asp0113 part 1 (council decision 2002/813/ec) summary notification

ASP0113 Summary Notification

16 Apr 2013 Astellas of 39

ASP0113

PART 1 (COUNCIL DECISION 2002/813/EC)

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

Astellas (Including, but not limited to, Astellas Pharma Global Development, Inc,

Astellas Pharma Europe B.V. and Astellas Pharma Inc.)

Date: 16 April 2013



Contents 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 ··············· 15

D. INFORMATION ON THE ORGANISM(S) FROM WHICH THE INSERT IS DERIVED ···························································································· 20

E. INFORMATION RELATING TO THE GENETICALLY MODIFIED ORGANISM ························································································· 22

F. INFORMATION RELATING TO THE RELEASE ········································ 27

G. INTERACTIONS OF THE GMO WITH THE ENVIRONMENT AND POTENTIAL IMPACT ON THE ENVIRONMENT, IF SIGNIFICANTLY DIFFERENT FROM THE RECIPIENT OR PARENT ORGANISM ·················· 30

H. INFORMATION RELATING TO MONITORING ········································ 33

I. INFORMATION ON POST-RELEASE AND WASTE TREATMENT ··············· 34

J. INFORMATION ON EMERGENCY RESPONSE PLANS ······························ 34

K. REFERENCES ······················································································ 36



A. GENERAL INFORMATION

1. Details of notification

(a) Member State of notification France

(b) Notification number B/FR/13/GT06

(c) Date of acknowledgement of notification 27/09/2013

(d) Title of the project

A Randomized, Double-Blind, Placebo-Controlled, Phase 3 Trial to Evaluate the Protective Efficacy and Safety of a Therapeutic Vaccine, ASP0113, in Cytomegalovirus (CMV)-Seropositive Recipients Undergoing Allogeneic, Hematopoietic Cell Transplant (HCT) (Study 0113-CL-1004)

(e) Proposed period of release

Each patient will receive a total of 5 injections of ASP0113 over a 6 month period. The dosing period is expected to take approximately 3 years to complete for all subjects.

2. Notifier

Name of institution or company

Astellas Pharma Global Development, Inc. 1 Astellas Way Northbrook, IL 60062, USA

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



The ASP0113 medicinal product is not a GMO per se. It is an investigational gene therapy medicinal product consisting of a 1:1 mass ratio of two purified plasmid DNA (pDNA) drug substances formulated with formulated with poloxamer CRL1005 and benzalkonium chloride (BAK) at a concentration of 5 mg/mL pDNA. The pDNA drug substances, designated VCL-6365 and VCL6368, are derived from the commonly used laboratory cloning vector pBR322. The pDNAs contain an antibiotic resistance gene and a bacterial origin of DNA replication, and are manufactured in the USA by growth in Escherichia coli (E. coli) cultures maintained under antibiotic selection with kanamycin. The VCL-6365 and VCL-6368 pDNA drug substances in ASP0113 are acellular products free of contaminating bacteria or viruses.

For the purposes of this notification, the VCL-6365 and VCL-6368 pDNA components of ASP0113 are considered to be the active component of the GMO.

(b) Identity of the GMO (genus and species)

The VCL-6365 and VCL-6368 pDNAs do not have a taxonomic classification.

(c) Genetic stability – according to Annex IIIa, II, A(10)

Annex IIIa, II, A(10): Verification of the genetic stability of the organisms and factors affecting it.

Available data indicate that pDNAs are genetically stable under the controlled conditions used for their propagation in E. coli during the manufacturing process, and will not become unstable via chromosomal integration of plasmid sequences in trial subjects treated with ASP0113 or via conjugative gene transfer between bacteria.

The VCL-6365 and VCL-6368 pDNAs do not encode a conjugative pilus or contain sequence elements such as oriT utilized in conjugative gene transfer between bacteria [Willetts, 1984]. The results of initial experiments performed with pBR322 indicate it is not detectably mobilised under conditions permissive for conjugative gene transfer [Bolivar, 1977]. The eukaryotic donor elements of VCL-6365 and VCL-6368 are not known or predicted to alter the stability of the pDNA relative to the pBR322 recipient vector in bacteria, or to effect pDNA mobilisation between bacteria.

The kanamycin resistance gene of VCL-6365 and VCL-6368 originates from the naturally occurring bacterial transposon Tn903 [Grindley, 1980] and includes the promoter and coding sequence for the 3´aminoglycoside phosphotransferase type I (APH-3´ type I) enzyme. The encoded gene product is expressed in bacteria and confers resistance to kanamycin or similar aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin, lividomycin, and gentamicin B) [Shaw, 1993]. The kanamycin resistance gene and flanking sequences of VCL-6365 and VCL-6368 do not contain any of the inverted repeat sequences necessary for classical transposition of the gene into bacterial chromosomes or other plasmids [Grindley, 1980].

Unpublished data from a nonclinical biodistribution study of ASP0113 performed in mice indicates the pDNAs do not persist in gonadal or other tissues following intramuscular injection. Data from biodistribution studies of other pDNA vaccines, including a highly similar trivalent vaccine (VCL-CT01) containing VCL-6365 and VCL-6368 formulated in poloxamer CRL1005 and BAK [Vilalta, 2005a], indicate that pDNA administered by intramuscular injection is progressively cleared from tissues



and does not integrate into host cell genomic DNA at the site of injection [Vilalta, 2005b; Sheets, 2006; Martin, 1999].

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)

Release is also planned in BE, DE, ES and SE for study 0113-CL-1004. The investigational product is not considered a GMO in these countries; therefore, notification for release does not apply and was not done for these countries.

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

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 (X) No (.)

If yes:

- Member State of notification Japan

- Notification number YAKUSYOKUHATSU 0718-13’

An application for Gene Therapy was approved on 18 July 2012 by the Biological Technology Committee of the Ministry of Health, Labour and Welfare (MHLW) in Japan.

The clinical trial will also be conducted in the USA, Canada, Australia, Taiwan and South Korea for study 0113-CL-1004. The investigational product is not considered a GMO in these countries; therefore, notification for release does not apply and was not done for these countries.

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



The data and considerations provided below support the following conclusions regarding the potential environmental impact of the ASP0113 medicinal product:

The ASP0113 medicinal product is not infectious or capable of replication in humans or mammals.

Based on the results of previous clinical and nonclinical studies, the ASP0113 medicinal product is not pathogenic to humans or animals.

The risk of uptake and sustained replication of the VCL-6365 or VCL-6368 pDNAs in resident human bacteria or bacteria present in the environment is remote.

The risk of dissemination of the VCL-6365 or VCL-6368 pDNAs via excretion from trial subjects or integration into human or mammalian chromosomal DNA is negligible.

The viral and bacterial DNA sequence elements of the VCL-6365 or VCL-6368 pDNAs are essentially similar to their naturally occuring parental forms, and as such pose no risk for homologous recombination events resulting in increased virulence or spread of the parental organisms.

The potential environmental risk of ASP0113 is therefore considered to be minimal and acceptable in view of the potential clinical benefit to hematopoietic cell transplant recipients at risk for CMV disease.

B. INFORMATION RELATING TO THE RECIPIENT OR PARENTAL ORGANISM FROM WHICH THE GMO IS DERIVED

1. Recipient or parental organism characterisation:

(a) Indicate whether the recipient or parental organism is a:

(select one only)

viroid (.)

RNA virus (.)

DNA virus (.)

bacterium (.)

fungus (.)

animal

- mammals (.)

- insect (.)

- fish (.)

- other animal (.)



(specify phylum, class) …

other, specify: pBR322

The VCL-6365 and VCL-6368 pDNAs are derived from the commonly used laboratory cloning vector pBR322 [Bolivar, 1977], an artificial bacterial plasmid which does not exist in the natural environment and does not have a taxonomic classification.

2. Name

(i) order and/or higher taxon (for animals) …

(ii) genus …

(iii) species …

(iv) subspecies …

(v) strain …

(vi) pathovar (biotype, ecotype, race, etc.) …

(vii) common name pBR322

3. Geographical distribution of the organism

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

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

The pBR322 cloning vector is used in research laboratories worldwide.

(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 ..

Mediteranean ..

Boreal ..

Alpine ..

Continental ..

Macaronesian ..

(ii) No (.)

(iii) Not known (.)

(Used in research laboratories.)

(c) Is it frequently used in the country where the notification is made?

Yes (x) No (.)




(d) Is it frequently kept in the country where the notification is made?

Yes (x) 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 systems (.)

other, specify

The pBR322 pDNA is not known to exist in the natural environment, but in theory could reside in E. coli or other members of the family Enterobacteriaceae, which can be found in soils, wastewater, and human or mammalian gastrointestinal flora.

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

…

5. (a) Detection techniques

Polymerase Chain Reaction (PCR) assays.

(b) Identification techniques

Polymerase Chain Reaction (PCR) assays, Southern blot.

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

(a) Generation time in natural ecosystems:

The generation time of pBR322 in natural ecosystems is not known. The pBR322 plasmid is capable of replication in permissive species of bacteria such as E. coli. In laboratory cultures, doubling times of 24-71 minutes have been reported [Bremer, 1986].

(b) Generation time in the ecosystem where the release will take place:

Relevant nonclinical data suggest that plasmids such as pBR322, VCL-6365, or VCL-6368 will not undergo episomal replication in mammalian cells [Wolff, 1990].

(c) Way of reproduction:

Sexual (.)

Asexual (x)

(d) Factors affecting reproduction:

The VCL-6365 and VCL-6368 pDNAs are capable of reproduction (i.e., replication) in bacteria such as E. coli that could be present on or in human recipients of ASP0113 or in the natural environment. In addition, the pDNAs could hypothetically be replicated via integration into chromosomal DNA in humans or animals. Replication of the VCL-6365 or VCL-6368 pDNAs in bacteria present on or in human recipients of ASP0113 is not expected to occur based on the following data and considerations:

The pDNA does not disseminate to sites where bacteria are present. Bacteria are

typically present in anatomic sites such as the large and small intestine, skin, eyes, ears, nose, throat and mouth. With the exception of the skin, these sites are generally distinct anatomical compartments separated from the injection site (deltoid muscle) used for the ASP0113 product. Data from a nonclinical biodistribution study of ASP0113 performed in mice indicate that, following intramuscular administration, the VCL-6365 and VCL-6368 pDNAs did not readily disseminate beyond the injection site tissue. However, rare instances of bacterial exposure to VCL-6365 or VCL-6368 pDNA on the skin of trial subjects



cannot be excluded (for example, within epidermal tissue directly in the path of the injection needle) and are addressed below.

The pDNA cannot gain entry into bacteria. It is estimated that 500 to 1000

different species of bacteria live in the human body. At least 40 bacterial species (about 1% of known bacterial species) are known to become naturally competent for receptor-mediated uptake of DNA under certain circumstances. Clinically pathogenic species known to become naturally competent include Campylobacter jejuni, Campylobacter coli, Haemophilus influenzae, Haemophilus parainfluenzae, Helicobacter pylori, Moraxella species, Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus sanguis, and Streptococcus mutans [Lorenz, 1994]. Expression of a specific set of genes is required for competence. Except in Neisseria gonorrhoeae, which has been reported to be constitutively competent, expression of competence genes is transient, and is tightly controlled by environmental signals including quorum-sensing and nutritional deprivation [Chen, 2004]. In some Gram-negative strains, such as Haemophilus influenzae [Sisco, 1979] and Neisseria species [Goodman, 1988], efficient DNA uptake occurs only if a specific DNA sequence is present. The process of DNA uptake by competent bacteria is accompanied by endonuclease cleavage and degradation of one strand of the DNA. Incoming single-stranded DNA can be integrated into the bacterial chromosome by a process that requires sequence homology between the incoming DNA and the bacterial chromosome [Lorenz, 1994]. In specific circumstances, plasmids can be reconstituted and maintained as episomal elements [Chen 2004]. However, the risk of pDNA encountering a pathogenic bacterial species in trial subjects in a competent state is low. In clinical isolates of the pathogenic strains listed above, transformation frequencies ranged from 10-2 to 10-6 in viable cells [Lorenz, 1994]. Bacterial cells that are not naturally competent for exogenous DNA uptake are otherwise impermeable to pDNA under normal physiologic conditions. For example, artificial transformation of the E. coli strains used in molecular biology laboratories is typically accomplished by the use of a procedure involving hypotonic swelling of the bacteria in an aqueous solution containing CaCl2, followed by a brief heat shock at 42°C. These or other extreme physical conditions (e.g., freezing and thawing) which may render bacteria permeable to pDNA are not expected to occur in trial subjects.

Wild-type bacteria do not support transformation by the plasmid. Strains of E. coli

used for molecular cloning, including the DH10B strain currently used for manufacture of ASP0113, are specifically maintained genotypes containing mutations that enhance transformation and plasmid replication [Durfee, 2008]. In some cases, wild-type bacterial species may possess species-specific DNA restriction and modification systems capable of destroying foreign DNA that is not methylated in the same manner as their own [Lorenz, 1994].



Wild-type bacteria do not support replication of the plasmid. Replication of the VCL-6365 and VCL-6368 pDNAs in bacteria is controlled by the Col E1 origin of replication sequence present in the plasmid backbone. The parental Col E1 plasmid from which the origin of replication sequence of VCL-6365 and VCL-6368 is derived is a narrow host-range plasmid that generally does not transform or replicate in bacterial hosts outside of the family Enterobacteriaceae. In addition, Col E1-based replicons require several E. coli host factors to support replication [Kues, 1989]. These considerations suggest that, with the exception of E. coli and other Enterobacteriaceae, replication of the VCL-6365 or VCL-6368 pDNAs is unlikely to occur in bacteria found in trial subjects, including the pathogenic species of bacteria listed above that are known to acquire natural competence. The risk of transformation and replication in E. coli or other Enterobacteriaceae is addressed below.

Replication of pDNA in bacteria would not occur in the absence of antibiotic. In

trial subjects or individuals inadvertently exposed to ASP0113, selection and outgrowth of rare bacterial transformants carrying the aminoglycoside 3´ phosphotransferase gene would require the continued presence of aminoglycoside antibiotics (kanamycin, neomycin, paromomycin, ribostamycin, lividomycin, and gentamicin B) known to be inactivated by the product of the kanamycin resistance gene expressed by VCL-6365 and VCL-6368 [Shaw, 1993]. In the absence of continued exposure of trial subjects to these antibiotics, rare instances of pDNA uptake would not confer any selective advantage on the bacterial host, and would be expected to negatively impact bacterial growth and survival due to the reduced availability of host cell factors for bacterial DNA replication [Bouma, 1988]. In the special case of bacteria transformed to kanamycin resistance through integration of the plasmid-derived kanamycin resistance gene fragment into their chromosomal DNA, the absence of antibiotic selection, the requirement for DNA sequence homology for integration into chromosomal DNA of the host, and the potential negative effects of integration on the host greatly reduce the risk of stable transformants will arise in trial subjects.

Antibiotics such as kanamycin are no longer widely used. Kanamycin or similar

aminoglycoside antibiotics are no longer widely used for treatment of human infections due to their toxicity, the development of resistant bacterial strains, and the availability of newer antibiotics that are less toxic and/or more effective against drug-resistant strains. According to the Merck Manual for Healthcare Professionals1, aminoglycosides are used for serious gram-negative infections, especially Pseudomonas aeruginosa. Aminoglycosides are used alone infrequently, typically for plague or tularemia. They can be used for treatment of severe infections due to suspected gram-negative bacteria in conjunction with a broad-spectrum β-lactam antibiotic. Because of increasing aminoglycoside

1http://www.merckmanuals.com/professional/infectious_diseases/bacteria_and_antibacterial_drugs/aminoglycosides.html?qt=aminoglycosides&alt=sh, accessed March 26, 2013



resistance, a fluoroquinolone can be substituted for the aminoglycoside in initial empiric regimens, or the aminoglycoside can be stopped after 2 to 3 days unless an aminoglycoside-sensitive P. aeruginosa is identified. While transformation of P. aeruginosa by VCL-6365 or VCL-6368 pDNA in a trial subject could be of concern, it should be pointed out that this species is not among those known to support plasmid replication via the Col E1 origin of replication, or to develop natural competence [Lorenz, 1994]. In addition, the use of an aminoglycoside antibiotic in conjunction with a broad-spectrum β-lactam antibiotic would be expected to eliminate strains of P. aeruginosa harboring resistance to only the aminoglycoside.

These data and considerations support the conclusion that replication of the VCL-6365 or VCL-6368 pDNAs will not occur in bacteria on or in trial subjects. While rare instances of bacterial uptake of plasmid cannot be ruled out, available evidence suggests that such rare instances would not result in pDNA replication or the creation and perpetuation of stable, antibiotic-resistant bacterial strains in the absence of antibiotic selection. Replication of the VCL-6365 or VCL-6368 pDNA in bacteria present in the natural environment is not expected to occur based on the data and considerations given above, and the following additional data and considerations:

Intact pDNA will not gain entry into bacterial cells. Bacteria in the natural environment can be rendered permeable to pDNA by extreme physical conditions such as freezing and thawing, exposure to an electrical charge, hypotonic swelling, or heating under conditions that result in a temporary loss of cell wall integrity without cell death. The possibility of replication of the VCL-6365 or VCL-6368 pDNAs in these cases is considered remote due to many of the reasons listed above, including the narrow host range of the plasmid’s Col E1-derived origin of replication element, the existence of bacterial host defense mechanisms such as restriction and modification systems, and the absence of antibiotic selection.

Intact pDNA will not gain entry into the mammalian digestive tract. In the case of

ingestion by humans or mammals, the risk of transformation of resident E. coli or other bacteria in the gastrointestinal tract is increased. However, ingestion of significant amounts of intact VCL-6365 or VCL-6368 pDNA secondary to its inadvertent release into the environment must be viewed as a highly unlikely circumstance due to the nearly ubiquitous presence of nucleases in the natural environment. Moreover, the likelihood of ingestion resulting in successful transformation of gastrointestinal bacteria would be diminished significantly due to degradation of the ingested pDNA by digestive system nucleases, depurination of the pDNA in the highly acidic environment of the stomach and upper intestine, and other reasons listed above. Rare instances of transformation of intestinal bacteria would not be expected to lead to significant plasmid replication in the absence of antibiotic selection. Antibiotics are not effective for



treatment of suspected cases of pathogenic E. coli infection in humans, and may increase the risk of hemolytic uremic syndrome [Walterspeil, 1992]. In addition, as stated above, aminoglycoside antibiotics are infrequently used alone for treatment of human infections, and are not present in the natural environment of mammals or among the antibiotics used to promote the growth of livestock. For these reasons, the likely extent of replication of the VCL-6365 or VCL-6368 pDNA following ingestion by humans or mammals and transformation of resident bacteria is considered negligible.

Replication of pDNA sequences via integration into chromosomal DNA is not expected to occur based on the following data and considerations:

Rapid degradation of pDNA in blood and tissues. A nonclinical biodistribution study of ASP0113 in mice found that, 2 days following a single intramuscular injection, pDNA was detectable in a range of tissues with the highest levels observed in injection site tissues. Plasmid levels declined in all tissues with time, and pDNA was cleared to undetectable levels (<10 plasmid copies per µg of tissue DNA) in many tissues by day 14. Analysis of tissue DNA purified from injection site tissues showed barely detectable plasmid copy numbers 2 months following administration. Similar findings were obtained in a biodistribution study of an investigational product highly similar to ASP0113 in mice [Vilalta, 2005a]. In a biodistribution study of another pDNA-based investigational product, pDNA was found to have a half-life of less than 5 minutes in blood following intravenous injection [Lew, 1995]. In humans, the mean half-life of circulating fetal DNA in maternal plasma has been reported to be 16.3 minutes [Lo, 1999]. In early developmental studies in mice involving intramuscular injection of a reporter pDNA encoding firefly luciferase, over 95% of the residual plasmid DNA recovered from injected muscles was found to be degraded by 90 minutes after injection [Manthorpe, 1993].

Lack of pDNA persistence in gonadal tissues. A biodistribution study of ASP0113

in mice found that pDNA was cleared to undetectable levels in gonadal tissues by day 14 after intramuscular injection. In a rabbit biodistribution study of a product highly similar to ASP0113, pDNA was undetectable in gonadal tissues by 30 days after intramuscular injection [Vilalta, 2005a]. The limit of detection for the PCR assays used in these studies was 10 plasmid copies per μg of tissue DNA. In other biodistribution studies involving pDNA-based investigational products administered by intramuscular injection, pDNA was not quantifiable by PCR in genomic DNA isolated from gonadal tissues at the final time points tested (no later than two months after injection). The lower limit of quantification of the PCR assays used in these studies was >10 plasmid copies (3 studies) or 50 plasmid copies (4 studies) per μg genomic DNA [Sheets, 2006]. Similarly, a tissue distribution study performed in support of a pDNA vaccine for anthrax found no evidence for pDNA persistence in gonadal tissues after intramuscular injection [Vilalta, 2005b].



Lack of evidence for pDNA integration. Nonclinical studies of pDNA vaccines administered intramuscularly by needle and syringe have found no evidence for pDNA integration into injection site tissues. An integration study of ASP0113 in mice found no evidence for integration of either pDNA into genomic DNA purified from injection site tissues 60 days after intramuscular injection. Similar results were obtained in an integration study of a product highly similar to ASP0113 performed in rabbits [Vilalta, 2005a]. The PCR assay used in this study had a lower limit of detection of 10 plasmid copies per µg of genomic DNA. Other studies of pDNA vaccines administered by intramuscular injection have also found no evidence for integration of pDNA in injection site tissues [Sheets, 2006; Martin, 1999]. The potential for integration into germline tissues has not directly been evaluated for ASP0113 or other pDNA-based investigational products due to the lack of pDNA persistence in gonadal tissues at later timepoints [Vilalta 2005a; Vilalta, 2005b; Sheets, 2006; Martin, 1999].

9. Survivability

(a) ability to form structures enhancing survival or dormancy:

(i) endospores (.)

(ii) cysts (.)

(iii) sclerotia (.)

(iv) asexual spores (fungi) (.)

(v) sexual spores (funghi) (.)

(vi) eggs (.)

(vii) pupae (.)

(viii) larvae (.)

(ix) other, specify

The pBR322, VCL-6365, or VCL-6368 pDNAs are not known to form any of the structures listed above. Instances of inadvertent environmental release would generally be expected to lead to rapid degradation of the pDNA due to the abundance of deoxyribonucleases in the natural environment. However, based on experimental findings related to the presence of free DNA in the environment [Lorenz, 1994], trace amounts of pDNA could persist in soils or wastewater.

(b) relevant factors affecting survivability:

See B(8)(d) above.

10. (a) Ways of dissemination

See B(8)(d) above.

(b) Factors affecting dissemination



See B(8)(d) above.

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)

A Summary Notification for Allovectin-7®, an investigational medicinal product containing a modified pBR322 recipient pDNA (VCL-1005), was submitted to AFSSAPS on May 5, 2008 as part of a clinical trial application for a Phase 3 clinical trial titled “Essai Clinique de Phase 3 pour evaluer la Tolerance et l’Efficacite de 2mg d’Allovectin-7® en Administration Intra-Lesionnelle versus Dacarbazine (DTIC) ou Temozolomide (TMZ) chez des Sujets en Recurrence de Melanoma Metastatique.” A positive opinion on the clinical trial application was issued by AFSSAPS on June 30, 2009. No notification number was received by the trial sponsor, Vical Incorporated.

C. INFORMATION RELATING TO THE GENETIC MODIFICATION

1. Type of the genetic modification

(i) insertion of genetic material (x)

(ii) deletion of genetic material (x)

(iii) base substitution (x)

(iv) cell fusion (.)

(v) others, specify …

2. Intended outcome of the genetic modification

The pBR322 vector has been modified to insert eukaryotic expression cassettes designed to express messenger RNAs (mRNAs) hCMV gB (VCL-6365) or pp65 (VCL-6368) . In addition, the prokaryotic backbone region of the pBR322 vector has been modified as follows:

• The antibiotic resistance genes (encoding ampicillin and tetracycline resistance) have been deleted and the kanamycin resistance gene was inserted to allow for antibiotic selection with kanamycin during plasmid manufacture.

• The sequences controlling replication in bacteria were modified by a single-base substitution to increase pDNA yield during manufacture.

3. (a) Has a vector been used in the process of modification?

Yes (.) No (x)

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

(a) Type of vector

plasmid (.)

bacteriophage (.)

virus (.)

cosmid (.)

transposable element (.)

other, specify …

(b) Identity of the vector

…

(c) Host range of the vector

…

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

Yes (.) No (.)

antibiotic resistance (.)

other, specify …

Indication of which antibiotic resistance gene is inserted

…

(e) Constituent fragments of the vector

…

(f) Method for introducing the vector into the recipient organism

(i) transformation (.)

(ii) electroporation (.)

(iii) macroinjection (.)

(iv) microinjection (.)

(v) infection (.)

(vi) other, specify …

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

VCL-6365 and VCL-6368 were constructed using standard recombinant DNA cloning methods.

6. Composition of the insert

(a) Composition of the insert

The eukaryotic insert of VCL-6365 consists of the human CMV Immediate Early (hCMV-IE) gene promoter and 5´ untranslated region (5´ UT), hCMV Intron A, a codon-optimized sequence encoding the extracellular domain of hCMV glycoprotein B (gB), and a synthetic transcriptional terminator element and polyadenylation signal derived from the rabbit β-globin gene (Synthetic RBG polyA).

A plasmid map of VCL-6365 is provided in Figure 1 below.

VCL-6365

6603 bps

hCMV-IE promoter+ 5' UT

hCMV Intron A

pUC18 ori

KanamycinResistance

Synthetic RBGpolyA

codon-optimized hCMV gBextracellular domain

pUCsequence

Figure 1. VCL-6365 plasmid map. Regions of the plasmid are depicted as boxes and coding sequences as arrows. The hCMV-IE promoter + 5´ UT and hCMV Intron A



regions control expression of a human codon-optimized mRNA encoding the extracellular domain of hCMV gB. The synthetic RBG polyA transcriptional terminator effects cleavage and polyadenylation of the primary RNA transcript to form the mRNA encoding gB. The kanamycin resistance gene is expressed in bacteria and is used as a plasmid selection marker during bacterial fermentation. Replication in permissive bacterial hosts such as E. coli is regulated by the sequences contained in the pUC18 ori region of VCL-6365, which includes a bacterial origin of DNA replication derived from pBR322 [Vieira, 1982].

The eukaryotic insert of VCL-6368 consists of the hCMV-IE promoter, 5´ UT, and hCMV intron A regions, a codon-optimized sequence encoding hCMV phosphoprotein 65 (pp65) with a deletion spanning the codons for amino acids 435-438, and the synthetic RBG polyA transcriptional terminator.

A plasmid map of VCL-6368 is provided in Figure 2 below.

Figure 2. VCL-6368 plasmid map. Regions of the plasmid are depicted as boxes and coding sequences as arrows. The hCMV-IE promoter + 5´ UT and hCMV Intron A regions control expression of a human codon-optimized mRNA encoding hCMV pp65 with a deletion spanning amino acids 435-438 of the wild-type protein sequence. The synthetic RBG polyA transcriptional terminator effects cleavage and polyadenylation of



the primary RNA transcript to form the messenger RNA encoding pp65. The kanamycin resistance gene is expressed in bacteria and is used as a plasmid selection marker during bacterial fermentation. Replication in permissive bacterial hosts such as E. coli is regulated by the sequences contained in the pUC18 ori region of VCL-6368, which includes a bacterial origin of DNA replication derived from pBR322 [Vieira, 1982].

(b) Source of each constituent part of the insert

The constituent parts of the insert and their location in base pair numbers within the VCL-6365 pDNA are shown in Table 1 below. The sources shown represent the ultimate natural or synthetic origin of the constituent parts.

Table 1. VCL-6365 insert domains.

Domain Location

(base pairs) Origin of Sequence

hCMV-IE promoter + 5´ UT 6595 - 796 Human CMV

Intron A 796 - 1621 Polylinker 1622 - 1687 Synthetic Kozak translation initiation sequence

1688 - 1693 Synthetic

Codon-optimized gB extracellular domain coding sequence

1694 - 3835 Human CMV

Polylinker 3836 - 3862 Synthetic Synthetic RBG polyA 3863 - 3926 Rabbit β-globin gene

The constituent parts of the insert and their location in base pair numbers within the VCL-6368 pDNA are shown in Table 2 below. The sources shown represent the ultimate natural or synthetic origin of the constituent parts.

Table 2. VCL-6368 insert domains.

Domain Location

(base pairs) Origin of Sequence

hCMV-IE promoter + 5´ UT 6127 - 796 Human CMV

Intron A 796 - 1621 Polylinker 1622 - 1687 Synthetic Kozak translation initiation sequence

1688 - 1693 Synthetic

Codon-optimized pp65 coding sequence with 435-438 deletion

1694 - 3367 Human CMV

Polylinker 3368 - 3394 Synthetic Synthetic RBG polyA 3395 - 3458 Rabbit β-globin gene

(c) Intended function of each constituent part of the insert in the GMO

The hCMV-IE promoter, 5´ UT and Intron A, and Kozak translation initiation elements function to promote high-level expression of the gB and pp65 coding sequences in mammalian cells transfected with the VCL-6365 and VCL-6369 pDNAs, respectively.

The synthetic polylinker elements flanking the coding sequences function to facilitate cloning of coding sequences into the plasmid using restriction endonucleases.



The synthetic RBG polyA transcriptional terminator effects cleavage and polyadenylation of the primary RNA transcript to form the messenger RNAs encoding gB and pp65.

(e) Location of the insert in the host organism

- on a free plasmid (.)

- integrated in the chromosome (.)

- other, specify

The eukaryotic inserts of both VCL-6365 and VCL-6368 were subcloned into the pBR322-derived vector backbone to form a double-stranded, circular pDNA molecule.

(f) 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 (x)

bacterium (.)

fungus (.)

animal

- mammals (x)

- insect (.)

- fish (.)

- other animal (.)

(specify phylum, class) …

other, specify …

2. Complete name

i) Order and/or higher-level taxon (for animals); Herpesvirales

ii) Family name; Herpesviridae



iii) Genus; Cytomegalovirus

iv) Species; Human cytomegalovirus

v) Subspecies;

vi) Strain;

vii) Cultivar/line;

viii) Pathovar;

ix) Common name. HHV-5 (strain AD169)

3. Is the organism significantly pathogenic or harmful in any other way (including its

extracellular products), either living or dead?

Yes (x) No (.) Not known (.)

If yes, specify the following:

(a) to which of the following organisms:

humans (x)

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):

Annex III A, point II(A)(11)(d): Pathogenicity: infectivity, toxigenicity, virulence,allergenicity, carrier (vector) of pathogen, possible vectors, host range including non-target organism. Possible activation of latent viruses (proviruses). Ability to colonise other organisms.

Based on the nonclinical safety data and the human clinical safety experience obtained with ASP0113, the hCMV-derived sequences of VCL-6365 and VCL-6368 are not pathogenic in humans or animals.

The relationship of the hCMV-derived elements used in VCL-6365 and VCL-6368 to pathogenic effects of the virus is poorly understood. Early in infection, the hCMV-IE promoter, 5´ UT, and Intron A sequences function to promote transcription of the IE-1 gene product, which regulates viral gene expression [Hennighausen, 1986]. The gB protein is a highly conserved envelope glycoprotein that influences virus binding to and entry into cells, fusion of adjacent cells, and release of progeny virus from cells. It has been proposed as a determinant of virulence based on reported associations between gB



genotype and disease in immunocompromised transplant patients [Fries, 1994]. The transmembrane and cytoplasmic domains comprising amino acids 714-906 of the wild-type AD169 Gb protein [Bradley, 2009] have been deleted from the gB protein expressed by VCL-6365. The deleted domains are necessary for membrane anchoring of the gB protein [Reschke, 1995] and may also mediate fusion of viral particles to cell membranes during infection [Bold, 1996]. The wild-type pp65 tegument protein of wild-type CMV has a putative kinase activity which has been eliminated from the VCL-6368 pp65 protein by deleting amino acids 435-438 encoding the putative ATP-binding site (RKRK) [Yao, 2001]. However, the wild-type pp65 protein has been reported to be dispensable for growth of the virus in cell culture [Schmolke, 1995]. These considerations suggest that isolated hCMV-derived sequences of VCL-6365 and VCL-6368 do not have intrinsic pathogenic potential outside of their specific roles in the propagation and pathogenicity of functional wild-type hCMV in a human host.

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 …

Cytomegalovirus (Herpesviridae) is listed in Annex III of Directive 2000/54/EC (which repealed and superseded Directive 90/679/EEC). It has a group 2 infection hazard classification.

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

Yes (.) No (.) Not known (x)

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

(a) is the GMO different from the recipient as far as survivability is concerned?


Specify …

Survival of pBR322, VCL-6365, or VCL-6368 in the natural environment has not been studied. Based on the data and considerations provided under A(3)(c) and B(8)(d) above, the potential for survival of the VCL-6365 or VCL-6368 pDNA in the natural environment as a consequence of the intended use is considered remote.



(b) is the GMO in any way different from the recipient as far as mode and/or rate of reproduction is concerned?

Yes (x) No (.) Unknown (.)

Specify

The bacterial origin of DNA replication of VCL-6365 and VCL-6368 is derived from pBR322 and confers the ability to replicate in E. coli. To increase pDNA yields during manufacture in E. coli, the pUC18-derived origin of replication used in VCL-6365 and VCL-6368 contains a single base change (G to A) relative to pBR322. This single-base change is located in the region controlling plasmid replication, and effects a modest increase in the plasmid copy numbers obtained in E. coli cultures grown under antibiotic selection [Lahijani, 1996]. Replication of the VCL-6365 and VCL-6368 pDNAs in E. coli is otherwise unaltered relative to pBR322.

(c) is the GMO in any way different from the recipient as far as dissemination is concerned?


Specify

Dissemination of pBR322 or the VCL-6365 or VCL-6368 pDNAs in the natural environment has not been studied. Based on the data and considerations provided under A(3)(c) and B(8)(d) above, the potential for dissemination of the VCL-6365 or VCL-6368 pDNAs in the natural environment as a consequence of the intended use is considered remote, and no greater than that of pBR322.

(d) is the GMO in any way different from the recipient as far as pathogenicity is concerned?


Specify

The pathogenicity of pBR322 in humans is not known. The modifications to pBR322 embodied in the VCL-6365 or VCL-6368 pDNAs are considered non-pathogenic based on the nonclinical and clinical safety experience with ASP0113 summarized below.

Nonclinical safety experience:

A biodistribution study in mice found that, 2 days following a single intramuscular injection, the plasmid component of ASP0113 was detectable in a range of tissues with the highest levels observed in injection site tissues. Plasmid levels declined in all tissues with time and were cleared to undetectable levels in many tissues by day 14. Samples from the injection site showed barely detectable plasmid copy numbers 2 months after injection. In an integration study performed in mice, high molecular weight DNA separation via field-inversion gel electrophoresis followed by quantitative PCR of the high-molecular weight (genomic DNA) fraction was used to assess the potential for genomic integration of injected pDNA in muscle tissues harvested at 61 days after injection. No evidence of integration was found.

Single-dose toxicity data from mice and repeated-dose toxicity studies in rabbits showed that bilateral injections of ASP0113 appeared to be well-tolerated and



resulted in no observable clinical signs of systematic toxicity due to injection of the vaccine. The administration of 5 mg/animal ASP0113 resulted in a reversible, 2.4-fold elevation of serum creatine phosphokinase (CPK) levels. There was also dose-related, minimal to moderate histologic inflammation of the muscle, skin and subcutis at the injection site. A reversible increase in CPK followed administration of the last injection; this finding may have been due to muscle response at the injection site, as inflammation was noted on histopathological examination of the muscle at the same time as CPK elevation, and was largely resolved during the recovery period. Importantly, injection of ASP0113 did not elicit production of antinucleic acid antibodies or antibodies for double-stranded DNA, indicating that the risk of production of anti-DNA antibodies and development of autoimmune illnesses following vaccination with ASP0113 is low.

Clinical safety experience:

Study VCL-CB01-101 was a phase 1, multicenter, randomized, open-label study to evaluate the safety and immunogenicity of intramuscular ASP0113 in CMV-seronegative and CMV-seropositive healthy adult volunteers [Wloch, 2008]. ASP0113 was administered intramuscularly at doses of 1 mg or 5 mg (Groups 1 and 2, respectively; 3 doses) on weeks 0, 2, and 8, and at a dose of 5 mg on days 0, 3, 7, and 28 (Group 3; 4 doses). Safety was evaluated by monitoring adverse event incidence and severity, measurement of clinical laboratory test and vital signs, review of reactogenicity 30 minutes after each injection, symptom-directed clinical evaluations, postinjection diaries and review of concomitant medication usage. A total of 44 (22 CMV-seropositive and 22 CMV-seronegative) volunteers were enrolled into the study and received vaccination; 4 volunteers discontinued the study prematurely.

No discontinuation due to a treatment-emergent adverse event (TEAE) occurred and there were no deaths or serious adverse events (SAEs). A total of 42 (95.5%) of volunteers experienced at least one TEAE. Overall, injection site pain, myalgia, headache and malaise were the most frequently reported TEAEs considered by the investigator to be related to vaccination. Although volunteers receiving the higher dose or accelerated vaccine schedule tended to experience more local reactogenicity, the severity of TEAEs did not increase with increasing dose and a more compressed vaccination schedule. Several cutaneous TEAEs were considered related to ASP0113.

Study VCL-CB01-202 was a phase 2, multicenter, randomized, double-blind, placebo controlled study of the safety, efficacy and immunogenicity of ASP0113 in donors and CMV-seropositive recipients undergoing hematopoietic cell transplantation (HCT) for the treatment of a hematologic disease [Kharfan-Dabaja, 2012]. Recipients or donor/recipient pairs were randomized in a 1:1 ratio to receive 3 (donor) or 4 (recipient) intramuscular injections of ASP0113 or placebo. ASP0113 (5 mg) was administered to matched, related or unrelated HCT recipients (between days -5 and -3 prior to HCT, between days 21 and 42 and at days 84 and 196 after the HCT) or to HCT donors (days -9, -6 and -2 prior to hematopoietic cell donation) and to the paired, related HCT recipients (between days -5 and -3 prior to the HCT, between days 21 and 42 and at days 84 and 196 after the HCT). Subjects were to be



followed for 1 year after transplant. Safety was monitored via safety labs and clinical evaluations. A total of 108 donors or recipients were enrolled in study VCL-CB01-202, including 14 donor/recipient pairs, and received at least one dose of vaccine.

In this study, all recipients developed at least one TEAE. The incidence of TEAEs considered by the investigator to be at least possibly related to the study drug was similar between treatment groups. The following SAEs were considered by the investigators to be at least possibly related to study drug: allergic reaction, worsening pericardial effusion with cardiac tamponade and subarachnoid hemorrhage from a known aneurysm in the ASP0113 group and CMV colitis in the placebo group. After review, the data and safety monitoring board regarded only the allergic reaction as possibly related to vaccine treatment, and that was the only patient in the trial who was discontinued due to a study drug related TEAE. Six recipients in the ASP0113 group and 5 recipients in the placebo group discontinued treatment/study due to a TEAE. Ten (20.8%) recipients from the ASP0113 group and 15 (32.6%) recipients from the placebo group died during the 1-year study follow-up. No recipients died due to study drug-related TEAEs and no deaths, SAEs or discontinuations due to TEAEs occurred among donors.

The pathogenicity of ASP0113 in plants has not been evaluated. The VCL-6365 and VCL-6368 pDNAs lack the structural genes and sequence elements of plasmid vectors typically used for stable transformation of plants via Agrobacterium tumefaciens [Zambryski, 1983], and are not predicted to replicate autonomously in plants or fungi based on theoretical and experimental data [Griffiths, 1995; Robinson, 2005]. Moreover, the likelihood of pDNA uptake by plant cells is low [Davey, 1989].

2. Genetic stability of the genetically modified organism

See A(3)(c) above.

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)



Annex III A, point II(A)(11)(d): Pathogenicity: infectivity, toxigenicity, virulence, allergenicity, carrier (vector) of pathogen, possible vectors, host range including non-target organism. Possible activation of latent viruses (proviruses). Ability to colonise other organisms.

Based on the clinical and nonclinical safety experience with ASP0113 summarized under E(1)(d) above, the VCL-6365 and VCL-6368 pDNAs are not considered pathogenic in humans or animals. Activation of latent proviruses in humans or animals has not been observed. Based on the data and considerations provided under A(3)(c), B(8)(d), and E(1)(d) above, the potential for colonisation of bacteria or other organisms is considered remote.

Annex III A, point II(C)(II)(i): Toxic or allergenic effects of the GMOs and/or their metabolic products.

Based on clinical trial data published to date [Wloch, 2008; Kharfan-Dabaja, 2012] and the clinical and nonclinical safety experience described under E(1)(d) above, no toxic or allergenic effects are expected as a consequence of treatment with ASP0113.

Comparison of the modified organism to the donor, recipient or (where appropriate) parental organism regarding pathogenicity.

Based on the clinical and nonclinical safety experience with ASP0113 summarized under E(1)(d) above, the VCL-6365 and VCL-6368 pDNAs are not considered pathogenic in humans or animals.

Human CMV infection is usually harmless, except in the fetus and the immunocompromised host. CMV is the most common intrauterine infection in the U.S., occurring in about 1 in 150 live-born infants and resulting in permanent disabilities in approximately 8,000 children per year. Most symptomatic congenital infections occur following a primary maternal infection during pregnancy. In the immunocompromised host, primary infection, reinfection or reactivation of latent virus can cause significant morbidity and mortality. Persons undergoing an HCT or solid organ transplantation (SOT) are particularly susceptible to both primary infection and reactivation of CMV as a result of the interventions and drugs that ablate or suppress their immune system [Ljungman, 2002; Paya, 2001;]. Both primary infection and reactivation are risk factors for CMV disease, including tissue-invasive disease for both HCT and SOT recipients and CMV viral syndrome for SOT recipients, and are also potential risk factors for other serious clinical outcomes which result in significant morbidity and increased mortality. The most common diseases in HCT recipients are pneumonitis and gastroenteritis [Boeckh, 2009]. The mortality associated with CMV pneumonitis remains > 50% [Winston, 1993]. CMV infection may also be a risk factor for fungal, bacterial, and non-CMV viral infections as well as acute graft versus host disease and chronic graft versus host disease [Boeckh, 2009]. Similarly, CMV is one of the most important viral infections occurring in SOT. Infection is a risk factor for CMV disease, including CMV viral syndrome and tissue-invasive disease, and an independent risk factor for secondary bacteremia, Epstein Barr Virus-mediated, post-transplant lymphoproliferative disorder, and allograft injury [Humar, 2009; Fishman, 2007].

Capacity for colonisation.



The VCL-6365 and VCL-6368 pDNAs of ASP0113 are not expected to colonise bacterial hosts for the reasons stated under A(3)(c) and B(8)(d) above.

If the organism is pathogenic to humans who are immunocompetent: diseases caused and mechanism of pathogenicity including invasiveness and virulence, communicability, infective dose, host range, possibility of alteration, possibility of survival outside of human host, presence of vectors or means of dissemination, biological stability, antibiotic-resistance patterns, allergenicity, availability of appropriate therapies.

The clinical safety experience with ASP0113 in 44 healthy volunteers enrolled in the VCL-CB01-101 phase 1 trial is reviewed above under E(1)(d) above. Based on the results, ASP0113 is not considered pathogenic to immunocompetent individuals.

Other product hazards.

None.

4. Description of identification and detection methods

(a) Techniques used to detect the GMO in the environment

PCR assays utilizing primers specific for VCL-6365 and VCL-6368 pDNA sequences have previously been developed for detection of these pDNAs in mammalian tissue samples and in principle could be employed for detection of the pDNAs in samples obtained from other organisms.

(b) Techniques used to identify the GMO

In addition to the PCR assays described above, the VCL-6365 or VCL-6368 pDNAs may be identified by Southern blot analysis with specific DNA probes.

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 phase 3 clinical trial titled “A Randomized, Double-Blind, Placebo-Controlled, Phase 3 Trial to Evaluate the Protective Efficacy and Safety of a Therapeutic Vaccine, ASP0113, in Cytomegalovirus (CMV)-Seropositive Recipients Undergoing Allogeneic, Hematopoietic Cell Transplant (HCT) (Study 0113-CL-1004).” No potential benefit to the environment is expected.

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 pBR322 recipient organism is an artificial laboratory cloning vector with no known natural habitat or ecosystem.



Human subjects that are seropositive for CMV, including subjects to be enrolled in the planned phase 3 trial, have previously been infected by the human CMV parental organism.

3. Information concerning the release and the surrounding area

The release will be conducted at the clinical study sites in France listed below.

Henri Mondor University Hospital Hematology Department 94000 Créteil - France

Hôpital Saint-Louis Service Hematologie Greffe de Moelle 1 avenue Claude Vellefaux 75475 Paris Cedex 10

CHU de Nantes-Hôpital Dieu Service d'hématologie clinique Place Alexis Ricordeau 44093 Nantes Cedex 1

CRLCC Henri Becquerel Service d'hématologie Rue d'Amiens 76038 ROUEN CEDEX 1

CHU de Nice Service d’hematologie Hôpital Archet 1 151 Route de Saint Antoine de Ginistière, BP 3079 06202 Nice Cedex 3

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

(b) Size of the site (m2): … m2

(i) actual release site (m2): … m2

(ii) wider release site (m2): … m2

Not relevant (see below).

(c) Proximity to internationally recognised biotopes or protected areas (including



drinking water reservoirs), which could be affected:

Not relevant (see below).

(d) Flora and fauna including crops, livestock and migratory species which may

potentially interact with the GMO

Sections (b)-(d) above are not considered relevant. Environmental release of the ASP0113 product is not intended beyond the treatment of trial subjects. Treatment will be on an outpatient basis. Proximity to internationally recognised biotopes or protected areas (including drinking water reservoirs) cannot be specified under these circumstances as the movement of treated subjects will not be restricted. Flora and fauna including crops, livestock and migratory species which may potentially interact with ASP0113 or the VCL-6365 or VCL-6368 pDNAs under these circumstances include bacteria present on or in human subjects as detailed under B(8)(d) above.

4. Method and amount of release

(a) Quantities of GMOs to be released:

Up to 65 mg of VCL-6365 and 65 mg of VCL-3638.

(b) Duration of the operation:

Each patient will receive a total of 5 injections of ASP0113 over a 6 month period. The dosing period is expected to take approximately 3 years to complete for all subjects.

(c) Methods and procedures to avoid and/or minimise the spread of the GMOs beyond the site of the release

Safety containment measures for handling of the ASP0113 product will follow institutional guidelines. Subjects will not be isolated or subjected to containment following treatment.

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

The product will be administered to trial subjects at clinical sites in an enclosed facility at ambient temperature.

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.

Potential environmental impacts of ASP0113 have not been assessed at clinical sites in the U.S. Based on the clinical and nonclinical experience reviewed under E(1)(d) above, ASP0113 poses no significant risk to human or animal health.



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 Hominidae

(iii) genus Homo

(iv) species H. 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)

ASP0113 is an immunotherapeutic vaccine, the detailed mechanism of action has not been established. The VCL-6365 and VCL-6368 pDNAs are designed to elicit specific immune responses to the encoded gB and pp65 antigens following cellular uptake and expression in trial subjects receiving ASP0113.

In the VCL-CB01-101 phase 1 study, the 1-mg and 5-mg doses of ASP0113 induced pp65 T-cell responses and gB antibody responses in CMV-seronegative volunteers. ASP0113 induced pp65 T-cell responses, but not gB antibody responses, in seropositive volunteers [Wloch, 2008].

In the VCL-CB01-202 phase 2 study, the mean gB IFN-gamma T-cell responses were numerically enhanced in the ASP0113 group relative to the placebo group at all time points after day 84, and there was no significant different between groups. The geometric mean gB antibody levels were significantly higher in recipients in the ASP0113 group compared with recipients in the placebo group by day 365 (P = 0.009, Wilcoxon rank sum test)[Kharfan-Dabaja, 2012]. In a post-hoc analysis of the CMV pp65 T-cell response, an ordinal logistic regression model was used because the CMV pp65 T-cell responses had a U-shaped distribution in both the ASP0113 group and the placebo group. Therefore, 3 categories of pp65 assay data, defined as < 750, 750 - 2999, and ≥ 3000 SFU/106 PBMC (with boundaries at ½ and 2 the mean of about 1500 SFU/106 PBMC defined in normal CMV+ individuals), were used for analysis of the data. The treatment effect P value of 0.022 for day 56 through day 365 suggests that the vaccine was immunogenic [Kharfan-Dabaja, 2013].



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

Other potentially significant interactions with other organisms in the environment are not expected for the reasons given under A(3)(c), B(8)(d), and E(1)(d) above.

4. Is post-release selection such as increased competitiveness, increased invasiveness for the

GMO likely to occur?


Give details

See A(3)(c), B(8)(d), and E(1)(d) above.

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

The VCL-6365 and VCL-6368 pDNA components of ASP0113 could potentially disseminate into soils and wastewater through accidents in transport or improper handling and disposal at clinical sites. The possibility of dissemination and establishment in soil or wastewater ecosystems (e.g., uptake and replication in permissive bacteria) in these cases is considered remote based on the data and considerations provided above under A(3)(c), B(8)(d), and E(1)(d) above.

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

(i) 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 …

No unintentional significant harm to non-target organisms is anticipated.

7. Likelihood of genetic exchange in vivo



(a) from the GMO to other organisms in the release ecosystem:

The likelihood of genetic exchange between VCL-6365 or VCL-6368 and bacteria present on or in treated human subjects is considered remote for the reasons stated under A(3)(c) and B(8)(d) above. The likelihood of recombination between human CMV and hCMV-derived elements of the VCL-6365 and VCL-6368 pDNAs is limited by the requirement that replicating CMV virus and the pDNAs must be present in the same cell for recombination to occur, and the low likelihood that this would occur. During acute infection, CMV replication may occur in fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages [Sinzger, 1995]. However, it has not been reported to replicate in skeletal muscle cells (i.e. myofibers) that are the primary site for expression of pDNA-encoded transgenes following intramuscular injection [Doh, 1997]. In addition, the DNA sequence homology of the gB and pp65 coding sequences with their wild-type hCMV counterparts is reduced as a consequence of the nucleotide sequence alterations resulting from the codon optimization process, which would limit the ability of the coding sequences to form stable heteroduplexes required for homologous recombination.

(b) from other organisms to the GMO:

See G(7)(a) above.

(c) likely consequences of gene transfer:

Based on the data and considerations provided under A(3)(c) and B(8)(d) above, the likely consequence of gene transfer of the VCL-6365 or VCL-6368 pDNAs or the kanamycin resistance gene to bacteria present in trial subjects or the natural environment is considered minimal. In the case of homologous recombination between the CMV-derived regulatory elements of VCL-6365 and VCL-6368, the likely consequences of gene transfer are considered minimal because the promoter, 5´ UT, and Intron A regions of the pDNAs are unmodified from their natural forms. In the case of homologous recombination of the gB and pp65 coding regions with their wild type counterparts, the likely consequences of gene transfer are considered to be neutral (in the case of pp65) or detrimental (in the case of gB). The wild-type pp65 tegument has a putative kinase activity which has been eliminated from the VCL-6368 pp65 protein by deleting amino acids 435-438 encoding the putative ATP-binding site (RKRK) [Yao, 2001]. However, the wild-type pp65 protein has been reported to be dispensable for growth of the virus in cell culture [Schmolke, 1995]. The deletion of the transmembrane and cytoplasmic domains of the gB protein, which anchor the full-length gB protein to lipid membranes and may contribute to the fusion of viral particles with cell membranes [Bold, 1996], would be expected to negatively impact the capacity of viral recombinants containing this deletion to form infectious viral particles.

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 studies of ASP0113 or the VCL-6365 or VCL-6368 pDNAs have been performed in simulated natural environments.

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

No significant interactions with biogeochemical processes are anticipated.

H. INFORMATION RELATING TO MONITORING

1. Methods for monitoring the GMOs

Methods for monitoring include PCR assays utilizing primers specific for VCL-6365 and VCL-6368 pDNA sequences that have previously been developed for detection of the pDNA in mammalian tissue samples. However, based on the nonclinical and clinical safety experience with ASP0113 described under E(1)(d) above, no monitoring is planned.

2. Methods for monitoring ecosystem effects

…

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

…

4. Size of the monitoring area (m2)

… m2

5. Duration of the monitoring

…

6. Frequency of the monitoring

…



I. INFORMATION ON POST-RELEASE AND WASTE TREATMENT

1. Post-release treatment of the site

No post-release treatment of study sites is planned. A clinical evaluation of trial subjects (physical exam and vital signs) will be performed on each administration visit. The subject may leave the clinic once the investigator deems the subject to be clinically stable. Periodic evaluations will be performed at scheduled visits throughout the duration of treatment. Subjects will continue visits to the site for up to 12 months after Hematopoietic Cell Transplantation, and will be followed for 4.5 years after their last dose by way of questionnaire for long-term safety related to the DNA vaccine. Items to be assessed by questionnaire and available patient records include; mortality, development of any new/recurrent cancer, development of infection requiring hospitalization or resulting in death, and erythema or induration at sites of study drug injection.

2. Post-release treatment of the GMOs

Disposal of used syringes, gloves or other items that may potentially contain residual amounts of medicinal product will be performed in accordance with institutional procedures of the clinical site.

3. (a) Type and amount of waste generated

The amount of medical waste generated on a daily basis at clinical sites will generally not exceed the contents of one vial (< 5 mg pDNA). The type of medical waste will generally include residual liquid ASP0113 medicinal product within used vials and syringes, and trace amounts potentially residing on used gloves and bandages applied to injection sites.

(b) Treatment of waste

Treatment of medical waste will be performed in accordance with institutional procedures of the clinical site.

J. INFORMATION ON EMERGENCY RESPONSE PLANS

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

Instructions on spill response procedures will be provided to clinical sites. Site personnel responsible for spill cleanup will be instructed to wear proper personal protective equipment (protective gloves, safety glasses, and clothing).



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

Spilled liquids will be absorbed with common absorbent materials and placed in appropriate containers for disposal.

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

Disposal of spilled liquids will be performed in accordance with institutional procedures. Disposal or sanitation of plants, animals, soils, etc. exposed to the product is not mandated due to the low risk of resultant harm.

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

Because the medicinal product does not appear to pose a significant risk to human health or the environment, specific plans for protection have not been prepared.



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asp0113 part 1 (council decision 2002/813/ec) summary notification

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