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Notification report


General information

Notification Number
B/ES/11/20

Member State to which the notification was sent
Spain

Date of acknowledgement from the Member State Competent Authority
04/02/2011

Title of the Project
Industrial production of the recombinant human enzyme acid β-glucosidase (GCase) for enzyme replacement therapy of Gaucher disease.

Proposed period of release:
01/04/2011 to 30/11/2011

Name of the Institute(s) or Company(ies)
Transactiva srl, ;


3. Is the same GMPt release planned elsewhere in the Community?
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 NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
ricepoaceaeoryzaoryza sativa

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
The rice plant was engineered to produce the therapeutic protein human acid β-glucosidase in the seed endosperm. For this purpose, the pTRS_GCase vector was obtained and employed for Oryza sativa genetic transformation.
pTRS_GCase has a single T-DNA harbouring the expression cassettes for the gene of interest (rhGCase), the selectable marker (PMI), and an interfering RNA (FxantiFX). Only the first two cassettes determine the presence of additional elements in the plant proteome, being the third cassette only devoted to raise specific siRNAs, i.e. small RNA molecules, interfering with the expression of the two endogenous genes, namely α(1,3)-fucosyltransferase and β(1,2)- xylosyltransferase.


Genetic modification

3. 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:
The genetic material inserted corresponds to a single T-DNA containing three cassettes for the expression of: 1. the gene of interest (hGCase); 2.the selective agent PMI (phosphomannose isomerase); the RNAi for specific silencing of α(1,3)-fucosyltransferase and β(1,2)-xylosyltransferase genes (FxantiFX). The molecular cassettes are separated each other by short spacers. The DNA sequence integrated in the plant genome has a total length of 11634 bp.

The GCase expression cassette includes the glutelin 4 rice promoter (GluB-4), the GCase gene sequence (both in its native form and in two artificial but synonymous versions) and the NOS terminator of Agrobacterium tumefaciens.
The cassette for PMI expression is composed by the 35S CaMV promoter, the E. coli manA gene coding for the phosphomannose isomerase, and the 3’ UTR CaMV terminator. Finally, the RNAi cassette has the following elements: maize ubiquitin 1 promoter (Ubi1), sense sequence of α(1,3)-fucosyltransferase and β(1,2)-xylosyltransferase genes of O. sativa, cre intron (artificial sequence), antisense sequence of α(1,3)-fucosyltransferase and β(1,2)-xylosyltransferase genes of O. sativa and Tml terminator (artificial sequence). These elements form an hairpin which induces RNAi silencing of the aforementioned enzymes.


6. Brief description of the method used for the genetic modification:
CR W3 rice genetic transformation was performed according to Hiei et al. (1994) with little modification. In particular, the engineered expression vector, pTRS_GCase, was introduced into EHA105 strain of Agrobacterium tumefaciens by electroporation. During and after shoot differentiation, the selection based on PMI/mannose system was performed according to Datta and Datta (2006).

7. 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

1. Purpose of the release:
The aim of the genetic modification is the conversion of rice plants into cell factories for the production of human acid β-glucosidase. The type and the function of the inserted exogenous sequences are consistent with an application of molecular farming, i.e. the use of plants for the production of novel compounds rather than for the production of food or livestock feed. For this reason, the project does not impact the agrosystem and the food industry.
So far, GCase rice has been grown and evalutated in growth chambers only.
The purpose of the release is the scale-up of grain production for technological platform completion and drug development. The availability of further GCase amounts is essential to allow a pharmaceutical company to perform the biotechnological, preclinical and clinical development activities required to obtain the Marketing Authorisation for the enzyme as replacement therapy for the Gaucher Disease.


2. Geographical location of the site:
The field planting will be located in the municipality of Vinaròs, province of Castellón. The plot of land is part of a farm of 115.805 m2, which is cultivated mainly with citrus fruits and where the owner has his residence. The proposed site of release is as far as 133 km and 22 km from the nearest rice fields of Albufera de Valencia and of the Ebro Delta, respectively. The whole farm is fenced and totally isolated.

3. Size of the site (m2):
The size of the release plot will be 2.000 square meters.

4. 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:
A review of the reproductive and vegetative fitness showed that GCase rice compares to its parental variety CR W3 in all aspects except GCase production in seed endosperm. The GCase protein does not influence the reproductive or vegetative fitness in rice, therefore the likelihood of the GMHP becoming more persistent than the recipient rice is negligible. The GCase protein does not confer a selective advantage to rice. Potential impact is the same as for other commercial rice varieties. GCase rice is not a “pesticide plant” and there is no “target” organism. There has been no intentional genetic change in the GM plants to affect their susceptibility to disease or insect damage and there is no reason to believe that these or similar characteristics are different between genetically engineered and non-engineered plants. Since red rice seeds often have dormancy and shatter easily, the gene could be harboured in a weedy population for a number of years. Nevertheless GM rice will be grown in a district, which is not a rice productive area. No impact on existing agricultural practices is expected. Agricultural practices consistent with healthy, certified seed production will be applied.

Brief description of any measures taken for the management of risks:
In order to prevent GM rice dispersion in the environment during growing, manufacturing and transport, specific measures will be adopted. The field trial will be conducted in a privately owned property. Seed sowing in rows and at low density will spare seeds and reduce dissemination. During the crop, the plot will be covered with an anti-hail net. The surrounding 400-m area will be monitored for the presence of red rice and rice volunteers. Flood irrigation will not be used. Red/weedy rice populations within the field test site will be immediately removed in order to preclude movement of transgenes to red rice via cross pollination. To minimize the risk of seed movement, equipment used for planting and harvesting, as well as other field equipment (e.g. tractors and tillage attachments, such as disks, plows, harrows, and subsoilers) used at any time, from the time of planting through the post-harvest monitoring period, will be carefully cleaned before they are moved off the test site. The field will be managed to allow detection and destruction of volunteer plants or sexually compatible plants with transgenic rice in the 400-m surrounding area. In the subsequent growing season, the production site and the 400-m surrounding zone will not be planted with rice unless transgenic rice is repeated. If the same crop does not follow in subsequent years, the site will be monitored for volunteer rice plants throughout the next 2 years. Any volunteer rice plants will be destroyed before flowering.

Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
Not applicable.

Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Not known