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GMO DOSSIER Summary
For biomedical research N° Eudract 2012‐000856‐33
A phase I/II, open‐label, study of intracerebral administration of adeno‐associated viral vector containing the human alpha‐N‐
acetylglucosaminidase gene for the treatment of Sanfilippo type B syndrome
Version number: 01 Date of the version: 10/10/2012
Institut Pasteur 25‐28 rue du Docteur Roux
75724 Paris Cedex 15 Phone: +33 (0) 1 44 38 91 01 Fax: +33 (0) 1 40 61 39 77
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Table of Content
A. General Information .............................................................................................................. 5 1. Details of notification ......................................................................................................... 5 2. Notifier ............................................................................................................................... 5 Name of institution or company ............................................................................................ 5 3. GMO characterisation ........................................................................................................ 5 4. Is the same GMO release planned elsewhere in the Community (in conformity with article 6(1)), by the same notifier?......................................................................................... 6 5. Has the same GMO been notified for release elsewhere in the Community by the same notifier? .................................................................................................................................. 6 6. Has the same GMO been notified for release or placing on the market outside the Community by the same notifier?.......................................................................................... 6 7. Summary of the potential environmental impact of the release of the GMOs................. 6
B. Information relating to the recipient or parental organisms from which the GMO is derived.................................................................................................................................................... 8 1. Recipient or parental organism characterisation: ............................................................. 8 2. Name .................................................................................................................................. 8 (i) order and/or higher taxon (for animals): NA ..................................................................... 8 (ii) genus: Dependovirus ........................................................................................................ 8 (iii) species: Adeno‐Associated Virus...................................................................................... 8 (iv) subspecies: Parvoviridae/Parvoviriae .............................................................................. 8 3. Geographical distribution of the organism ........................................................................ 9 4. Natural Habitat of the organism ........................................................................................ 9 5. Techniques ....................................................................................................................... 10 6. Is the recipient organism classified under existing Community rules relating to the protection of human health and/or the environment?....................................................... 10 7. Is the recipient organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead? ................................................................ 10 8. Information concerning reproduction ............................................................................. 10 9. Survivability ...................................................................................................................... 10 10. Ways of dissemination and factors affecting dissemination ......................................... 11 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) ..... 11
C. Information relating to the genetic modification................................................................. 11 1. Type of the genetic modification ..................................................................................... 11 2. Intended outcome of the genetic modification ............................................................... 11 3. Use of a vector.................................................................................................................. 12 4. If the answer to 3(b) is Yes, supply the following information ........................................ 12 5. If the answer to question B.3(a) and (b) is no, what was the method used in the process of modification? ................................................................................................................... 12 6. Composition of the insert................................................................................................. 12
D. Information on the organism(s) from which the insert is derived....................................... 13 1. Indicate whether it is a:.................................................................................................... 13 Viroid ...................................................................................................................... 13 RNA virus ...................................................................................................................... 13
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2. Complete name ................................................................................................................ 13 3. Is the organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead?..................................................................... 14 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?...................... 14 5. Do the donor and recipient organism exchange genetic material naturally? ................. 14
E. Information relating to the genetically modified organism ................................................. 14 1. Genetic traits and phenotypic characteristics of the recipient or parental organism which have been changed as a result of the genetic modification?.................................... 14 2. Genetic stability of the genetically modified organism ................................................... 15 3. Is the GMO significantly pathogenic or harmful in any way (including its extracellular products), either living or dead? .......................................................................................... 15 4. Description of identification and detection methods ...................................................... 16
F. Information relating to the release ....................................................................................... 16 1. Purpose of the release (including any significant potential environmental benefits that may be expected) ................................................................................................................. 16 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? ...................... 17 3. Information concerning the release and the surrounding area ...................................... 17 4. Method and amount of release ....................................................................................... 18 5. Short description of average environmental conditions (weather, temperature, etc…) 18 6. Relevant data regarding previous release carried out with the same GMO, if any, specially related to the potential environmental and human health impacts from the release .................................................................................................................................. 19
G. Interactions of the GMO with the environment ad potential impact on the environment, if significantly different from the recipient or parent organism .................................................. 19 1. Name of target organisms (if applicable)......................................................................... 19 2. Anticipated mechanism and result of interaction between the released GMOs and the target organism (if applicable) ............................................................................................. 19 3. Any other potentially interactions with other organisms in the environment................ 20 4. Is post‐release selection such as increased competitiveness, increased invasiveness for the GMO likely to occur?...................................................................................................... 20 5. Types of ecosystems to which the GMO could be disseminated from the site of release and in which it could become established ........................................................................... 20 6. Complete name of non‐target organisms which (taking into account the nature of the receiving environment) may be unintentionally harmed by the release of the GMO ........ 20 7. Likelihood of genetic exchange in vivo ............................................................................ 20 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):.................................................................................... 21 9. Possible environmentally significant interactions with biogeochemical processes (if different from the recipient or parental organism) ............................................................. 21
H. Information relating to monitoring...................................................................................... 21 1. Methods for monitoring the GMOs ............................................................................. 21 2. Methods for monitoring ecosystem effects..................................................................... 21
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3. Methods for detecting transfer of the donated genetic material from the GMO to other organisms ............................................................................................................................. 21 4. Size of the monitoring area (m2) ...................................................................................... 21 5. Duration of the monitoring .............................................................................................. 21 6. Frequency of the monitoring ........................................................................................... 22
I. Information on post-release and waste treatment ................................................................. 22 1. Post‐release treatment of the site ............................................................................... 22 2. Post‐release treatment of the GMOs........................................................................... 22 3. Waste............................................................................................................................ 22
J. Information on emergency response plans ........................................................................... 22 1. Methods and procedures for controlling the dissemination of the GMO(s) in case of unexpected spread............................................................................................................... 22 2. Methods for removal of the GMO(s) of the areas potentially affected .......................... 23 3. Methods for disposal or sanitation of plants, animals, soils, etc…that could be exposed during or after the spread .................................................................................................... 23 4. Plans for protecting human health and the environment in the event of an undesirable effect .................................................................................................................................... 23
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SUMMARY NOTIFICATION INFORMATION FORMAT FOR THE RELEASE OF GENETICALLY ORGANISMS OTHER THAN HIGHER PLANTS IN ACCORDANCE
WITH ARTICLE 11 OF DIRECTIVE 2001/18/EC
A. General Information
1. Details of notification (a) Member State of notification : FRANCE (b) Notification Number: B/FR/13/GT02 (c) Date of acknowledgement of notification : 25/04/2013 (d) Title of the project A phase I/II, open‐label, study of intracerebral administration of adeno‐associated viral vector containing the human alpha‐N‐acetylglucosaminidase gene for the treatment of Sanfilippo type B syndrome (e) Proposed period of release Between Q2 2013 and Q2 2015
2. Notifier
Name of institution or company Institut Pasteur 25‐28 Rue du Docteur Roux 75724 Paris Cedex 15
3. GMO characterisation (a) Indicate whether the GMO is a:
Viroid RNA virus
DNA virus Bacterium Fungus Animal
‐ Mammals ‐ Insect ‐ Fish ‐ Other animal specify phylum, class
Other, specify (b) Identify of the GMO (genus and species)
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AAV2.5‐hNaGlu is an Adeno‐Associated Virus serotypes 2 and 5 carrying the human alpha‐N‐acetylglucosaminidase gene. Genus: Dependovirus Family: Parvoviridae (c) Genetic stability – according to Annex IIIA, II, A(10) The GMO is derived from an AAV (Parvoviridae, Dependoviruses) but deficient for replication. Therefore the stability in terms of genetic traits is expected to be equivalent to wild‐type AAV. DNA of wild type AAV and of AAV‐based vectors persists in transduced cells as circular (extrachromosomal) episomal concatemers in human tissues (Chen et al. 2005, Schnepp et al. 2005, Schnepp et al. 2009). However, due to the lack of viral Rep and Cap genes, AAV2.5‐hNaGlu will remain in the cells as episomes and will not replicate and produce viral particles. The expression cassette will be transcribed and translated by host cell enzymes leading to expression of NaGlu. The genetic stability of the baculovirus seeds used for manufacturing is confirmed for each passage by Q‐PCR, calculating the ratio between the genetic insert, the ORF closest to the insert and a specific baculovirus region (HR3). This ratio should be stable for releasing the baculovirus seeds for product manufacturing.
4. Is the same GMO release planned elsewhere in the Community (in conformity with article 6(1)), by the same notifier? Yes No If Yes, insert the country code (s):
5. Has the same GMO been notified for release elsewhere in the Community by the same notifier? Yes No If Yes:
‐ Member State of notification ‐ Notification Number
6. Has the same GMO been notified for release or placing on the market outside the Community by the same notifier? Yes No If Yes:
‐ Member State of notification ‐ Notification Number
7. Summary of the potential environmental impact of the release of the GMOs. AAV2.5‐hNaGlu is a replication deficient adeno‐associated viral vector based on a serotype 2 AAV genome packaged into a chimeric serotype 5 / serotype 2 capsid, derived from AAV
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virus. Viral genes Rep and Cap were removed and the human NaGlu cDNA was inserted in the expression cassette allowing the expression of the NaGlu protein by infected cells. The final GMO is replication deficient. Due to the lack of viral Rep and Cap genes, the rAAV will persist as episome and won’t replicate and produce viral particles. Expression cassette will be transcribed and translated by host cell enzymes leading to expression of NaGlu. This protein is a non harmful human natural protein and is not known to induce cytotoxic effect. In natural conditions, wild type AAV in the presence of a helper virus (adenovirus or herpes virus) is found to infect human and non human primates through the respiratory tract. AAV is capable of infecting non‐dividing and dividing cells. In presence of helper virus, AAV undergoes productive infection characterized by genome replication, viral gene expression and virion production. In absence of a helper virus co‐infection, wild type AAV may integrate into the host cell genome or remain as an extrachromosomal form and in both situations the virus appears to remain latent. Site‐specific integration on human chromosome 19 has not been observed in naturally infected subjects and seems to be a unique feature of AAV adaptation to cultured cells. AAV has never been associated with any disease or pathological conditions in humans. From the donor, AAV2.5‐hNaGlu vectors contain the coding sequence of the human NaGlu. There is no unused or unknown sequence. In the final GMO, the viral rep and cap genes are deleted. It is therefore anticipated that the recombinant AAV2.5‐hNaGlu will not replicate or disseminate from transduced cells. In the proposed release, recombinant AAV will be injected into the brain of the patient, which is not the common route of infection. The neurosurgical procedure has been designed to minimize the risk of viral particles spreading from the cerebral tissue to the systemic compartment. The amount of delivered recombinant particles reaching the blood and other biological fluid is expected to be low. The mobilization of AAV2.5‐hNaGlu genomes present in patient brain cells is highly unlikely to occur since wild type AAV does not replicate in brain tissue. Some vector DNA may be shed via body fluids and excreta for some periods of time after injection but are not expected to be infectous. Natural exposure to AAV can result in production of antibodies. Many adults have antibodies reactive against one or more AAV serotypes which is consistent with early and repeated exposures to AAV. Despite these exposures, AAV has never been associated with any disease or pathological conditions in humans. Toxicology studies performed on rats after intracerebral injection of AAV2.5‐hNaGlu have shown no toxic or allergenic effect. Management strategies to reduce the risk of contamination and dissemination will be applied. The product will be tested for the absence of replication competent AAV upon co‐infection with adenovirus prior to release of the batch for human use.
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To further reduce the risk of dissemination to other human subjects, a restricted access to the patient will be required during the days following AAV2.5‐hNaGlu administration. Only the necessary medical staff in charge of the patient will be authorized around him. Handling of the GMO will be limited to the Pharmacy laboratory and operating room where appropriate measures are taken to prevent shedding and release in the environment. All the medical staff would have been previously informed about the presence of a GMO in all biological samples and educated on the way to handle them. The medical staff will have to wear a mask and gloves. In case of spill, splashing or accidental injection, procedures of decontamination will be applied. Waste will be decontaminated before incineration or sterilization. Dissemination of the GMO will be monitored by qPCR in patient urine and blood after administration. The patient will be authorized to leave the hospital only after this test indicates 2 times undetectable number of genome copies. Based on the characteristics of AAV2.5‐hNaGlu vector and recipient organism, the modification performed, administration route and environmental protection procedures, unwanted exposure to AAV2.5‐hNaGlu is unlikely and AAV2.5‐hNaGlu is not expected to cause harm to humans, animals or plants.
B. Information relating to the recipient or parental organisms from which the GMO is derived
1. Recipient or parental organism characterisation: (a) Indicate whether the recipient or parental organism is a:
Viroid RNA virus DNA virus Bacterium Fungus Animal
‐ Mammals ‐ Insect ‐ Fish ‐ Other animal specify phylum, class
Other, specify
2. Name (i) order and/or higher taxon (for animals): NA (ii) genus: Dependovirus (iii) species: Adeno‐Associated Virus (iv) subspecies: Parvoviridae/Parvoviriae
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(v) strain: NA (vi) pathovar (biotype, ecotype, race, etc) : NA (vii) common name : Adeno‐Associated Virus or AAV
3. Geographical distribution of the organism (a) Indigenous to, or otherwise established in the country where the notification is made: Yes No Not known (b) Indigenous to, or otherwise established in, other EC countries:
(i) Yes
If Yes, indicate the type of ecosystem in which it is found
Atlantic Mediterranean
Boreal Alpine Continental Macaronesian
(ii) No (iii) Not known
(c) Is it frequently used in the country where the notification is made? Yes No (d) Is it frequently kept in the country where the notification is made? Yes No
4. Natural Habitat of the organism (a) If the organism is a microorganism Water Soil, free‐living Soil in association with plant‐root systems In association with plant leaf/stem sytems Other, specify: infectious of humans and non human primates (b) If the organism is an animal: natural habitat or usual agroecosystem: NA
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5. Techniques (a) Detection techniques Quantitative PCR (b) Identification techniques Quantitative PCR with specific primers
6. Is the recipient organism classified under existing Community rules relating to the protection of human health and/or the environment? Yes No If Yes, specify: GMO Class 2
7. Is the recipient organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead?
8. Information concerning reproduction (a) Generation time in natural ecosystems: NA (b) Generation time in the ecosystems where the release will take place: NA (c) Way of reproduction: sexual asexual (d) Factors affecting reproduction: NA
9. Survivability (a) ability to form structures enhancing survival or dormancy: (i) endospores (ii) cysts (iii) sclerotia (iv) asexual spores (fungi) (v) sexual spores (fungi) (vi) eggs (vii) pupae (viii) larvae (ix) Other, specify: co‐infection of transduced cells with a helper virus (adenovirus, herpes‐simplex virus, cytomegalovirus or human herpes virus‐6). (b) relevant factors affecting survivability: Replication of AAV is dependent on co‐infection of helper viruses such as adenovirus or herpes‐simplex virus. In presence of helper virus, AAV undergoes productive infection characterized by genome replication, viral gene expression and virion production.
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In absence of a helper virus co‐infection, the virus may integrate into the host cell genome or remain as an extrachromosomal form and in both cases the virus appears to remain latent.
10. Ways of dissemination and factors affecting dissemination (a) Ways of dissemination: air way (b) Factors affecting dissemination: Co‐infection with a helper virus (adenovirus, herpes‐simplex virus, cytomegalovirus or human herpes virus‐6).
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) For 15 years, AAV vectors are used and modified to express deficient proteins and to study gene transfer in animal models and more recently to evaluate safety and efficacy in clinical trials. More than 2800 PubMed references are published on AAV. In a search of “AAV gene therapy”, 1980 PubMed references were presented (until February 2012). Adeno‐associated virus vectors are currently among the most frequently used viral vectors for gene therapy. Fifty‐two adult patients received deposits of AAV2‐derived vector in the brain in gene therapy trials for Parkinson disease. AAV2‐derived vector was administrated to the brain of 24 children with genetic diseases (Janson et al. 2002, Carter BJ. 2005). Adverse event associated to vector administration was not reported. According to the Journal of Gene Medicine Clinical trial site, AAV vectors represent 4.7% of the vectors used in Gene therapy clinical trials that corresponds to 81 clinical trials (http://www.wiley.co.uk/genmed/clinical). Currently, several clinical trials evaluate the use of AAV vectors for disease treatment. According to the GMO register of the European Commission AAV vector release was repeatedly notified in France (data from http://gmoinfo.jrc.ec.europa.eu/gmo_browse.aspx).
C. Information relating to the genetic modification
1. Type of the genetic modification (i) Insertion of genetic material (ii) Deletion of genetic material (iii) Base substitution (iv) cell fusion (v) other, specify
2. Intended outcome of the genetic modification The final GMO, AAV2.5‐hNaGlu is an AAV viral vector deficient for replication due to the removal of Rep and Cap genes.
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Genetic modifications lead to the deletion of viral sequences (Rep and Cap) and to the insertion of a transgene expression cassette. Genetic modifications allow loss of replication and integration ability and allow synthesis of the protein NaGlu by infected cells. The objective of the intracerebral injection of the GMO, AAV2.5‐hNaGlu vector, is to deliver the functional human alpha‐N‐acetylglucosaminidase gene encoding for the enzyme into the brain of Sanfilippo B patients to correct the course of their disease.
3. Use of a vector (a) Has a vector been used in the process of modification?
Yes No ‐ rep baculovirus ‐ cap baculovirus ‐ hNaGlu baculovirus
If No, go straight to question 5. (b) If Yes, is the vector wholly or partially present in the modified organism?
Yes No If No, go straight to question 5.
4. If the answer to 3(b) is Yes, supply the following information
5. If the answer to question B.3(a) and (b) is no, what was the method used in the process of modification? (i) transformation (ii) microinjection (iii) microencapsulation (iv) macroinjection (v) other, specify: It consists of a triple infection of SF+ insect cells with baculovirus encoding 1) Rep protein needed for packaging of DNA and replication; 2) Cap protein, encoding the coat protein specific for the AAV serotype used; 3) transgenic DNA in between ITRs.
6. Composition of the insert (a) composition of the insert Inverted Terminal Repeats of AAV2 (ITRs) Mouse Phosphoglycerate kinase promoter (muPGK) Human NaGlu cDNA (NaGlu) Bovine Growth Hormone polyA unit (pA bGH) (b) Source of each constituent part of the insert ITRs from AAV2
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muPGK from mouse NaGlu from human pA bGH from bovine growth hormone gene. (c) Intended function of each constituent part of the insert in the GMO ITRs: elements required for genome replication and packaging. muPGK: promoter, to enhance transgene expression. NaGlu: required for the stepwise degradation of a family of glycosaminoglycans called heparan sulfate. NaGlu is involved in the sixth step of heparan sulfate degradation. pA bGH: for mRNA translation. (d) Location of the insert in the host organism ‐ on a free plasmid ‐ integrated in the chromosome ‐ other, specify: the insert is cloned into the AAV genome, remain as an extrachromosomal form. (e) Does the insert contain parts whose product or function are not known?
Yes No 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 Fongus Animal
‐ mammals ‐ insects ‐ fish ‐ other animal (please specify phylum, class)
Other, specify: human for NaGlu cDNA
2. Complete name (i) order and/or higher taxon (for animals): primates (ii) family name for plants: NA (iii) genus: Homo (iv) species: Sapiens
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(v) subspecies: Sapiens (vi) strain: NA (vii) cultiva/breeding line: NA (viii) pathovar: NA (ix) common name: Human
3. Is the organism significantly pathogenic or harmful in any other way (including its extracellular products), either living or dead? Yes No 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 Not Known If Yes, give the relevant information under Annex IIIA, 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 No If Yes, specify
5. Do the donor and recipient organism exchange genetic material naturally? Yes No Not Known AAV is known to infect human and non human primates.
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?
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(a) is the GMO different from the recipient as far as survivability is concerned? Yes No Not Known The final GMO is an AAV viral vector deficient for replication due to the removal of Rep and Cap genes. (b) is the GMO in any way different from the recipient as far as mode and/or rate of reproduction is concerned? Yes No Not Known The final GMO is an AAV viral vector deficient for replication due to the removal of Rep and Cap genes. (c) is the GMO in any way different from the recipient as far as dissemination is concerned? Yes No Not Known The GMO is non replicative; ie, it cannot enter an infectious cycle event in the presence of helper function. (d) is the GMO in any way different from the recipient as far as pathogenicity is concerned? Yes No Not Known Wild type AAV is non pathogenic. AAV2.5‐hNaGlu vector is replication deficient and non pathogenic. Injection of concentrations of AAV2.5‐hNaGlu in rats didn’t induce any toxic effect.
2. Genetic stability of the genetically modified organism The final GMO is genetically stable. Due to the lack of viral Rep and Cap genes, AAV2.5‐hNaGlu will stay as episome and won’t replicate and produce particles. Expression cassette will be transcribed and translated by host cell enzymes leading to the expression of NaGlu.
3. Is the GMO significantly pathogenic or harmful in any way (including its extracellular products), either living or dead? Yes No Not Known If yes, specify the following:
(a) to which of the following organisms? Humans Animals Plants
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Other …
(b) give the relevant information under Annex III A, point II(A)(11)(d) and II(C)(2)(i): Humans are likely infected through the respiratory tract, sexual and gastrointestinal route. AAV is capable of infecting either non‐dividing or dividing cells. In the presence of helper virus (adenovirus or herpes virus), AAV undergoes productive infection characterized by genome replication, viral gene expression and virion production. In the absence of a helper virus co‐infection, AAV DNA may integrate into the host cell genome, or remain extrachromosomal. In both situations the virus remains latent. AAVs are weakly immunogenic. AAV‐induced immune reaction is seemingly restricted to the generation of neutralizing antibodies, while they induce minimal clearly‐defined cytotoxic responses. AAV has never been associated with any disease or pathological conditions in humans. AAV is not known to be associated to plants. AAV2.5‐hNaGlu is not expected to be pathogenic and does not interfere with any prophylactic or therapeutic treatments since it does not contain any sequences (no antibiotic‐resistance genes) that could affect prophylaxis or treatment of pathogenic microorganism infection. Wild type AAV was not found associated with any pathology in humans or animals, even in immunosuppressed hosts. Preclinical studies in immuno‐competent or immunosupressed animals did not reveal pathology induced by AAV2.5‐hNaGlu.
Toxicology studies performed on rat species after intracerebral injection of AAV2.5‐hNaGlu did not show any toxic or allergenic effect.
The NaGlu cDNA present in the vector is a naturally occurring sequence in healthy humans. Expression of this protein by infected cells does not induce cytopathic effect.
4. Description of identification and detection methods
(a) Techniques used to detect the GMO in the environment The number of vector genomes is determined by quantitative PCR with primers specific for vector sequences.
(b) Techniques used to identify the GMO The vector is identified by quantitative PCR with primers specific for vector sequences.
F. Information relating to the release
1. Purpose of the release (including any significant potential environmental benefits that may be expected) The considered therapeutic application consists in AAV2.5‐hNaGlu vector‐based deposits in the brain parenchyma.
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The proposed clinical trial is a non controlled, non randomized open label phase I/II monocentric study of intracerebral administration of AAV2.5‐hNaGlu. Endpoints are related to the evaluation of treatment safety in children with Sanfilippo type B syndrome. The subjects will be enrolled in a single arm. Enrolment and treatment of patients will be performed in a sequential manner. Pre‐inclusion and inclusion visits will be performed for patient recruitment by the Principal Investigator at Hôpital Bicêtre. Two days before the surgery, the patient will be hospitalized at Hôpital Bicêtre. One day before surgery the patient will be transferred to Hôpital Necker where intracerebral injection will be performed in the operating room of the Department of Paediatric Neurosurgery. The patient will return to the Hôpital Bicêtre 36 hours after the surgery and will stay 6 days at Hôpital Bicêtre for observation in the unit of Paediatric Neurology. The treatment plan consists of intracerebral deposits of AAV2.5‐hNaGlu, the vector containing the medicinal gene specific to Sanfilippo Type B syndrome (NaGlu), performed in a single injection session. Viral vector preparation will be administered directly into the brain by intracerebral injection. This technique allows targeting selective neuro‐anatomical sites to control vector delivery. Upon injection into the brain parenchyma, AAV vector particle will diffuse locally and may also be transported along axons to remote anatomical brain structures. The vector particles are internalised by neuronal, glial or microglial cells. Each of these cell types are deficient for the NaGlu enzyme in MPSIIIB patients and suffer from the toxic accumulation of undegraded heparan sulphate metabolites. Upon entry into the cells, the recombinant genome encoding NaGlu protein is transported into the nucleus where it undergoes a series of molecular transformations that result in its stable establishment as a double stranded deoxyribonucleic acid (DNA) molecule. This DNA is actively transcribed into messenger ribonucleic acids (mRNAs) by the cellular machinery. The mRNAs are translated into the NaGlu protein that will complement genetic deficiency in target cells.
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 AAV virus is known to infect the airways of human and non human primates. In the proposed release, AAV2.5‐hNaGlu will be injected into the brain of patients.
3. Information concerning the release and the surrounding area (a) Geographical location (administrative region and where appropriate grid reference): Necker Hospital 149 rue de Sèvres 75015 Paris France
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(b) Size of the site (m2): (i) actual release site (m2) : (ii) wider release site (m2) : The release of the final GMO will be limited to the brain’s patient. Handling of the GMO will be limited to the Pharmacy laboratory and operating room of Necker Hospital, Paris, France where appropriate measures are taken to prevent release in the environment (c) Proximity to internationally recognised biotopes or protected areas (including drinking water reservoirs), which could be affected: Not Applicable (d) 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: 4 patients will be treated. Each patient will receive 960 µL of vector suspension as 16 simultaneous vector deposit each containing 2,4. 1011 vector genomes (4.1012 vector genomes in total). 16. 1012 vector genomes will be released during the clinical trial (4.1012vg x 4 patients). (b) Duration of the operation: Clinical trial: Q2 2013 to Q2 2015 The neurosurgery should last 3 hours approximately for each patient. Patients will only be treated once. The expression of NaGlu is expected to be sustained. (c) Methods and procedures to avoid and/or minimise the spread of the GMOs beyond the site of the release After the neurosurgery, all treated patients will be transferred in a single room in the same hospital service. Each urine morning after vector administration will be measured individually until vector presence is undetectable twice successively before the patient leaves the hospital. Biological samples possibly containing, AAV2.5‐hNaGlu genome will be collected, and decontaminated as recommended by the “Haut Conseil des Biotechnologies”.
5. Short description of average environmental conditions (weather, temperature, etc…) Hospital environment
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6. Relevant data regarding previous release carried out with the same GMO, if any, specially related to the potential environmental and human health impacts from the release This GMO was previously used in non clinical study 20110053TRP. Uses in experimental were previously declared to the Commission du Génie Génétique (CGG) and obtained the agreement n°4397 on November 7th, 2006. This GMO, AAV2.5‐hNaGlu, has never been used in clinical trial. However, previous releases were performed with recombinant AAV in animal models and human clinical trials. To date, no involuntary dissemination has been observed.
G. Interactions of the GMO with the environment ad potential impact on the environment, if significantly different from the recipient or parent organism
1. Name of target organisms (if applicable)
(i) order and/or higher taxon (for animals): primates (ii) family name (for plants): NA (iii) genus: Homo (iv) species: Sapiens (v) subspecies: Sapiens (vi) strain: NA (vii) cultiva/breeding line: NA (viii) pathovar: NA (ix) common name: Human
2. Anticipated mechanism and result of interaction between the released GMOs and the target organism (if applicable) Upon injection into the brain parenchyma, the AAV vector particle will diffuse locally and may also be transported along axons to remote anatomical brain structures. The vector particles are internalised by neuronal, glial or microglial cells. Each of these cell types are deficient for the NaGlu enzyme in MPSIIIB patients and suffer from the toxic accumulation of un‐degradated heparan sulphate metabolites. Upon entry into the cells, the recombinant genome encoding NaGlu protein is transported into the nucleus where it undergoes a series of molecular transformations that result in its stable establishment as a double stranded deoxyribonucleic acid (DNA) molecule. This DNA is actively transcribed into messenger ribonucleic acids (mRNAs) by the cellular machinery. The mRNAs are translated into protein, NaGlu which will complement the cell deficiency. Enzyme complementation and correction of lysosomal storage occurs by two different mechanisms:
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‐ The enzyme may reach the lysosome of cells which contain and express the AAV‐borne transgene and degrade the accumulated metabolites, or, ‐ The enzyme can be released outside these cells, recaptured by distant cells and routed towards their lysosome. Hence, a limited group of genetically modified cells allows for the correction of extended brain territories. Transduced cells will express and deliver the enzyme continuously, thus constituting an intracerebral permanent source of enzyme production.
3. Any other potentially interactions with other organisms in the environment No
4. Is post‐release selection such as increased competitiveness, increased invasiveness for the GMO likely to occur? Yes No Not Known Give details
5. Types of ecosystems to which the GMO could be disseminated from the site of release and in which it could become established The GMO could be disseminated to human and non human primate populations in case of co‐infection by wild type AAV and a helper virus (adenovirus, herpes‐simplex virus, cytomegalovirus or human herpes virus‐6).
6. Complete name of non‐target organisms which (taking into account the nature of the receiving environment) may be unintentionally harmed by the release of the GMO Not Applicable
7. Likelihood of genetic exchange in vivo (a) from the GMO to other organisms in the release ecosystem: Highly unlikely. Due to the low numbers of vector DNA copies potentially released into the environment through shedding, horizontal gene transfer is highly unlikely. Even if horizontal gene transfer occurred, the sequences would not confer a selective advantage to other organisms such as bacteria since AAV2.5‐hNaGlu does not contain any prokaryotic promoters, any antibiotic‐ or other types of resistance genes or any genes, which would enhance or constrain their growth. Therefore, it is unlikely that AAV2.5‐hNaGlu would interfere with the control of pathogenic microorganisms or that it would have an effect on the natural dynamics of microbial populations or the biogeochemical cycles at any given site in the environment.
(b) from other organisms to the GMO: Highly unlikely
Since AAV2.5‐hNaGlu contains the ITR‐sequences of AAV2, there is a (remote) possibility of homologous recombination of the vector with wild type AAV2 in case of a co‐infection
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in exposed persons. The result of such a recombination would be that AAV2.5‐hNaGlu would gain functional genes of the AAV2 required for replication and encapsidation, but, in turn, would lose the transgenes. Hence, recombination would lead to the formation of viruses that are identical to the starting material and replication incompetent. Furthermore, the genetic material from the rep and cap genes together with the transgene would be too large in size to be packed in an AAV capsid. Thus it is highly unlikely that the recombination would result in a replication‐competent vector containing transgenes.
(c) likely consequences of gene transfer: expression of NaGlu by infected cells.
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): No available references
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 Tracing the GMO will be performed by quantitative PCR of vector DNA in blood and urine.
2. Methods for monitoring ecosystem effects Effects on ecosystem will not be monitored.
3. Methods for detecting transfer of the donated genetic material from the GMO to other organisms Method for detecting transfer of the donated genetic material to other organisms will be qPCR. However transfer of donated genetic material from the patient to other organisms is impossible. The batch that is going to be used in the trial is indeed tested and manufactured in order to rule out the presence of replication‐competent viruses.
4. Size of the monitoring area (m2) This clinical trial is a monocentric trial that takes place in the neurosurgery service of Necker hospital, and the neuropediatric service of Bicêtre hospital, Paris.
5. Duration of the monitoring The duration of follow‐up for each patient is one year. Therefore, the maximum time of the trial will be 2 years. Moreover, an extension study will be proposed after the end of one year follow‐up for the long term safety evaluation of the proposed treatment. The initial duration of the extension phase will be of one year and be repeated each year.
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6. Frequency of the monitoring There will be 23 visits (17 on site‐clinical visits and 6 phone contacts) allowing evaluation of safety and biodistribution. In order to allow patient to leave the hospital, urine samples will be taken from treated patients each morning after administration of the GMO until vector presence is undetectable twice successively. Blood samples will be taken from treated patients after the end of vector administration at +3minutes, +1 hour, +12hour and until undetectable.
I. Information on post-release and waste treatment
1. Post‐release treatment of the site Decontamination of the operating room by standard procedures will be used after surgical operation. In case of contact with AAV2.5‐hNaGlu, any material or surface will be decontaminated with 10% bleach solution allowing contact for at least 10 minutes or with autoclave.
2. Post‐release treatment of the GMOs Unused GMO will be stored in Necker Pharmacy at ‐80°C until dispatch to the manufacturer defined by the sponsor.
3. Waste (a) Type and amount of waste generated Type of waste generated during the treatment: ‐surgical materials (surgery tools, lines) ‐surgery waste (gloves, compresses) ‐injection system (syringes, needles, tubing and catheters) The amount of waste will be very small. 20 injection sytems are prepared and filled with the therapeutic product under sterile conditions (16 for injection and 2 extra pair as a backup). (b) Treatment of waste Syringes, tubing and catheters will be decontaminated by immersion in a 10% bleach solution for at least 30 minutes in the surgery room before incineration. All the surgical materials (surgery tools, linens) and surgery waste (gloves, compresses) will be collected and autoclaved before washing and sterilization or incineration.
J. Information on emergency response plans
1. Methods and procedures for controlling the dissemination of the GMO(s) in case of unexpected spread AAV2.5‐hNaGlu is susceptible to appropriate virucidal disinfectants with activity for non‐enveloped viruses such as 10% sodium hypochlorite (for at least 10 minutes) or heat
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(>80°C for 60 minutes). A spillage kit containing disposable absorbent and an appropriate virucidal disinfectant should be present during the preparation, transport to the operation room and administration.
2. Methods for removal of the GMO(s) of the areas potentially affected
Method for decontamination consists of immersion or washes with a fresh solution of 10% bleach for 30 minutes.
For splashes to the eye of the GMO, rinse eye at eyewash for 15 minutes then report to hospital emergency room for evaluation.
In case of accidental injection of material containing the GMO, wash area well with soap and water then report to hospital emergency room for evaluation.
In case of spills: allow aerosols to settle 15 minutes; wear protective clothes and gently cover the spill with adsorbent paper towel and apply freshly prepared 10% bleach starting at the perimeter and working towards the center; allow at least 30 minutes contact time before clean up.
3. Methods for disposal or sanitation of plants, animals, soils, etc…that could be exposed during or after the spread No dispersal or sanitation of plants, animals, soils can be planned. All possibly contaminated material will be decontaminated with 10% bleach solution before destruction.
4. Plans for protecting human health and the environment in the event of an undesirable effect Not Applicable