Remissvar till Läkemedelsverket rörande GMO

Läkemedelsverket

registrator@mpa.se

 Diarienummer 5.1-2017-97435

Remissvar rörande

Klinisk prövning med genmodifierad organism - tillfälle att lämna synpunkter

den 11 januari 2018

Den aktuella prövningen avser studieläkemedlet CPK850 som består av en rekombinant adeno-associerad virus 8 (AAV8)-vektor som innehåller den humana RLBP1-genen.

 https://lakemedelsverket.se/Alla-nyheter/NYHETER---2018/Klinisk-provning-med-genmodifierad-organism---tillfalle-att-lamna-synpunkter1/

 

After reading the application there are some questions that are not answered in the application

 

Page 4 middle section states:

3. Geographical distribution of the organism (a) Indigenous to, or otherwise established in, the country where the notification is made:

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

(b) Indigenous to, or otherwise established in, other EC countries:

         (i) Yes                 (X)

If yes, indicate the type of ecosystem in which it is found:

         Atlantic              (X)

         Mediteranean     (X)

         Boreal Alpine     (X)

         Continental        (X)

         Macaronesian    (X)

                                    (X)

 

         (ii)    No                                  (.)

         (iii)    Not known                     (.)

 

 

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

 

Qustions:

  1. What region is not included below Macronesianin the text of p 4 above?

 

  1. How is the AAV8 recombinant vector propagated/replicated?
    It is stated that the vector is not capable of replication. Then how is the vector replicated/propagated?
    On p 8 it states: Transfection of HEK293 cells with three different plasmids carrying the various components needed to produce the vector. Residual plasmid DNA is then removed by Benzonase treatment and downstream processing.
    This is a too short description of how the vector is replicated/produced. The efficiency of this three vector approach seems to be a complicated and uncertain way to produce the complex vector. There are at least three plasmid DNA sequences that should be concatenated in the right direction and in the right order. There are 3! ways to put the pieces together when there is equal concentrations of each piece. Then the pieces can be concatenated in variable right or wrong orientations. This means a yield of 1/4!= 1/24th of the vectors is the correct one. 23 out of 24 are a false vector that can not be used.
    How do they retrieve the right vector? Please give a clear and full description of the procedure including how to produce and pack the DNA in the viral capsid. Otherwise what is the carrier and cell transmembrane transporter of the vector DNA? Please give a clear and full description of the procedure.
    How much of the HEK293 cell DNA or RNA is a contaminant of the Plasmid DNA? Please give a clear and full description of the amounts of contaminating DNA and RNA in actual values in M (moles/L with appropriate prefix).

 

  1. How is the AAV8 recombinant vector selected without antibiotic or other resistant factor(s)?
    Please give a clear and full description of the procedure.

 

  1. How many base pairs outside the vector gene(s) are included in the expression cassette?
    Any extra base pairs may give rise to scRNA and snRNA that can interfere with the normal cell regulation, see e.g. http://bioscience.jbpub.com/cells/MBIO5245.aspx
    Please give a full DNA sequence of the expression cassette with the position of the different genes clearly marked from BP to BP. Are there any splice donor/acceptor sites in the sequence?

 

  1. How many base pairs are included outside the expression cassette?
    Any extra base pairs may give rise to scRNA and snRNA that can interfere with the normal cell regulation, see e.g. http://bioscience.jbpub.com/cells/MBIO5245.aspx Please give a full DNA sequence outside of the expression cassette with the position of the different genes clearly marked from BP to BP and reading frame(s).

 

Page 6

  1. The text states: Wild-type AAVs and AAV-based gene therapy vectors have the ability to form extrachromosomal concatemers that remain episomal for extended periods of time.
    How long is extended periods of time? Seconds, hours, weeks, years? Please specify.   

 

Page 12

  1. The application states:
    Receiving environment for the shed vector particles is most likely waste water and ambient temperature.             
    How can ambient temperature be a recipient of shed vector particles? Please specify.

 

Summary

This application is way too insufficient to be accepted. There is no description how the plasmid is produced, there is no information on scRNA and snRNA contamination, nor any information on HEK293 cell DNA and RNA contamination.

 

How does the vector infect the retinal target cells as AAV 8 has higher affinity to liver cells than retinal cells? The vector has to pass through at least one cell membrane that is usually impenetrable for most molecules and particles.

 

There are no scientific literature sources to all the allegations made in the application.
Every statement and procedure must be confirmed by published scientific literature posts. Otherwise the allegations are just some kind of unproven hypothesises.

 

I recommend that the present application will be rejected as there is insufficient information and some errors and omissions that indicates an inferior level of sufficient knowledge for the investigators to be able to accomplish the intended trial.

 

 

Mora 2018-01-15

 

 

Björn Hammarskjöld
Assistant professor in Pediatrics
Ph.D in Biochemistry
Independent Senior Molecular Biologist
bjorn@hammarskjold.nu

 

 

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