated since 25 years are : (i) the DNA micro-injection, (ii) the viral transduction, (iii) the sperm-mediated gene transfer, (iv) the nuclear transfer, the cell-based transfer using (v) primordial germ cells, or (vi) embryonic stem cells. In the chick, only the DNA micro-injection approach (Love et al, 1994 Biotechnology 12:60-63) and the viral transduction (Bosselman et al, 1990 J. Reprod. Fertil. Suppl. 41: 183-195) have been validated for germ line transmission of the transgene. However, these two technologies suffer from several 35 limitations; they are cumbersome and laborious and have a low efficacy, partly due to random integration and silencing of the transgene. Viral transduction technology has the additional limitation of the transgene size to be incorporated into the viral vector. These two technologies did not allow today to reach protein expression level compatible with commercial developments. [0006] Injection into the developing chick embryo of primordial germ cells (PGC) or Embryonic Stem (ES) cells, 40 previously in vitro genetically engineered are among the promising technologies of avian transgenesis especially because these technologies allow targeting transgene integration to specific sites within the genome which should allow high expression levels of the transgene. However, in order to use this approach to produce transgenic chicks, an important prerequisite must be fulfilled: cells must survive to in vitro manipulations, while still maintaining their ability to be incor- porated within a recipient embryo, to colonize the germ-line and then to transmit the modification to the progeny. 45 [0007] In the past, many attempts were made to overcome the different technical hurdles at each process steps and today, somatic and germ-line chimera had been obtained by injection of freshly isolated blastodermal cells isolated from un-incubated embryos into the sub-germinal cavity of freshly laid embryos. Donor and recipient cells contribution was assessed in (i) the melanocyte population through examination of black and white pigmentation (Barred Rocks or Brown Leghorns have a recessive allele at the I locus while the White Leghorns have a dominant allele at the I locus); (ii) the 50 erythrocyte population through analysis of DNA fingerprints; (iii) the gonads through the analysis of the progeny with a donor-derived phenotype (Petitte et al, 1990 Development 108:185-189; Carsience et al, 1993 Development 117: 669-675; Thoraval et al, 1994 Poultry Science 73:1897-1905; Pain et al, 1996 Development 122:2339-2348). Chimera with contributions to both somatic tissues and the germline were observed when blastodermal cells were injected after 48 hours (Etches et al, 1996 Mol. Reprod. Dev. 45:291-288) up to 7 days of culture (Pain et al, 1996). Etches et al, 55 1996, had demonstrated that significantly more somatic chimeras were observed following the injection of cells co- cultured with mouse fibroblasts. Pain et al (1996) seeded avian blastodermal cells on STO mouse fibroblast cell line. The ES status of cells maintained in culture relied on the expression of the ECMA-7 and SSEA-1 epitopes and the telomerase activity (Pain et al, 1996). Proliferation in the absence of differentiation of blastodermal cells was stimulated 2 EP 2 500 417 A1 by the presence of Leukemia Inhibitory Factor (LIF) and other factors, II-11, SCF, bFGF, IGF-1 and differentiation was inhibited by exposure to anti-retinoic acid monoclonal antibody (Pain et al, 1996). It had been shown that colonization of the embryo by donor-derived cells was facilitated when the recipient embryo was compromised by exposure to irradiation prior to injection of the donor cells (Carsience et al, 1993). 5 [0008] However, blastodermal cells maintained in culture yielded fewer chimeras that exhibit reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Moreover, even so it was demonstrated that each of the component parts of the cell-based avian trans- genesis strategy could be accomplished; no transgenic animal had been described that were obtained with the ES cell technology. 10 [0009] It remains a need for efficient methods of generating transgenic chickens. This is the object of the instant invention. [0010] To achieve this object and in accordance with the purpose of the invention,