Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1997-03-07
1999-01-12
Guzo, David
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4353201, 435366, 435372, 4353721, C12N 1511, C12N 1563, C12N 510
Patent
active
058587446
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/EP95/03175, filed Aug. 10, 1995.
The invention concerns retroviral vector hybrids and their use for gene transfer in particular for gene transfer into haematopoietic stem cells (ES).
Retroviral vectors with replication defects are at present a standard in gene transfer and gene therapy applications on cells of the human blood-forming system (A. D. Miller (1992) (1), R. C. Mulligan (1993) (2), R. Vile and S. J. Russell (1994) (3)). Their main advantages are: sequences transferred by it into the host genome of mitotically active cells infection conditions complications have so far not occurred in numerous applications on humans.
The most frequently cited advantage of retroviral vectors, their high gene transfer efficiency, only partly applies to applications on the blood-forming system. Only late maturing blood-forming precursor cells can preferably be infected with conventional vectors based on the Moloney murine sarcoma virus (MoMuSV); 30-95% of the precursor cells transduced with these vectors exhibit either no or an inadequate expression of the transferred genes due to primary silencing mechanisms (4); moreover after a longer residence period in vivo the retrovirally controlled gene expression is weakened in a high percentage of the initially expressing cells up to the point of non-function due to secondary silencing (Palmer et al. (1991) (5), Brenner et al. (1993) (6)).
It has been demonstrated that also in embryonic cells such as embryonic carcinoma cells (myeloid (non-lymphatic) derivatives of haematopoietic stem cells) the viruses are indeed integrated but the expression of the integrated provirus is blocked.
Mutations and derivatives of MoMuSV are known from Stocking et al. (1993) (21) which can be used to improve the retroviral gene expression in such cells. Such hybrids are obtained by point mutations in the LTRs especially in the U3 region. For example the binding of the ECF-1 repressor can be reduced by a point mutation at -345 of MoMuLV. A point mutation which creates a binding site for SP-1 (e.g. point mutation at -166 (S. McKnight and R. Tijan (1986) (16)) is equally advantageous. It is furthermore advantageous to also introduce mutations outside the LTR regions. It has been shown that an 18 bp region which adjoins the 5'-LTR directly downstream is a negative regulation element (NRE; silencer binding element). Point mutations in this element enable the retroviral transcription to be improved (murine embryonic stem cell virus, MESV; M. Grez et al (1990) (22)).
The object of the invention is to provide retroviral vector hybrids which can be used to further improve retroviral gene expression especially in haematopoietic stem cells and their myeloid (non-lymphatic) derivatives.
The invention concerns retroviral vector hybrids which are characterized in that they MoMuSV in the leader region as a U5 region and/or tRNA primer binding site and (F-MuLV) as U3 and R regions in 3'-LTR.
The vector hybrids according to the invention preferably contain the leader region of MESV as the leader region. In a further embodiment of the invention the U5 region and/or preferably the tRNA primer binding site can also be derived from MoMuSV.
The vector hybrids according to the invention also preferably contain the LTR from a Friend murine leukemia virus (F-MuLV) as the 3'-LTR and thus also the U5 region from F-MuLV in addition to the U3 and R regions from MoMuLV and MESV.
The vector hybrids can be replication-defective as well as replication-competent.
A replication-defective vector is understood as a vector which contains no retroviral gene functions (gag, env, pol) and therefore cannot independently form virus particles. However, the vector usually contains a packaging function (psi). In order to form infectious retroviral particles a packaging cell line which contains the gene functions gag, env and pol in a stable form (as an episome or integrated into the genome) is transfected with the replication-defective DNA vector according to the invention. RNA transcripts are formed which
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Baum Christopher
Ostertag Wolfram
Stocking-Harbers Carol
Boehringer Mannheim GmbH
Guzo David
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