Conditional gene vectors regulated in cis

Reference Number TO 01-00624

Challenge

Most gene vectors encompass viral genetic components which provide the necessary cis-acting elements (i.e. promoters, enhancers) to express genes of therapeutic interest. In the recipient cell, gene vectors can be transiently present or maintained for a long period of time through chromosomal integration. Such integration disrupts the genetic integrity of the host chromosome. As a result, integrating viruses, viral vectors, and DNA-based gene vectors can act as insertional mutagens. This problem is avoided with gene vector systems which are maintained as extrachromosomal units in the recipient cell. However, no state of the art system teaches the regulation of vector maintenance in a target cell.

Technology

The Epstein-Barr virus (EBV) plasmid replicon (oriP), consists two essential elements (FR repeats and DS elements) and supports efficient DNA replication in cells selected to retain the plasmid at several copies if EBNA1 is provided. EBNA1 binds specifically and promotes the attachment of the oriP plasmids to chromatin via so-called AT-hook domains. Such AT-hook domains are also found in HMGA1a which binds to mitotic and interphase chromatin throughout the cell cycle.

The invention presents a combinatorial approach of the oriP replicon and the well known tet-regulated gene expression system. (1) The AT-hook containing regions of HMGA1a are fused to TetR (trans-acting). Hybrid plasmids can be maintained extrachromosomally for long time and can be regulated upon doxycycline treatment. (2) Replacing the DS elements of the oriP (cis-acting) with tetO sites make viral factors entirely dispensable. Fusion proteins harboring the TetR DNA binding domain can recruit the pre-replicative complex to the origin of replication as well. As a consequence, new gene vectors can be designed which carry all the elements needed and necessary within one plasmid.

Commercial Benefit and Opportunity

Such a regulation of vector maintenance could bring about the advantage to allow for reversible transfer of genetic information into a target cell by adding an agent which is specifically adapted to remove the vector from the target cell.

The technology is available for non-exclusive licensing. Parties interested in collaborative research and development are highly welcomed.

Patent Situation

A European patent has been granted (EP1634955), a US application is pending (2007/0184028).

Developmental Status

An in vitro proof-of-concept has been provided demonstrating that EBV-antigen specific CD4+ T cells are efficiently expanded from peripheral blood of EBV-positive donors using PBMC pulsed with EBV-VLP as stimulators. Experiments to use such EBV-VLP as a vaccine are ongoing.

Relevant Publication

Thomae et al. (2008), PNAS 105, 1692-1697.