The success of gene therapy techniques depends largely on the ability to achieve a combination of stable chromosomal integration and high-level, regulated expression of transferred genes. Regulated gene expression is most easily achieved by means of large DNA fragments containing extensive cis-acting regulatory regions. For example, gene therapy for xcex2-globin disorders may require high-level, position-independent expression of extended gene and LCR sequences.
Many current techniques allow efficient transient transfection of cells in vitro and in vivo with large DNA fragments. However, subsequent chromosomal integration is very inefficient. To overcome low levels of integration, retroviral vectors which integrate very efficiently in permissive cells can be used. However, such vectors are greatly limited by constraints of size and sequence composition.
There are also many other techniques available for stable integration of transgenes in mammalian cells (Kriegler, M. (1990) Gene Transfer and Expression. A Laboratory Manual, Stockton Press, New York); and (Wolf, J. A. (1994) Gene Therapeutics: Methods and Application of Direct Gene Transfer, Birkhauser, Boston). However, these methods result in integration at random chromosomal locations of an uncontrolled number of transgene copies that express at levels that generally cannot be predicted or reproduced with precision because of position-effects. The inability to control the site of integration, the number of integrated copies and the level of expression of transgenes has impeded progress in studies of both gene expression and the physiological effects of transgenes.
Systems which can perform site-specific chromosomal integration efficiently therefore have wide utility. The first site-specific chromosomal integrations in mammalian cells were based on integration of a single Lox or FRT site on a chromosome followed by trapping of rare integration events (O""Gorman et al. (1991) Science 251:1351-1355; and Sauer, B. (1994) Current Opinion in Biotechnology 5:521-527). These pioneering methods had three limitations: 1) they were quite inefficient, 2) the entire plasmid was integrated, and 3) a positive selectable marker was left in the chromosome after the integration. The low efficiency of these methods is due to the reversibility of the recombination reaction: after integration the transgene is re-excised if the two identical Lox or FRT sites that flank the transgene recombine with each other. Since the excision reaction is intra-molecular while the insertion reaction is inter-molecular, excisions are favored.
U.S. Pat. No. 4,959,317 discloses the use of Cre-Lox site-specific recombination to achieve gene transfer in eukaryotic cells (Sauer et al. (1993) Methods in Enzymology 225: at 898). The target site of the CRE recombinase is a 34 bp sequence that consists of two inverted 13 bp CRE-binding sites separated by an eight base spacer within which the recombination occurs (Hoess, et al. (1984) Proc. Nat. Acad. Sci. (USA) 81:1026-1029).
Additional site-specific DNA recombination systems which provide more efficient and stable integration of transgene sequences into genomic DNA, preferably without the use of a positively selectable marker, would be greatly beneficial.
The present invention provides methods and compositions for achieving efficient and stable site-specific DNA recombination using a recombinase/lox system, such as the Cre/lox system or the Flp/frt system. In one embodiment, the method comprises contacting a recombinase (e.g., Cre or Flp) with (a) an acceptor vector comprising two incompatible lox sequences, L1 and L2, and (b) a donor vector comprising a selected DNA flanked by the L1 and L2 sequences, or sequences which are compatible with the L1 and L2 sequences, thereby causing transfer of the selected DNA from the donor vector into the acceptor vector by recombination at the compatible lox sequences. In a preferred embodiment, the acceptor vector is a retroviral vector or an adeno-associated vector.
In another embodiment, the invention provides a method of transforming a cell with a selected DNA comprising, in any order, the steps of introducing into the cell an acceptor vector which integrates into the genome of the cell, the acceptor vector comprising two incompatible lox sequences, L1 and L2, (b) introducing into the cell a donor vector comprising the selected DNA flanked by the L1 and L2 sequences, or sequences which are compatible with the L1 and L2 sequences, and (c) contacting L1 and L2 with a recombinase, such as Cre or Flp, thereby causing transfer of the selected DNA from the donor vector into the acceptor vector. The recombinase can be introduced into the cell in the form of a protein or a gene encoding the protein.
In another embodiment, the invention provides a vector selected from the group consisting of retroviral vectors and adeno-associated vectors comprising two incompatible lox sequences, L1 and L2.
In another embodiment, the invention provides a method of achieving site-specific recombination by providing a donor DNA comprising two inverted lox sequences, and an acceptor DNA comprising the same two inverted lox sequences contained in the donor DNA, and then contacting the donor and acceptor DNA with a recombinase (e.g., Cre or Flp). Preferably, the acceptor DNA is integrated into the genome of a host cell prior to contact with the recombinase (e.g., by homologous recombination), so that recombination results in site-specific genomic integration of a desired transgene or other polynucleotide. In another preferred embodiment, the donor DNA is present in excess of the acceptor DNA. Suitable lox sequences comprise the nucleotide sequences of SEQ ID NO:1, SEQ ID NO:2 and inversions thereof. The donor and acceptor DNAs can further contain selectable markers which are generally positioned between the two inverted lox sequences to help select for cells which have undergone the desired recombination.
The methods and compositions of the invention can be used in methods of in vivo and In vitro gene transfer (e.g., gene therapy) to cause efficient and stable site-specific (targeted) integration of transgene sequences. By controlling the site or position of integration of a transgene into the genome of a cell, expression levels can be predictably determined. For example, in cells, the invention can be used to produce desired proteins (e.g., drugs) by insertion of transgenes at pre-selected chromosomal locations where expression of the transgene will be high. Similarly, the invention can be used to develop xe2x80x9cdesigner proteinsxe2x80x9d by insertion of multiple versions of a gene or DNA (e.g., related variants) at the same locus to test the various versions of proteins produced in a context in which the proteins are all produced in the same amount. The invention can also be used to study and to identify genetic elements that control position effects.
In addition, the invention can be used in vivo to create transgenic mammals and/or plants. For example, animal models of human disease can be generated, particularly if multiple genes have to be expressed at well regulated levels. Animals and/or plants can be created which contain target lox sequences (e.g., inverted or incompatible) at chromosomal locations that are not subject to position effects or to desired position effects for directing expression of a gene of interest. This allows for the generation of animal models and/or plants with, for example, higher resistance to disease or improved physical/functional characteristics.