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Development of gene transfer vectors for recessive dystrophic epidermolysis bullosa (RDEB)

Project leadAlessandra Recchia, PhD
Organisation IMBA Institute of Molecular Biotechnology, Vienna
Project budgetEUR 74,000.00
Start date / Duration01. Feb 2011 / 30 months
Funder(s) / Co-Funder(s) DEBRA Austria, MSAP/EBEP Recommended
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the human type VII collagen gene (col7A1). Individuals with RDEB lack type VII collagen and anchoring fibrils, structures that attach epidermis and dermis. The current lack of treatment for RDEB is an impetus to develop gene therapy strategies that efficiently transfer and stably express genes delivered to skin cells in vivo. Several vector platforms exist for the delivery of therapeutic gene constructs into cells. Virus-based vectors are widely used in gene therapy applications, because they allow efficient delivery of the therapeutic gene into a target cell population. Gene therapy, or the transplantation of cultured skin that has been genetically modified by the introduction of a therapeutic gene, might provide a permanent correction of some forms of EB, as indicated by correction of skin fragility in a patient with junctional EB (JEB). However, current gene transfer technology based on modified integrating retroviruses carries a significant potential for insertional mutagenesis and activation of protooncogenes by elements in the long terminal repeats of the virus. The objective of this project is the development of a safer technology as an alternative to viral vectors that aims at directly correcting the mutated, disease-causing gene. The technology is based on the Sleeping Beauty (SB)-derived transposon. The transposon-based gene vectors represent a non-viral approach with an integrating feature.

Scientific summary

The Sleeping Beauty, a reconstructed transposon from fish, has been widely used in gene transfer studies and represents a milestone in the application of transposition-mediated gene delivery invertebrates. SB vectors can provide long-term gene expression in vivo, and there has been a steady growth in interest in applying the SB system for the treatment of a number of diseases. In its natural configuration, the SB transposon consists of a single gene encoding the SB transposase that catalyzed the strand-cleavage and strand-transfer reactions involved in the transposition process. The interest of the scientific community was converted in an extensive engineering effort in order to ameliorate the transposition activity modifying both components of the SB system, the transposon IR and the transposase enzyme. The last generation is based on conventional (T2) and sandwich (SA) shape of transposon and on hyperactive (SB100X) transposase.
For gene delivery purposes, SB is typically used as a two-component vector system, in which a genetic cargo (therapeutic gene) is flanked by the transposon IRs, and the transposase is supplied in trans. Transposition is catalyzed by the transposase which excises the element from its donor site, and reintegrates it into a target site in a process called cut-and-paste transposition. The transposon provided into a plasmid vector can be integrated into the human genome thanks to the activity of the transposase SB100X provided in trans by a second plasmid.
Basically, co-transfection of transposon/transposase carrying plasmid could result in the integration of the therapeutic gene into the human genome. This procedure is called gene addition strategy. We have generated a panel of transposon vectors carrying the Venus reporter gene to compare the conventional T2 and the sandwich SA transposon because data from literature reported that the SA transposon has an increased capacity for large gene (as the collagen VII gene) integration. The experiments performed in the first year of project demonstrate that although the sandwich transposon mediates a genuine cut and paste mechanism of transposition with reasonable efficiency in human cells, the transposition efficiency of reporter expression cassette in SA transposon is not better than the T2 transposon, so we decided to utilize the T2 transposon. We have constructed a SB transposon carrying the col7A1 gene between the IR sequences required for the transposition (pT2Col7).
In presence of the transposase (SB100X) plasmid, stable integration of the corrected collagen VII gene could be obtained in the human genome.
We started to molecularly characterize the collagen VII gene addiction by pT2Col7 in HeLa cells that do not express the endogenous collagen VII gene. We demonstrated that the col7A1 cassette was correctly integrated into the genome without rearrangements previously observed with retroviral and lentiviral vectors. Moreover the vector copy number of the exogenous collagen VII gene integrated into the genome is comparable with a MLV-derived retroviral vector carrying the same cassette. Lastly the collagen VII protein level produced by several clones is comparable to the wild type primary keratinocytes. To better address this issue we have translate the experiment in a proper in vitro cell model, the immortalize Col7A1-deficient keratinocytes obtained from RDEB patients.

What did this project achieve?

We have confirmed the genuine cut and paste mechanisms of the pT2Col7 transposon in these immortalized Col7A1-deficient keratinocytes, the absence of rearrangements and the “physiological” amount of collagen VII protein. Based on these encouraging results we moved to primary Col7A1-deficient keratinocytes from donor to test the transposition efficiency with the pT2Col7. Unfortunately, the transfection efficiency of primary cells was very low, lower than 10% although the transposition efficiency was around 40%.
Despite the enormous potential of the Sleeping Beauty transposon, the plasmid transfection procedure of the transposon/transposase integrating system in RDEB keratinocytes remains an obstacle to its practical application in gene therapy. To overcome this limitation we incorporated the pT2.Col7 transposon/transposase system by using adenoviral vector able to efficiently and transiently infect the primary keratinocytes.

In the last extended 6 months of the project, we have restored the collagen VII protein production in immortalized RDEB keratinocytes. Experiments performed in primary RDEB keratinocytes showed that this strategy based on pT2.Col7 transposon/transposase system carried by adenoviral vectors is feasible and leads to a significant increase of collagen VII expression.


gene therapy
IMBA Vienna
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