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Patents on basic co-integrated vectors and methods – Patents granted to Max-Planck

Specific Patent Information

Patent Number Title, Independent Claims and Summary of Claims Assignee
EP 116718 B2

  • Earliest priority – 13 January 1983
  • Filed – 22 December 1983
  • Granted – 8 May 1996
  • Expected expiry – 21 December 2003
 Title – Process for the introduction of expressible genes into plant cell genomes and agrobacterium strains carrying hybrid Ti plasmid vectors useful for this process

Claim 1A vector combination consisting of:

A) an acceptor Ti plasmid which is substantially free of internal T-DNA sequences of wild-type Ti plasmid and incapable of inducing tumors in plants comprising:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA sequence derived from a cloning vehicle, located between the two border sequences; and
(iii) a DNA segment of a wild type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes, and

B) an intermediate cloning vector, said cloning vector comprising:
(i) at least one gene of interest under the control of a promoter capable of directing gene expression in plants; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous with the DNA sequence (ii) in said acceptor Ti plasmid permitting a single crossover event.
Claim 5A vector combination consisting of:

A) an acceptor Ti plasmid which is incapable of inducing tumors in plants being free of border sequences and internal T-DNA sequences of a wild-type Ti plasmid, comprising:
(i) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(ii) a DNA sequence derived from a cloning vehicle, and

B) an intermediate cloning vector comprising:
(i) a cloning vehicle segment containing the two border sequences of the T-region of a wild-type Ti plasmid; and
(ii) a DNA sequence located between said two border sequences which is homologous with the DNA sequence (ii) in said acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(iii) at least one gene of interest under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome.
Claim 15A hybrid Ti plasmid vector obtained by cointegration either between

A) an acceptor Ti plasmid which is incapable of inducing tumors in plants comprising:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA sequence devoid of oncogenic internal T-DNA regions of the wild-type T-DNA, derived from a cloning vehicle, located between the two border sequences, and containing a DNA sequence which is homologous with at least a part of a DNA sequence in an intermediate cloning vector permitting a single crossover event; and
(iii) a DNA segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes; and

B) an intermediate cloning vector comprising
(i) at least one gene of interest under the control of a promoter capable of directing gene expression in plants; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous with the above DNA sequence (ii) in the acceptor Ti plasmid; or between
I) an acceptor Ti plasmid comprising:
(a) a DNA segment of the wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence derived from a cloning vehicle; and II) an intermediate cloning vector comprising:
(a) a cloning vehicle segment containing the two border sequences of the T-region of a wild-type Ti plasmid and a DNA sequence located between said two border sequences which is homologous with the above DNA sequence (b) in the acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(b) at least one gene of interest under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome; III) said hybrid Ti plasmid vector comprising at least:
(1) the two border sequences in the T-region of a wild-type Ti plasmid;
(2) non-oncogenic DNA sequences derived from a cloning vehicle;
(3) a DNA segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of wild type Ti plasmid by Agrobacterium into plant cell genomes; and
(4) at least one gene of interest under the control of a promoter capable of directing gene expression in plants which is located between the two border sequences.

Designated contracting States at the time of grant are: Austria, Belgium (patent lapsed as reported on INPADOC), Switzerland (patent lapsed as reported on INPADOC), Germany, France, United Kingdom (patent lapsed as reported on INPADOC), Italy, Liechtenstein, Luxembourg, Netherlands (patent lapsed as reported on INPADOC), Sweden (patent lapsed as reported on INPADOC).

The invention claimed in patent EP 116 718 B2 relates to vector combinations consisting of acceptor Ti plasmids and intermediate cloning vectors that result in the formation of a hybrid Ti plasmid. The hybrid plasmid is also claimed.

In certain claims, the region between border sequences is said to be substantially free of internal T-DNA sequences of a wild type Ti plasmid.  How free “substantially free” is, is not defined in the disclosure.  Further, the claims do not exclude the presence of sequences from a mutated Ti plasmid.

The co-integration of both plasmid types is achieved through a single crossover event. The homologous region derives from a cloning vector and is present in both the acceptor plasmid and the intermediate cloning vector.

The resultant hybrid plasmid contains at least:

  • a gene of interest under the control of a promoter, located between two T-DNA borders which must be from a wild-type Ti plasmid; and
  • a DNA sequence from Ti plasmid that is essential for T-DNA transfer into the plant genome.

Max-Planck-Gesellschaft zur Förderung der Wissenschaften E.V.
AU 546542 B

  • Earliest priority – 13 January 1983
  • Filed – 13 January 1984
  • Granted – 5 September 1985
  • Patent expired – 12 August 2004
 Title – Introduction of exressible genes into plant genomes and Agrobacterium strains carrying hybrid Ti plasmid

Claim 1Acceptor Ti plasmid comprising: (i) two border sequences of the T-region of the wild-type Ti plasmid; and
(ii) a non-oncogenic DNA segment derived from a cloning vehicle located between the two border sequences containing a DNA sequence which is homologous with at least a part of a DNA in an intermediate cloning vector permitting a single crossover event; and
(iii) a segment of the wild type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes.
Claim 2Acceptor Ti plasmid comprising: (i) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(ii) a DNA sequence derived from a cloning vehicle which is homologous with a DNA sequence of an intermediate cloning vector which contains the two border sequences of the T-region of the wild-type Ti plasmid.
Claim 9Hybrid Ti plasmid vector comprising: (i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) non-oncogenic DNA segments derived from a cloning vehicle;
(iii) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmids into plant cell genomes; and
(iv) at least one gene of interest which is located between the two border sequences.

The patent AU-B-546 542 claims acceptor Ti plasmids and intermediate cloning vector molecules that through a single crossover event form a co-integrated or hybrid Ti plasmid. The hybrid plasmid contains a gene of interest located between two border sequences.

The resulting hybrid plasmid disclosed in both of the patents is incapable of inducing tumors in plants transformed via Agrobacterium;however, the “comprising” language used in the claims doesn’t exclude the addition of oncogenic sequences.
EP 290799 B1

  • Earliest priority – 13 January 1983
  • Filed – 22 December 1983
  • Granted – 26 November 2003
  • Reprinted (B9) – 1 September 2004
  • Expected expiry – 21 December 2003
 Title – Transgenic dicotyledonous plant cells and plants

Claim 1
A cell of a dicotyledonous plant, obtainable by Agrobacterium transformation, which contains stably integrated into its genome a foreign DNA which is characterised in that:(a) it does not contain T-DNA genes that control neoplastic growth and it is substantially free of internal T-DNA sequences of a wild-type Ti-plasmid; and
(b) it comprises at least one gene of interest containing:
(i) a coding sequence; and
(ii) a promoter region that contains a promoter sequence other than the natural promoter sequence of said coding sequence, and wherein said promoter sequence regulates transcription of downstream sequences containing said coding sequence to produce an RNA in said cell.

This granted patent is a divisional of now granted EP 116718 (see above).

Designated contracting States at the time of grant are: Austria, Belgium (patent lapsed as reported on INPADOC), Switzerland (patent lapsed as reported on INPADOC), Germany, France, United Kingdom, Italy, Liechtenstein, Luxembourg, Netherlands (patent lapsed as reported on INPADOC), Sweden (patent lapsed as reported on INPADOC).

Independent claim 1 of granted EP 290799 B2 does not claim a co-integrated vector system (see application EP 290799 A2 below), but a transformed cell of a dicotoledonous plant that:

  1. is substantially free of T-DNA from a wild-type Ti plasmid (as above, the definition of term “substantially free” is unclear, and the construct could include T-DNA from a non-wild-type Ti plasmid),
  2. contains a gene of interest that has a coding sequence (so might not cover a siRNA-producing sequence), and
  3. a promoter that regulates expression of the gene of interest.
EP 320500 B1

  • Earliest priority – 13 January 1983
  • Filed – 22 December 1983
  • Granted – 17 November 2004
  • Expected expiry – 21 December 2003
 Title – Non-oncogenic Ti plasmid vector system and recombinant DNA molecules for the introduction of expressible genes into plant cell genomes

Claim 1
A cloning vector which comprises:(a) a cloning vehicle segment (3′) containing a left border sequence (1) and a right border sequence (2) of a T-region of a wild-type Ti-plasmid, and
(b) a DNA segment which is located between said border sequences in a manner allowing its integration into a plant genome, wherein said DNA segment does not contain T-DNA genes that control neoplastic growth and wherein said DNA segment is substantially free of internal T-DNA sequences of a wild-type Ti-plasmid, except for promoter sequences, and which contains at least one gene of interest which comprises:
(i) a coding sequence, and
(ii) a promoter region that contains a promoter sequence other than the natural promoter sequence of said coding sequence, and wherein said promoter sequence regulates transcription of downstream sequences containing said coding sequence to produce an RNA in a cell of a plant.
Claim 9

A vector combination consisting of
(i) an acceptor Ti plasmid, which is incapable of inducing tumors in plants being free of border sequences and intemal T-DNA sequences of a wild-type Ti plasmid, comprising:
(a) a DNA segment (4) of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence (3) derived from a cloning vehicle, and
(ii) an intermediate cloning vector comprising:
(c) a cloning vehicle segment (3′) containing the two border sequences of the T-region of a wild-type Ti plasmid (1; 2) and a DNA sequence located outside of said two border sequences which is homologous with the DNA sequence (b) in said acceptor Ti plasmid permitting a single cross-over event wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and
(d) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome.
Claim 16

A hybrid Ti plasmid vector obtained by co-integration between an acceptor Ti plasmid comprising:
(a) a DNA segment (4) of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence (3) derived from a cloning vehicle; and an intermediate cloning vector comprising:
(a’) a cloning vehicle segment (3′) containing the two border sequences of the T-region of a wild-type Ti plasmid (1; 2) and a DNA sequence located outside of said two border sequences which is homologous with the above DNA sequence (b) in the acceptor Ti plasmid permitting a single crossover event, wherein the region between said border sequences is substantially free of internal T-DNA sequences of a wild-type Ti plasmid; and

(b’) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants located between the two border sequences in a manner allowing its integration into the plant genome;

said hybrid Ti plasmid vector comprising at least:

(1) the two border sequences (1; 2) of the T-region of a wild-type Ti plasmid;

(2) non-oncogenic DNA sequences (3; 3′) derived from a cloning vehicle;

(3) a DNA segment (4) of the wild-type Ti plasmid containing DNA sequences essential for the transfer of the T-region of wild-type Ti plasmids by Agrobacterium into plant cell genomes; and

(4) at least one gene of interest (5) under the control of a promoter capable of directing gene expression in plants which is located between the two border sequences (1; 2).

This granted patent is a divisional of now granted EP 116718 (see above).

Designated contracting States at the time of grant are: Austria (patent lapsed as reported on INPADOC), Belgium (patent lapsed as reported on INPADOC), Switzerland (patent lapsed as reported on INPADOC), Germany, France, United Kingdom, Italy, Liechtenstein, Luxembourg, Netherlands (patent lapsed as reported on INPADOC), Sweden.
EP 290799 A2

  • Earliest priority – 13 January 1983
  • Filed – 22 December 1983
  • Granted as EP 290799 B1 (see above)
 Title – Process for the production of expressible genes into plant cell genomes and Agrobacterium strains carrying hybrid Ti plasmid vectors useful for this process

Claim 1
Recombinant plant DNA genome being free of oncogenic internal T-DNA regions of the wild-type Ti plasmid containing an integrated gene of interest foreign to said plant DNA obtainable by infecting a plant cell with an Agrobacterium harboring a hybrid Ti plasmid vector created by homologous recombination between:A) an acceptor Ti plasmid which is incapable of inducing tumors in plants and comprises:
(i) the two border sequences of the T-region of the wild-type Ti plasmid;
(ii) a DNA segment devoid of oncogenic internal T-DNA regions of the wild-type T-DNA, derived from a cloning vehicle, located between the two border sequences, and containing a DNA sequence “A” which is homologous with at least a part of a DNA sequence in an intermediate cloning vector permitting a single crossover event; and
(iii) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmid into plant cell genomes, and

B) an intermediate cloning vector which comprises:
(i) at least one gene of interest; and
(ii) a cloning vehicle segment containing a DNA sequence which is homologous to the DNA sequence “A” in said acceptor Ti plasmid,
or a hybrid Ti plasmid vector created by homologous recombination between:
I) an acceptor Ti plasmid which is incapable of inducing tumors in plants and which comprises:
(a) a DNA segment of a wild-type Ti plasmid without the T-region and without the two border sequences of the T-region; and
(b) a DNA sequence derived from a cloning vehicle which is homologous with at least a part of a DNA sequence of an intermediate cloning vector which contains the two border sequences of the T-region of the wild-type Ti plasmid, and II) an intermediate cloning vector which comprises:
(a) a cloning vehicle segment containing the two border sequences of the T-region of the wild-type Ti plasmid and a DNA sequence which is homologous with the DNA sequence (b) in said acceptor Ti plasmid; and
(b) at least one gene of interest located between the two border sequences in a manner allowing its integration into the plant genome; said hybrid Ti plasmid comprising at least:
1) the two border sequences of the T-region of the wild-type Ti plasmid;
2) non-oncogenic DNA segments derived from a cloning vehicle;
3) a segment of the wild-type Ti plasmid containing DNA sequences essential for the transfer by Agrobacterium of the T-region of wild-type Ti plasmid into plant cell genomes, and
4) at least one gene of interest which is located between the two border sequences.

The European application EP 290 799 A2 claims a recombinant plant genome obtained after infection of plant cells with an Agrobacteriumstrain having a hybrid Ti plasmid. The recombinant genome contains an exogenous gene of interest and is free of oncogenic sequences. The hybrid plasmid is the product of homologous recombination between an acceptor plasmid and an intermediate cloning vector. The elements of the combined acceptor plasmids and intermediate cloning vectors are set out in Claim 1.
EP 320500 A2

  • Earliest priority – 13 January 1983
  • Filed – 22 December 1983
  • Granted as EP 320500 B1 (see above)
 Title – Non-oncogenic Ti plasmid vector system and recombinant DNA molecules for the introduction of expressible genes into plant cell genomes

Claim 1
A non-oncogenic Ti plasmid vector system free of T-DNA genes controlling neoplastic growth of transformed plants which comprises:(i) DNA sequences coding for functions which are essential for the transfer of the T-region of a wild-type Ti plasmid by Agrobacterium into a plant cell genome; and
(ii) at least one gene of interest which has been inserted into said vector system and which is capable of being expressed in plants which are susceptible to infection by Agrobacterium,
said gene being under the control of at least one promoter capable of directing the expression of said gene in the plant and associated with at least the right border sequence of the T-region of a wild-type Ti plasmid, said border sequence allowing the integration of said gene into the plant genome.
Claim 4
An intermediate cloning vector comprising:(i) a cloning vehicle segment containing the right border sequence of the T-region of a wild-type Ti plasmid and a DNA sequence which is homologous with a DNA sequence in an acceptor Ti plasmid; and
(ii) at least one gene of interest under the control of a promoter capable of directing gene expression in plants which is associated with said border sequence in a manner allowing its integration into the plant genome.

The European application EP 320 500 A2 claims a non-oncogenic Ti plasmid vector containing a gene of interest and its controlling promoter associated with at least the right border of the wild type T-region. The border allows the integration of the gene into the plant genome. Also, the Ti plasmid contains the sequences needed for transfer of T-region into plant genome (claim 1).

In another independent claim, a gene of interest and its promoter that is associated with a right T-border are contained in an intermediate cloning vector (claim 4).
Remarks
  1. Related patent of EP 116718 B2 in Canada (CA 1341419) has been granted.
  2. Related patent of EP 116718 B2 in Japan and their status are:
  • JP 1633546 C – granted and expired
  • JP 2726267 B2 – granted and expired
  • JP 2769539 B2 (divisional of now granted JP 1633546) – granted and expired
  • JP (H)03/108478 A (divisional of now granted JP 2769539) – application rejected
  • JP (H)06/105629 A (divisional of now granted JP 2769539) – application rejected
  • JP 2001/029092 A (divisional of JP (H)06/105629 A) – application rejected

Note: Patent information on this page was last updated on 28 February 2006.