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General information Notification Number B/ES/19/07 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 15/04/2019 Title of the Project Field production (under net confinement) of rice plants accumulating three microbicide components in the endosperm. Proposed period of release: 01/06/2019 to 31/10/2019 Name of the Institute(s) or Company(ies) University of Lleida
Is the same GMPt been notified elsewhere by the same notifier? No Has the same GMPt been notified elsewhere by the same notifier? No Genetically modified plant Complete name of the recipient or parental plant(s):

Common Name	Family Name	Genus	Species	Subspecies	Cultivar/breeding line
rice	poaceae	oryza	oryza sativa		EYI105 (Japonica)

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The transgenic rice line H136 accumulates three microbicide molecules in the endosperm: the antibody 2G12, and the lectins Griffitsin and Cyanovirin N. This line has the EIY105 Japonica genetic background. It contains 2 genes (heavy and light chain) that encode for the monoclonal antibody 2G12, one gene for the lectin Griffitsin (GRFT) and one for the lectin Cyanovirin N (CV-N).

This line accumulates 17.2 µg/g 2G12; 1.2 µg/g GRFT and 6.4 µg/g CV-N when compared to the wild type EYI105 (near isogenic line, control).

This line has not been designed to affect any live organism, other than binding of the HIV virus to its target receptors in human cells.

The line H136 also contains the hpt marker gene conferring resistance to the antibiotic hygromicin. Genetic modification Type of genetic modification: Insertion; In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted: Production of the 2G12 antibody and function
2G12 is a monoclonal antibody that was first isolated from humans and has been synthesized by the company Polymun (https://www.polymun.com/research-reagents/hiv-antibodies/). The antibody 2G12 recognizes the epitope gp120 and neutralizes HIV in vitro.

Production of the lectin GRIFFITSIN and function
Griffithsin (GRFT) is a lectin isolated from the red algae Griffithsia sp. found in the coastal waters off New Zealand. It is a 121 amino acid dimeric protein with a domain-swapped structure. This lectin neutralizes HIV-1 by binding to mannose-rich glycans on the envelope glycoproteins. Both native and recombinant GRFT display potent antiviral activities against laboratory-adapted strains and primary isolates of M- and Tropic HIV-1. GRFT was also shown to be active against a broad range of HIV-1 strains including 4 sub-type C viruses.

Only small amounts of this lectin can be isolated from the alga which limits the development of this molecule as a microbicide. Our group has expressed this lectin in rice endosperm and demonstrated its activity in vitro against HIV. Our transformation plasmid was named pgZ63-Griff.

Production of the lectin CYANOVIRIN N and function
The lectin Cyanovirin-N (CV-N) was isolated from the blue green algae Nostoc ellipsosporum. It is a 101 amino acid protein that has been shown to exist either as a quasi-symmetric two-domain monomer or as a domain-swapped dimmer. Like GRFT, CV-N binds to mannose-rich glycans and both native and recombinant CV-N have shown potent anti-HIV-1 activity in vitro. We also demonstrated that this molecule could be expressed in rice. Out transformation plasmid was named pRPS-CV-N.

As a result of their ability to block HIV-1 entry in vitro, GRFT and CV-N have been proposed as potential microbicides to prevent the sexual transmission of HIV-1. Although these compounds are not HIV specific, they target high-mannose arrays that are present on all HIV envelope glycoproteins. Since such arrays are very uncommon in mammalian cells, these molecules are not predicted to be toxic to human cells in vivo even at relatively high concentrations. Furthermore, a recombinant GRFT produced in the tobacco-like plant Nicotiana benthamiana was shown to be non-toxic in a rabbit vaginal irritancy model and in human cervical explants. Although GRFT and CV-N have not yet been tested in human clinical trials it is noteworthy that CV-N was shown to be effective in protecting pigtailed macaques after vaginal and rectal challenges with high dose of SHIV 86.9P.

Selectable marker
The selectable marker we used was hpt, with the following components in the transforming plasmid:
Promoter 35S Cauliflower mosaic caulimovirus (CaMV), conferring constitutive expression.
Gene: hpt from Escherichia coli – Hygromycin B. Conferring resistance to hygromycin..
Terminator: pA35S from Cauliflower mosaic caulimovirus NOS polyA terminator. Brief description of the method used for the genetic modification: Rice transformation: Seven-day-old mature rice zygotic embryos (Oryza sativa cv. EYI105) were transferred to osmotic medium 4 h before bombardment with 10 mg gold particles coated with the 2G12 heavy and light chain, GRFT and CV-N constructs and the selectable marker hpt at a 6:6:3:3:1 ratio. The embryos were returned to osmotic medium for 12 h before selection on MS medium supplemented with 50 mg/l hygromycin and 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) in the dark for 2–3 weeks. Transgenic plantlets were regenerated and hardened off in soil. Plants were grown in the growth chamber at 28/20°C day/night temperature with a 12-h photoperiod and 80% relative humidity.

Molecular analysis. Genomic DNA was isolated from rice leaves by phenol extraction and 50-ng aliquots were used for PCR with three primers designed to generate overlapping products to confirm transgene integrity. PCR reactions were carried out under standard conditions with 100 ng genomic DNA and 0.5 units from GoTaq DNA polimerasa enzyme in a total volume of 20 microliters. DNA and primers were denaturalized at 94 °C during 3 min, followed from 35 cycles reactions at 94 °C during 45 s, 60 °C during 45 s, 72 °C during 3 min, and a final extension cycle of 72 °C during 10 min.

Seeds from line H136 T0 contain: 17.2 µg/g de 2G12; 1.2 µg/g de GRFT y 6.4 µg/g de CV-N when compared to the wild type control EYI105. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Not applicable Experimental Release Purpose of the release: The main objective of this release is to grow in a field (under net confinement) transgenic rice plants that accumulated 3 microbicides molecules in their endosperm. The event to be tested has been designated H-136 (T3)

The molecules are a monoclonal antibody 2G12 and two lectins GRFT y CV-N, able to neutralize HIV.

Secondary objectives are:
a) To amplify the material for future in Vitro neutralization studies.
b) To compare the line to the control wild type (near isogenic line).
o Agronomic characteristics of the plant.
o Effects of biotic and abiotic stress on the stability of the produced molecules. Geographical location of the site: Only Lleida municipality Size of the site (m2): 10 m2 surrounded by nets. Relevant data regarding previous releases carried out with the same GM-plant, if any, specifically related to the potential environmental and human health impacts from the release: Not applicable. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: • In consecutive multiplication generations no morphological, physiological or agronomic differences were detected between transformed plants and their conventional counterparts except for the presence of the three microbicides.
• The genetic modification introduced into the rice plant consists on the incorporation of different genes involved in the synthesis of the three microbicides, in order to accumulate this microbicides in seeds. No negative effect on human health is anticipated.
• The genetic modification does not target any organism (other than the intended HIV); therefore no toxic effects on other organisms within the ecosystem are anticipated.
• As cultivated species, rice varieties have been bred to germinate in the consecutive crop cycle when the appropriate environmental conditions are present. Their survival out of the cropping system is very unlikely since they are highly adapted to crop conditions. Moreover, in the region where the field trial will be carried out, any seeds that germinate after the harvest period will not survive due to autumn and winter temperatures (rice is extremely sensitive to low temperatures), or due to lack of water. Therefore, no selective advantage of the transformed plants under natural environment is predicted or anticipated.
• There are no predicted effects on biogeochemical cycles since the microbicides will be degraded like other organic compounds. Brief description of any measures taken for the management of risks: The following safety measures will be implemented:
- The field will be placed in an urban zone where rice is not cultivated and will be surrounded by a net.
- The transportation of the biological material between the field and the lab will be done with hermetically closed plastic boxes.
- The rice seed will be collected manually and placed in sealed bags.
- The grain harvested from the transgenic rice will be manipulated, transported and packaged by qualified staff.
- After collecting the seeds, the plants will be collected, placed in plastic bugs and autoclaved. Once autoclaved they will be discarded in the biological residues container.
- Water left in the container will be collected, filtered and autoclaved before been discarded.
- Next year the field will be used to grow a different crop.
- The trial will be periodically checked in order to register any information on adverse effects on the environment and/or food safety, which will be notified to the corresponding authority.
- In the end of the trial a report will be sent to the corresponding authority. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: Not applicable Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
06/25/2019
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