Work package 3

Fig. 1: WP 3 Partner Participation (WP Lead Underlined)

Optimization, Validation and Definition of Release Criteria of the Gene Therapy Medicinal Product

Work package 3 deals with the assessment, the optimisation and the validation of the quality and safety profile for each component, implemented in the manufacturing process of the Gene Therapy Medicinal Product (GTMP) as well as the final patients’ surgery procedure.

These components are realised by partners (Fig. 1) as following:

Component	Partner
Iris (IPE) and retinal pigment epithelial (RPE) cells extracted in analogy to patient`s autologous pigment epithelial cells from Human biopsies / iridectomies; Ex vivo human ocular donor tissue; Ex vivo bovine ocular cadaver tissue (due to availability rates of human material).	UD-G UKA UN UNIGE
The plasmid components applied via electroporation transfection technology PEDF gene encoding transposon plasmid DNA SB transposase plasmid DNA	CNRS
The transfection equipment Electroporation device Transfection/ storage buffer	IGEA 3P

Prior to the animal testing it was demonstrated by in vitro experiments that freshly isolated IPE as well as RPE cells can be transfected with the Sleeping Beauty transposon system with high efficiency and cell viability. Transfection efficiency and long-term protein expression (proofed via Western Blot analysis, Fig. 2) as well as immunohistochemistry and gene expression analysis were used to analyse morphological and genomic stability. Additionally, quantity and quality of the isolated cells were evaluated by microscopy, standardised cell viability assays and cell culture analysis. The consistency of the final product in adequate low cell amounts to the prospective patients’ biopsy is a key element of this WP.

Fig. 2: Illustration of Transfection Procedure — Fig. 2: Illustration of the Transfection Procedure with Pre-Cultured (A) or Isolated and Freshly Transfected (B) Iiris Pigment Epithelial (IPE) Cells Using the Sleeping Beauty Transposon System (Transfection Device (in vitro) (C), e.g. Neon® Transfection System, Invitrogen®. (D) Transfection efficiency and longterm protein expression is proofed by Western Blot analysis. The main focus in the experimental set-up is announced in issue 1-3.

The electroporation technique, a standard technique in gene delivery, is evaluated in relation to transfection efficiency and cell compatibility. An electroporation device with cell holding cuvettes, electrodes and buffers will be adapted to the electroporation procedure of the required low cell amount (5’000 to 10’000 cells) yielding by the patient`s biopsies. The optimal transfection conditions are used for the transfection of the primary human IPE cells.

The validation of defining release criteria and in-process testing will be an important guideline for the training of the personnel responsible for GTMP (Gene Therapeutic Medicinal Product) production during the clinical trial in Switzerland.

Work Package 3 will terminate with Milestone 2, the manufacturing authorisation for transfected IPE and RPE cells as GTMP (Gene Therapeutic Medicinal Product).