SUMMARY NOTIFICATION INFORMATION FORMAT FOR THE … · Treatment of X-linked Chronic Granulomatous Disease (CGD) Clinical trial code: G1XCGD.02 . EudraCT number: 2014‐002222‐12

Genethon SNIF- Ex-vivo Gene therapy of X-CGD with a Lentiviral Vector February 2015

G1XCGD.02 clinical trial Page 1 of 26

SUMMARY NOTIFICATION INFORMATION FORMAT FOR THE RELEASE OF GENETICALLY MODIFIED ORGANISMS

OTHER THAN HIGHER PLANTS IN ACCORDANCE WITH ARTICLE 11 OF DIRECTIVE 2001/18/EC

Genetically-modified Investigational Medicinal Product (IMP): Autologous CD34+ cells transduced ex vivo with the G1XCGD lentiviral vector containing the codon-optimized cDNA of the human GP91-PHOX gene (CYBB) for the Treatment of X-linked Chronic Granulomatous Disease (CGD)

Clinical trial code: G1XCGD.02

EudraCT number: 2014‐002222‐12

Sponsor: GENETHON

1 bis rue de l’Internationale 91000 Evry France



Table of content

A. General information ........................................................................................................ 3

B. Information relating to the recipient or parental organism from which the GMO is derived .................................................................................................................................... 6

C. Information relating to the genetic modification ......................................................... 11

D. Information on the organism(s) from which the insert is derived ............................ 14

E. Information relating to the genetically modified organism .......................................... 16

F. Information relating to the release ............................................................................... 18

G. Interactions of the GMO with the environment and potential impact on the environment, if significantly different from the recipient or parent organism ................... 21

H. Information relating to monitoring ........................................................................... 23

I. Information on post-release and waste treatment ...................................................... 25

J. Information on emergency response plans .................................................................. 26



A. General information

1. Details of notification

Member State of notification France Notification number B/FR/15/GT05Date of acknowledgement of notification 09/04/2015Title of the project A PHASE I/II, NON RANDOMIZED, MONOCENTRIC OPEN-LABEL STUDY OF AUTOLOGOUS CD34+ CELLS TRANSDUCED WITH THE G1XCGD LENTIVIRAL VECTOR IN PATIENTS WITH X-LINKED CHRONIC GRANULOMATOUS DISEASE Proposed period of release From 2015 until Q4 2019 (date of study completion).

2. Notifier

Name of institution or company: GENETHON (sponsor) 1 bis rue de l’Internationale- BP60 91002 Evry Cedex France

3. GMO characterisation

(a) Indicate whether the GMO is a: viroid (.) RNA virus () DNA virus (.) bacterium (.) fungus (.) animal mammals (X) (Genetically-modified Autologous CD34+ cells) insect (.) fish (.) other animal (.) Specify, phylum, class … (b) Identity of the GMO (genus and species) Genus: Human Species: Homo Sapiens Sapiens



The GMO/IMP consists of patient-autologous CD34+ cells transduced ex vivo with the G1XCGD lentiviral vector containing the codon-optimized cDNA of the human CYBB gene for the treatment of X-linked Chronic Granulomatous Disease (CGD). The genetically-modified Investigational Medicinal Product (IMP) is intended to restore the NADPH-oxidase function in granulocytes and other phagocytes after hematopoietic reconstitution. The cells will be used only for therapeutic purposes in the same patient from whom the cells were obtained (autologous application). (c) Genetic stability – according to Annex IIIa, II, A(10) A recombinant lentiviral vector (LV) derived from HIV-1 has been chosen to exploit some advantageous properties of lentiviruses: the resulting LV are genetically stable, permanently integrate into the genome of transduced cells and provide long-term gene expression in vitro and in vivo. The transduction of Hematopoietic Stem Cells (HSC) with such LV can be achieved after limited preactivation of the cells in short-term cultures with cytokines, in conditions that are compatible with the preservation of the self-renewing properties of these cells. These properties make these LV suitable for ex-vivo gene therapy strategies using HSC. The identity and sequencing of G1XCGD viral batches produced is tested and verified against a reference as part of release testing of the viral vector. Tests are performed to verify the identity and integrity of lentiviral vector and transduced patients’ cells. The genetic stability of the final Investigational Medicinal Product (i.e. autologous transduced CD34+ cells) is also assessed in order to:

- confirm the presence of the vector integration (assessment of vector copy number), - confirm the absence of Replication Competent Lentiviruses (RCL), and - determine the vector’s genomic integration profile.

4. Is the same GMO release planned elsewhere in the Community (in conformity with Article 6(1)), by the same notifier?

Yes (X) No (.) If yes, insert the country code(s) DE, GB

5. Has the same GMO been notified for release elsewhere in the Community by the same notifier?

Yes (.) No (X) If yes: - Member State of notification … - Notification number B/../../… Please use the following country codes: Austria AT; Belgium BE; Germany DE; Denmark DK; Spain ES; Finland FI; France FR; United Kingdom GB; Greece GR; Ireland IE; Iceland IS; Italy IT; Luxembourg LU; Netherlands NL; Norway NO; Portugal PT; Sweden SE



6. Has the same GMO been notified for release or placing on the market outside the Community by the same or other notifier?

Yes (.) No (X) If yes: - Member State of notification - Notification number B/../../…

7. Summary of the potential environmental impact of the release of the GMOs.

The GMO consists of genetically-modified CD34+ cells and are intended for a single application. These cells are used as a source of progenitor cells and in particular of hematopoietic stem cells (HSC) that will ensure a durable hematopoietic reconstitution after administration. Upon intravenous infusion, genetically-modified CD34+ cells will behave as any CD34+ cell: they will mainly migrate to lymphoid organs and to the bone marrow and will therefore not spread from the patient to another person or to the environment. In the case the cells would be exposed to the environment e.g. accidentally released from their container, they would not be viable, thereby destroying the vector sequences. In addition, due to the multiply deleted nature of the vector and the Self-Inactivating (SIN) configuration, the generation of RCL is highly unlikely. Lentiviral supernatant and patient samples will nevertheless be tested for the presence of RCL throughout the study. Particular attention will be paid on the first blood sample collected after IMP infusion given that patients’ blood samples should be negative for RCL prior to discharge the patient from the hospital. Patient blood samples will be banked at 3, 6, 12 and 24 months post gene therapy to allow a retrospective analysis of RCL in the case of a severe adverse event occurrence. If in the unlikely event that post-treatment samples are positive, further analysis will be undertaken to characterize the RCL. Also an excretion of live product or its progeny (“shedding”) by the patient is not possible. Vector sequences are unlikely to be mobilized.



B. Information relating to the recipient or parental organism from which the GMO is derived

The GMO is derived from the peripheral blood of humans. The recipient organism is the patient’s autologous CD34+ hematopoietic stem cells. The CD34+ cells are to be transduced ex vivo with the G1XCGD lentiviral vector encoding the codon-optimized cDNA for the human CYBB gene. The transduced CD34+ cells constitute the final GMO to be administered by intravenous infusion into X-linked CGD patients conditioned by a cyto-reductive chemotherapy treatment.

1. Recipient or parental organism characterisation:

The parental organism is the Human Immunodeficiency Virus (HIV), a replication‐competent Human virus. The recipient organisms are autologous CD34+ cells transduced ex-vivo with the G1XCGD lentiviral vector. Indicate whether the recipient or parental organism is a: (select one only) viroid (.) RNA virus (X) DNA virus (.) bacterium (.) fungus (.) animal mammals (.) insect (.) fish (.) other animal (.) (specify phylum, class) … other, specify

2. Name

order and/or higher taxon (for animals) Retroviridae genus Lentivirus species Human immunodeficiency virus (HIV) subspecies HIV-1 strain … pathovar (biotype, ecotype, race, etc.) … common name Lentiviral vectors



3. Geographical distribution of the organism

(a) Indigenous to, or otherwise established in, the country where the notification

is made: Yes (.) No (X) Not known (.)

(b) Indigenous to, or otherwise established in, other EC countries: Yes (.) No (X) (i) If yes, indicate the type of ecosystem in which it is found: Atlantic .. Mediteranean .. Boreal .. Alpine .. Continental .. Macaronesian .. (ii) No (.) (iii) Not known (.) (c) Is it frequently used in the country where the notification is made? Yes (.) No (X)

(d) Is it frequently kept in the country where the notification is made? Yes (.) No (X)

4. Natural habitat of the organism

Parental organism HIV “natural habitat” is the human being. Virus particles can be found in body fluids such as blood and sperm. HIV can therefore be transmitted during sexual contact, direct blood contact (sheering needles, blood transfusions) or from mother to child (before or during birth or through breast milk). It is unable to infect other host than Human. It is not naturally found in the environment. Recipient organism Human CD34+ cells are mainly found in the bone marrow and peripheral blood. After ex-vivo transduction with the G1XCGD vector and intravenous infusion to the patients, the cells are expected to home back to the bone marrow and reconstitute the blood and immune system.



(a) If the organism is a microorganism

Not applicable

water (.) soil, free-living (.) soil in association with plant-root systems (.) in association with plant leaf/stem systems (.) other, specify

(b) If the organism is an animal: natural habitat or usual agroecosystem:

Not applicable

5. Detection and identification techniques

(a) Detection techniques

Parental organism HIV-1 can be detected in human blood samples by mainly 2 methods:

- Enzyme-Linked Immunosorbent Assay (ELISA) quantifying the presence of anti-HIV-01 antibodies in the serum (indirect detection of the organism), and

- Polymerase Chain Reaction (PCR) or nucleic acid testing (NAT) used to detect HIV‐1 proviral DNA (integrated in white blood cells) after its amplification.

Recipient organism A sample of the active substance (CD34+ cells transduced ex vivo with the 1XCGD lentiviral vector) is collected at the end of the manufacturing process in order to perform a number of Quality Controls (QC). In particular, a transduction efficiency analysis is conducted by measuring the vector copy number integrated, by q-PCR.

(b) Identification techniques q-PCR using primers specific to some sequences included in the G1XCDG viral vector.

6. Is the recipient organism classified under existing Community rules relating to the protection of human health and/or the environment?

Yes (.) No (X) If yes, specify …

7. Is the recipient organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead?

Yes (.) No (X) Not known (.)



If yes: (a) to which of the following organisms: humans (.) animals (.) plants (.) other (.) (b) give the relevant information specified under Annex III A, point II. (A)(11)(d) of

Directive 2001/18/EC

8. Information concerning reproduction

After administration to the patients, genetically-modified CD34+ cells will proliferate via mitosis as any non-transduced CD34+ would do.

(a) Generation time in natural ecosystems: … (b) Generation time in the ecosystem where the release will take place: …

(c) Way of reproduction: Sexual .. Asexual .. Factors affecting reproduction: …

9. Survivability

(a) ability to form structures enhancing survival or dormancy: endospores (.) cysts (.) sclerotia (.) asexual spores (fungi) (.) sexual spores (funghi) (.) eggs (.) pupae (.) larvae (.) other, specify …

(b) relevant factors affecting survivability: It is not possible for CD34+ cells to survive in environment.

10. Dissemination

(a) Ways of dissemination The genetically-modified CD34+ cells are not able to survive, disseminate in and/or displace other organisms.



(b) Factors affecting dissemination The factor that could lead to the dissemination of lentiviruses is the emergence of RCL. This risk has been minimized with modification made on the vector construct and the use of 4 plasmids during the manufacturing process. The absence of RCL is nevertheless assessed:

- At the end of the manufacturing process of the G1XCGD vector, - At the end of the manufacturing process of the IMP (transduced CD34+ cells), and - On patients’ blood samples throughout the clinical trial.

11. Previous genetic modifications of the recipient or parental organism already notified for release in the country where the notification is made (give notification numbers)

Not applicable



C. Information relating to the genetic modification

1. Type of the genetic modification

insertion of genetic material (X) deletion of genetic material (.) base substitution (.) cell fusion (.) others, specify …

2. Intended outcome of the genetic modification

Insertion of the GP91 transgene in CD34+ hematopoietic cells’ DNA. After hematopoietic reconstitution, the expected result is the expression of the wild-type gp91-phox protein in myeloid cells and in particular in neutrophil granulocytes.

3.

(a) Has a vector been used in the process of modification? Yes (X) No (.) If no, go straight to question 5. (b) If yes, is the vector wholly or partially present in the modified organism? Yes (X) No (.) If no, go straight to question 5.

4. If the answer to 3(b) is yes, supply the following information

(a) Type of vector plasmid (.) bacteriophage (.) virus (X)) cosmid (.) transposable element (.) other, specify (b) Identity of the vector G1XCGD lentiviral vector as described in section A), 3. (c) Host range of the vector Patient’s autologous CD34+ cells.



(d) Presence in the vector of sequences giving a selectable or identifiable phenotype

Yes () No (X) antibiotic resistance (.) Kanamycin resistance gene only in production

plasmid, not in the GMO administered other, specify Human GP91 Transgene Indication of which antibiotic resistance gene is inserted The proviral insert that will become integrated into the host cell genome does not contain any antibiotic resistance gene. The plasmids used for the preparation of the G1XCGD vector particles contain the kanamycin resistance gene selectable marker (aminoglycoside 3’phosphotransferase) which confers resistance to kanamycin. The choice of this selection marker was made to facilitate the production of the plasmids while minimizing the risk of transferring bacterial resistance since these antibiotics are no longer used in patients. (e) Constituent fragments of the vector The proviral cassette integrated into the CD34+ cells’ genome is schematized below: Figure 1: Schematic Representation of the Integrated Cassette

Provirus G1XCGD

5487 bp

GP91cPPTcts

HIV1 psi W PRE* (PRE4)

deltaU3

deltaU3

HIV1 R

HIV1 RRE

HIV1-polyA

HIV1 PBS

Chimeric promoter

HIV U5 HIV1 R

HIV U5

HIV1: human immunodeficiency virus type 1 Psi: Encapsidation sequence PBS: primer binding site sequence LTR: Long Terminal Repeat 5’ or 3 HIV R: HIV-1 LTR, R region HIV delta U3: U3 region of the HIV-1 LTR, in which a 400 bp deletion was introduced RRE: Rev responsive element cPPT cts: central polypurine tract – central terminal sequence Chimeric promoter: heterologous sequence composed of c-fes and Cathepsin G 5’ minimal flanking regions GP91: codon-optimized cDNA sequence of the CYBB gene translated into the human cytochrome b-245, beta polypeptide also known as GP91-PHOX gene protein product. PRE4: mutated woodchuck hepatitis virus posttranscriptional regulatory element HIV1 polyA: polyadenylated region HIV1 U5: HIV1 LTR U5 region



(f) Method for introducing the vector into the recipient organism transformation (.) electroporation (.) macroinjection (.) microinjection (.) infection (.) other, specify ex vivo transduction into CD34+ autologous hematopoietic stem cells.

5. If the answer to question B.3(a) and (b) is no, what was the method used in the process of modification?

transformation (.) microinjection (.) microencapsulation (.) macroinjection (.) other, specify …

6. Composition of the insert

(a) Composition of the insert G1XCGD provirus includes a chimeric promoter designed to regulate the transgene expression in myeloid cells and a transgene called GP91 which is a codon-optimized CYBB (GP91-PHOX gene) cDNA. (b) Source of each constituent part of the insert Human (c) Intended function of each constituent part of the insert in the GMO Transgene: codon-optimized sequence of the human CYBB gene. The intended function is to produce a wild-type gp91-phox protein (cytochrome b-245 heavy chain) upon expression of the transgene in myeloid cells. Promoter: regulation of the transgene expression. The woodchuck hepatitis post regulatory element (WPRE): enhances transgene expression. A mutated version has been used that lacks the viral X protein open-reading frame.

(d) Location of the insert in the host organism - on a free plasmid (.) - integrated in the chromosome (X) - other, specify …

(e) Does the insert contain parts whose product or function are not known? Yes (.) No (X) If yes, specify …



D. Information on the organism(s) from which the insert is derived

1. Indicate whether it is a:

viroid (.)

RNA virus (.) DNA virus (.) bacterium (.) fungus (.) animal

mammals (.) insect (.) fish (.) other animal (.) (specify phylum, class) …

other, specify Homo sapiens sapiens The donor organism is the self-inactivating G1XCGD lentiviral vector containing the codon-optimized cDNA of the human CYBB gene. This is a non-replicative lentiviral vector.

2. Complete name

order and/or higher taxon (for animals) … (ii) family name for plants … (iii) genus … (iv) species … (v) subspecies … (vi) strain … (vii) cultivar/breeding line … (viii) pathovar … (ix) common name … Please see answer above. The viral genome of HIV-1 comprises genes and non-coding cis-acting gene regulatory sequences which map outside of the viral coding sequences. This segregation has been exploited to generate non-replicative recombinant viral vectors derived from HIV-1.

3. Is the organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead?

Yes (.) No (X) Not known (.) If yes, specify the following: (a) to which of the following organisms: humans (.) animals (.) plants (.) other ..



(b) are the donated sequences involved in any way to the pathogenic or harmful properties of the organism

Yes (.) No (X) Not known (.) If yes, give the relevant information under Annex III A, point II(A)(11)(d): …

4. Is the donor organism classified under existing Community rules relating to the protection of human health and the environment, such as Directive 90/679/EEC on the protection of workers from risks to exposure to biological agents at work?

Yes (X) No (.) If yes, specify The GMO G1XCGD lentiviral vector used in this project is classified as group 2, class 2 and containment L2 (Biosafety Level). (See description of donor organism in section D.1). Genethon has obtained an agreement from the French research minister to prepare recombinant lentiviral vectors. The insert has been classified as type A (without potential danger) by the French national advisory committee for GMOs (“Haut Conseil des Biotechnologies”) and the French Ministry of Research. The gene therapy for X-CGD is based on the ex vivo infection of patient autologous CD34+ hematopoietic stem/progenitor cells with the G1XCGD vector. The washed cells are reinfused after 3-4 days of culture. Human CD34+ cells infected with the G1XCGD vector for gene therapy are classified as class 1 risk. The containment L1 measures are applicable.

5. Do the donor and recipient organism exchange genetic material naturally?

Yes (.) No (X) Not known (.)



E. Information relating to the genetically modified organism

1. Genetic traits and phenotypic characteristics of the recipient or parental organism which have been changed as a result of the genetic modification

Is the GMO different from the recipient as far as survivability is concerned? Yes (.) No (X) Not known (.) Specify : The GMO consists of genetically modified CD34+ cells. No influence on cell survival is expected from the vector or the insert contained in the vector. The only phenotypic modification is the expression of the transgene by the corrected cells. Is the GMO in any way different from the recipient as far as mode and/or rate of

reproduction is concerned? Yes (.) No (X) Unknown (.) Specify : The GMO consists of genetically modified CD34+ cells. No influence on reproduction is expected from the vector or the insert contained in the vector. Is the GMO in any way different from the recipient as far as dissemination is

concerned? Yes (.) No (X) Not known (.) Specify : The GMO consists of genetically modified CD34+ cells. No influence on dissemination is expected from the vector or the insert contained in the vector. Is the GMO in any way different from the recipient as far as pathogenicity is

concerned? Yes (.) No (X) Not known (.) Specify : The GMO consists of genetic modified CD34+ cells. The genetic modification of hematopoietic stem cells with this vector will not result in any changes in the pathogenicity of the genetically modified cells for the environment, as determined by the experimental assays currently available.

2. Genetic stability of the genetically modified organism

A non-replicative and integrative lentiviral vector is used to deliver the correct CYBB gene into the patient’s CD34+ cells. Lentiviral vectors derived from HIV provide stable long-term gene expression in vitro and in vivo. These vectors are genetically stable and have not yet shown evidence of pathological consequences ascribed to the vector. So, G1XCGD lentiviral vector is stable and does not have capacity for replication. Moreover, the transduced cells are immediately delivered to the patient following batch release.



3. Is the GMO significantly pathogenic or harmful in any way (including its extracellular products), either living or dead?

Yes (.) No (X) Unknown (.) (a) to which of the following organisms? humans (.) animals (.) plants (.) other … (b) give the relevant information specified under Annex III A, point II(A)(11)(d) and

II(C)(2)(i) Genetically modified CD34+ cells can survive ex-vivo only under special cell culture conditions in a CO2 incubator at 37°C. Outside of the incubator the GMOs are not viable. Thus the environmental risk conferred by inappropriate disposal of waste or unused product or the accidental dissemination during product handling is considered to be negligible. Also an excretion of live product or its progeny (“shedding”) by the patient is unlikely for several reasons: (a) the vector has a SIN configuration limiting the possibility for replication (b) CD34+ cells are transduced ex vivo with infectious particles. (c) the cultured cells are extensively washed before reinfusion into the patient therefore limiting passive administration of infectious particles. The risk of passive transfer of vector sequences such as VSVg to the host is very low based on QC tests of the G1XCGD vector showing undetectable transfer of viral sequences to a permissive indicator cell line following infection with the vector. Donation of blood or an organ is prohibited for X‐linked CGD patients after gene therapy.

4. Description of identification and detection methods

(a) Techniques used to detect the GMO in the environment The final GMO is not released to the environment, and is not stable under uncontrolled environmental conditions. It is infused into the patient from whom the autologous cells were originally obtained.

(b) Techniques used to identify the GMO Functionality tests on the vector take place after transduction. They include qualitative and quantitative expression of the transgene. To identify proviral genomes in transduced cells the genomic DNA of patient´s cells is extracted and submitted to several PCR amplifications using oligonucleotide sets specific of the proviral vector sequence. Confirmation of the vector sequence identity is given according to the fidelity of amplicon sizes. A specific quantitative PCR method for detection of gene modified CD34+ cells and their progeny is established. Primers used in the qPCR are specific for the cDNA encoded by the G1XCGD vector. For the safety, replication competent lentiviruses testing (Method: RCL Assay (P24 decrease and absence of integrated wild-type HIV sequences following transduction and amplification of indicator cells (C8166))



F. Information relating to the release

1. Purpose of the release (including any significant potential environmental benefits that may be expected)

The purpose of the release is a therapeutic application of the GMO for patients suffering from X-linked CGD in the context of a gene therapy clinical application by reinfusing their autologous genetically modified T-CD34+ cells. The medicinal product is patient-specific and corresponds to autologous CD34+ cells transduced ex vivo with the G1XCGD lentiviral vector containing the human CYBB gene (coding for GP91-PHOX protein) in final formulation and container closure system, ready for intended medical use. The cells are expected to engraft durably and to generate the various types of blood cells that will express the correct non-mutated GP91-PHOX protein over long periods of time. The manufacturing of G1XCGD lentiviral vector, transduction of CD34+ cells by G1XCGD lentiviral vector and infusion of the IMP containing the GMO are done in restricted areas only with specific measures to avoid dissemination, under containment “L2”. The release, e.g. infusion of the IMP containing the GMO in X-linked CGD patients, will take place at the clinical site under the responsabilty of the Principal Investigator at Hôpital Necker enfants-malades Département d’Immuno-hémato-rhumatologie pédiatrique, 149 rue de Sèvres 75015 Paris. The deliberate release consists of a single infusion in a volume of 50-100 mL intravenously over 30-45 minutes. So, the final GMO is not released in the environment but it is infused to patient enrolled in a clinical trial.

2. Is the site of the release different from the natural habitat or from the ecosystem in which the recipient or parental organism is regularly used, kept or found?

Yes (.) No (X) If yes, specify The final GMO is not released in the environment; it is released under highly controlled conditions, in a limited number of patients (up to 5 patients). The final GMO is not viable outside the body of the specific recipient. It would be destroyed in any other recipient.

3. Information concerning the release and the surrounding area

(a) Geographical location (administrative region and where appropriate grid

reference): The final GMO is not released in the environment but infused to a patient in a restricted controlled area (isolation patient’s room) at the bone marrow transplantation unit in the following hospital: Hopital Necker- Enfants malades Département d’Immuno-hémato-rhumatologie pédiatrique Bâtiment Hamburger 149 rue de Sèvres 75015 Paris (b) Size of the site (m2): The hospital room has a size of less than 30 m2.



(i) actual release site (m2): … m2 (ii) wider release site (m2): … m2

Proximity to internationally recognised biotopes or protected areas (including drinking water

reservoirs), which could be affected: Not applicable Flora and fauna including crops, livestock and migratory species which may potentially

interact with the GMO Not applicable

4. Method and amount of release

(a) Quantities of GMOs to be released: The GMO will be infused as a single injection in a volume of 50-100 mL intravenously (at least 3.106 viable transduced CD34+ cells/kg). (b) Duration of the operation: The final GMO infusion lasts 30 to 45 minutes. (c) Methods and procedures to avoid and/or minimise the spread of the GMOs beyond

the site of the release The final GMO and G1XCGD lentiviral vector are not released in the environment. The final GMO is administered intravenously into the patient under standard controlled conditions for hematopoietic stem cell transplant at the clinical site. All waste is destroyed according to hospital bio-hazard disposal procedures, so called DASRI procedure. The GMO will be prepared at the “Laboratoire de Thérapie Génique et Cellulaire, Département Biothérapie”, Hospital Necker and transported to “Département d’Immuno-hémato-rhumatologie pédiatrique” by a dedicated carrier in a sealed S1 container. The actual release will occur within a closed area with restricted personnel access. The staff involved in the release of the GMO has wide experience in stem cell transplantation and will follow Good Clinical Practice rules as in any clinical trial. The GMO is only intended for clinical use and will be administrated immediately after preparation by experienced personnel. Personnel responsible for the administration of the GMO will wear disposable gloves and masks. Standard rules for spraying, wiping, gowning up and entry of personnel/goods into the room are followed. Remaining waste will be transported in sealed containers labelled with appropriate stickers to be inactivated.

5. Short description of average environmental conditions (weather, temperature, etc.)

Not applicable (Hospital environment)



6. Relevant data regarding previous releases carried out with the same GMO, if any, specially related to the potential environmental and human health impacts from the release.

A clinical trial with the same GMO is currently ongoing in the UK, Germany and Switzerland. Similar GMOs generated after transduction of CD34+ cells with a similar vector backbone but a different transgene cassette of have been used for the treatment of patients with severe immunodeficiency Wiskott-Aldrich Sydroma (WAS) or neurodegenerative conditions such as adrenoleukodystrophy or metachromatic leukodystrophy. No adverse effects for the environment or third parties have been described. Moreover the GMO cannot survive outside the patient or without special culture conditions.



G. Interactions of the GMO with the environment and potential impact on the environment, if significantly different from the recipient or parent organism

1. Name of target organism (if applicable)

(i) order and/or higher taxon (for animals) Primates (ii) family name for plants Hominidae (iii) genus Homo (iv) species Homo sapiens (v) subspecies … (vi) strain … (vii) cultivar/breeding line … (viii) pathovar … (ix) common name …

2. Anticipated mechanism and result of interaction between the released GMOs and the target organism (if applicable)

The GMO was obtained from the target organism (human patient) and after genetic modification will be reintroduced into the same target organism. The purpose of this is to provide a therapeutic advantage to patients suffering from CGD. The GMO will reconstitute hematopoiesis in the patients after homing and engrafting in the bone marrow of patients. The GMO will then contribute to the generation of all hematopoietic lineages including granulocytes which now will have the ability to protect the patients from severe and life-threatening infections. Thus, the reconstitution of function in cells derived from the GMO should reconstitute immune responses towards infections in treated patients.

3. Any other potentially significant interactions with other organisms in the environment

None

4. Is post-release selection such as increased competitiveness, increased invasiveness for the GMO likely to occur?

Yes (.) No (X) Not known (.) Give details The GMO consists of genetically modified CD34+ cell. No increased competitiveness or increased invasiveness is expected from the vector, the genetic modification or the insert contained in the vector

5. Types of ecosystems to which the GMO could be disseminated from the site of release and in which it could become established

None



6. Complete name of non-target organisms which (taking into account the nature of the receiving environment) may be unintentionally significantly harmed by the release of the GMO

order and/or higher taxon (for animals) … (ii) family name for plants … (iii) genus … (iv) species … (v) subspecies … (vi) strain … (vii) cultivar/breeding line … (viii) pathovar … (ix) common name … None. This section is not applicable.

7. Likelihood of genetic exchange in vivo

(a) from the GMO to other organisms in the release ecosystem: None

(b) from other organisms to the GMO: None

(c) likely consequences of gene transfer:

None

8. Give references to relevant results (if available) from studies of the behaviour and characteristics of the GMO and its ecological impact carried out in stimulated natural environments (e.g. microcosms, etc.):

Not applicable

9. Possible environmentally significant interactions with biogeochemical processes (if different from the recipient or parental organism)

Not applicable



H. Information relating to monitoring

1. Methods for monitoring the GMOs

The GMO will be infused to patients suffering from X-CGD. No other application is intended. Monitoring of the direct and indirect effects of the GMO in patients will be achieved using the following clinical assessments at regular intervals as defined in the treatment protocol: physical examinations, adverse event reporting, clinical laboratory assessments throughout the clinical study for all patients.

Essentially nucleic acid amplification by PCR and immunological detections (flow cytometry) will be used to detect the presence of the GMO or its progeny in the patient organism. The follow-up of the patient is designed to detect problems and to adapt the control measures of such situation, should it arise.

A fraction of cells after transduction will be retained for replication competent lentivirus (RCL) analysis.

Peripheral blood will be tested regularly for the presence of HIV gene expression. Peripheral blood cells will also be stored at 3, 6, and 12 months post gene therapy, and thereafter annually for retrospective analysis if necessary.

2. Methods for monitoring ecosystem effects

Not applicable. The final GMO and G1XCGD lentiviral vector are not released into the environment. As there is no risk for the ecosystem outside the patient’s body there is no need to assess for ecosystem effects.

3. Methods for detecting transfer of the donated genetic material from the GMO to other organisms

Not applicable. The final GMO i.e. genetically-modified cells are not able to donate their genetic material to any other organism. The GMO was modified with a replication defective lentiviral vector. No shedding of vector particles is expected from this GMO. Therefore the probability for a transfer of the donated genetic material from gene modified mammalian cells to other organisms is extremely unlikely.

4. Size of the monitoring area (m2)

Not applicable

5. Duration of the monitoring

Patient will be monitored 24 months after infusion of the GMO.



6. Frequency of the monitoring

Patients will be monitored up to two years Notably the RCL testing will be performed at 3, 6, 12, 24 months after infusion, then is performed on a yearly basis.



I. Information on post-release and waste treatment

1. Post-release treatment of the site

The site of the GMO administration will be cleaned according to standard cleaning methods for handling of biological hazard materials and according to the directives ruling the cleaning and decontamination of isolation rooms at Hôpital Necker enfants-malades Département d’Immuno-hémato-rhumatologie pédiatrique.

2. Post-release treatment of the GMOs

All waste, as well as any material that came into contact with the GMO will be destroyed according to the hospital facility bio-hazard disposal procedures, so called DASRI procedure. The waste is to be deactivated by autoclaving or utilisation of a disinfectant prior to incineration.

3. Waste

(a) Type and amount of waste generated At the site of the GMO administration to the patient, waste is generated such as: bags, tubings, gloves, paper towels, needles, syringes, cotton balls, dry adhesives, and disposable garments. Sharps (needles etc.) will be stored in different specific containers appropriately labelled. The estimated total amount of waste is expected to be minimal (less than 1000 ml). (b) Treatment of waste GMO containing waste is collected in special containers labeled as “S1 biological waste”. Waste will be collected and transported by trained staff. The transportation inside the hospital and to the final inactivation site will be performed in sealed containers identified with appropriate stickers using specific circuits. All waste will be destroyed by incineration according to Necker’s Hospital procedures.



J. Information on emergency response plans

1. Methods and procedures for controlling the dissemination of the GMO(s) in case of unexpected spread

The GMO consists of gene modified CD34+ cells. The GMO is not able to survive outside of the human body and does not release replication competent viruses. Moreover, the GMO will be released in an isolation ward at the bone marrow transplantation unit of the Necker Hospital. Thus any accidental spread will be limited to this space (30 m2) or the personnel involved in the production or administration of the GMO. Therefore an unexpected spread of the GMO is highly unlikely and the risk of dissemination is very limited. Nevertheless decontamination of the spread site will be done as estimated in the standard procedures ruling the cleaning and decontamination of isolation rooms at the Necker’s Hospital. And all of the people involved in the clinical trial will be trained about the procedures and measures to be taken in case of accidental release. Accidental stick injury with a needle contaminated with the GMO will not lead to a spread of the GMO as the immune system of the affected person will recognize the GMO as stranger component.

2. Methods for removal of the GMO(s) of the areas potentially affected

The site will be cleaned with a decontaminating solution according to the directives ruling the cleaning and decontamination of isolation rooms at Necker’s Hospital.

3. Methods for disposal or sanitation of plants, animals, soils, etc. that could be exposed during or after the spread

Not applicable

4. Plans for protecting human health and the environment in the event of an undesirable effect

The only undesirable effect the GMO may cause will affect only the patient to whom the GMO was administrated. The GMO contains a replication incompetent vector. Patients treated with the GMO will be monitored regularly. Depending on the results, decisions will be taken by the investigator responsible of the trial in order to protect the health of the patient. Moreover, the application will take place in the isolation ward for bone marrow transplantation. Here only experienced and specifically trained personnel are employed. Also regular meetings between physicians and caretakers for discussion of organizational issues and training are held. Within these conferences the personnel will be trained about the GMO and specific issues concerning patients treated with gene-modified cells. In brief, staff handling the drug product will wear area-specific clothing (for the BMT-ward), gloves and a surgical mask. Standard rules for spraying, wiping, gowning up and entry of personnel/goods into the room are followed. Due to the administration modalities the risk of accidental release is considered negligible.

Documents

SUMMARY NOTIFICATION INFORMATION FORMAT FOR THE … · Treatment of X-linked Chronic Granulomatous Disease (CGD) Clinical trial code: G1XCGD.02 . EudraCT number: 2014‐002222‐12