Title: Circular DNA molecule with conditional origin of replication, method for preparing the same and use thereof in gene therapy
Abstract: A circular DNA molecule, useful for gene therapy, comprising at least one nucleic acid sequence of interest, characterized in that the region allowing the replication thereof has an origin of replication with a functionality in a host cell that requires the presence of at least one specific protein foreign to said host cell. A method for preparing same, cells incorporating said DNA molecules and uses thereof in gene therapy are also described.
Patent Number: 6,977,174 Issued on 12/20/2005 to Crouzet, et al.
| Inventors:
|
Crouzet; Joël (Sceaux, FR);
Soubrier; Fabienne (Thiais, FR)
|
| Assignee:
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Centelion (Vitry-sur-Seine, FR)
|
| Appl. No.:
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043193 |
| Filed:
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September 13, 1996 |
| PCT Filed:
|
September 13, 1996
|
| PCT NO:
|
PCT/FR96/01414
|
| 371 Date:
|
March 13, 1998
|
| 102(e) Date:
|
March 13, 1998
|
| PCT PUB.NO.:
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WO97/10343 |
| PCT PUB. Date:
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March 20, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
435/320.1; 435/69.1; 435/325; 435/455; 536/23.1 |
| Intern'l Class: |
C12N 015/64; C12N 015/70; C12N 015/79 |
| Field of Search: |
435/6,914,455,471,320.1,252.3,252.33,325
536/231,242
|
References Cited [Referenced By]
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| 4654307 | Mar., 1987 | Morgan.
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| 4761367 | Aug., 1988 | Edgell et al.
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| 5434065 | Jul., 1995 | Mahan et al.
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| 5510099 | Apr., 1996 | Short et al.
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| 5656481 | Aug., 1997 | Baetge et al.
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| 5693622 | Dec., 1997 | Wolff et al.
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| 5700657 | Dec., 1997 | Beaudry et al.
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| 5714323 | Feb., 1998 | Ohshima et al.
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| 5773246 | Jun., 1998 | Keene et al.
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| 5859208 | Jan., 1999 | Fiddes et al.
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| 5955056 | Sep., 1999 | Short et al.
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| 5985644 | Nov., 1999 | Roseman et al.
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| 6254874 | Jul., 2001 | Mekalanos et al.
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| 6573100 | Jun., 2003 | Seeber et al.
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| Foreign Patent Documents |
| WO 95/3076/2 | Nov., 1995 | WO.
| |
| WO 96/0189/9 | Jan., 1996 | WO.
| |
Other References
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|
Primary Examiner: Lacourciere; Karen A.
Assistant Examiner: Gibbs; Terra C.
Attorney, Agent or Firm: Finnegan, Henderson, Farabow, Garrett & Dunner LLP
Claims
1. An extrachromosomal DNA molecule comprising a heterologous gene encoding a
protein and a conditional origin of replication whose functionality in a prokaryotic
host cell requires a replication initiating protein specific for said origin of
replication and foreign to the prokaryotic host cell, a suppressor transfer tRNA
for an amber codon, and a cer fragment from ColE1, wherein said DNA molecule does
not encode the replication initiating protein.
2. The extrachromosomal DNA molecule according to claim 1, wherein the heterologous
gene encodes acidic fibroblast growth factor.
3. The extrachromosomal DNA molecule according to claim 2, wherein the heterologous
gene is under the control of a bacteriophage T7 promoter.
4. A prokaryotic recombinant host cell comprising a heterologous replication
initiating protein that activates a conditional origin of replication and an extrachromosomal
DNA molecule comprising a heterologous gene encoding a protein, a selection gene
encoding a suppressor transfer RNA for a specific codon, and a conditional origin
of replication whose functionality in the prokaryotic recombinant host cell requires
a replication initiating protein specific for said origin of replication and foreign
to the host cell, wherein said DNA molecule does not encode the replication initiating protein.
5. A method for producing an extrachromosomal DNA molecule comprising a heterologous
gene encoding a protein, a selection gene encoding a suppressor transfer RNA for
a specific codon, and a conditional origin of replication whose functionality in
a prokaryotic host cell requires a replication initiating protein specific for
said origin of replication and foreign to the host cell, wherein said DNA molecule
does not encode the replication initiating protein, comprising:
a) culturing a prokaryotic recombinant host cell according to claim 4; and
b) isolating the extrachromosomal DNA molecule produced by the host cell.
6. The method according to claim 5, wherein the prokaryotic recombinant host
cell is
Escherichia coli.
7. The method according to claim 5, wherein the conditional origin of replication
originates from a bacterial plasmid or a bacteriophage.
8. The method according to claim 7, wherein the bacterial plasmid or the bacteriophage
is selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010,
P1, P4, lambda, Phi82 and Phi80.
9. The method according to claim 7, wherein the bacterial plasmid is R6K.
10. The method according to claim 9, wherein the conditional origin of replication
comprises SEQ ID No. 1.
11. The method according to claim 5, wherein the prokaryotic recombinant host
cell expresses a π initiator protein.
12. The method according to claim 11, wherein the π initiator protein is
encoded by gene pir-116.
13. The method according to claim 11, wherein the π initiator protein is
encoded by SEQ ID No. 2.
14. The method according to claim 5, wherein the selection gene is a suppressor
of an amber transfer RNA, and the host cell contains a gene essential under certain
culture conditions comprising an amber mutation.
15. The method according to claim 5, wherein the extrachromosomal DNA molecule
further comprises a target region for a site-specific recombinase.
16. The method according to claim 15, wherein the target region for the site-specific
recombinase is a cer fragment from ColE1.
17. The method according to claim 5, wherein the extrachromosomal DNA molecule
is pXL2774.
18. The prokaryotic recombinant host cell according to claim 4, wherein the cell
is
Escherichia coli.
19. The prokaryotic recombinant host cell according to claim 4, wherein the conditional
origin of replication is from a bacterial plasmid or a bacteriophage.
20. The prokaryotic recombinant host cell according to claim 19, wherein the
conditional origin of replication is from a bacterial plasmid or a bacteriophage
selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010, P1,
P4, lambda, Phi82 and Phi80.
21. The prokaryotic recombinant host cell according to claim 19, wherein the
conditional origin of replication is from bacterial plasmid R6K.
22. The prokaryotic recombinant host cell according to claim 4, wherein the host
cell expresses a π initiator protein.
23. The prokaryotic recombinant host cell according to claim 22, wherein the
π initiator protein is encoded by gene pir-116.
24. The prokaryotic recombinant host cell according to claim 22, wherein the
π initiator protein is encoded by SEQ ID No.: 2.
25. The prokaryotic recombinant host cell according to claim 4, wherein the host
cell further comprises a gene comprising an amber mutation, wherein said gene is
essential under certain culture conditions.
26. The extrachromosomal DNA molecule of claim 2, wherein the heterologous gene
is under the control of a CMV promoter.
27. The extrachromosomal DNA molecule of claim 3, which is designated pXL3056.
28. An extrachromosomal DNA molecule comprising:
a gene encoding acidic fibroblast growth factor operably linked to a cytomegalovirus
(CMV) promoter and an SV40 polyadenylation sequence;
a conditional origin of replication from plasmid R6K whose functionality in a
prokaryotic host cell requires a replication initiating protein specific for said
origin of replication and foreign to the prokaryotic host cell;
a cer fragment; and
a phenylalanine suppressor tRNA gene;
wherein the extrachromosomal DNA molecule does not contain a gene encoding the
replication initiating protein foreign to the prokaryotic host.
29. An extrachromosomal DNA molecule comprising a heterologous gene encoding
acidic fibroblast growth factor and a conditional origin of replication whose functionality
in a prokaryotic host cell requires a replication initiating protein specific for
said origin of replication and foreign to the prokaryotic host cell, wherein said
DNA molecule does not encode the replication initiating protein.
30. The extrachromosomal DNA molecule according to claim 29, wherein the conditional
origin of replication originates from a bacterial plasmid or a bacteriophage.
31. The extrachromosomal DNA molecule according to claim 30, wherein the bacterial
plasmid or the bacteriophage is selected from the group consisting of RK2, R6K,
R1, pSC101, Rtsl, F, RSF1010, P1, P4, lambda, Phi82 and Phi80.
32. The extrachromosomal DNA molecule according to claim 30, wherein the bacterial
plasmid is R6K.
33. The extrachromosomal DNA molecule according to claim 32, wherein the conditional
origin of replication comprises the gamma origin of replication of plasmid R6K.
34. The extrachromosomal DNA molecule according to claim 33, wherein the conditional
origin of replication comprises SEQ ID No: 1.
35. The extrachromosomal DNA molecule according to claim 29, further comprising
a selection gene, wherein the selection gene does not impart resistance to an antibiotic.
36. The extrachromosomal DNA molecule according to claim 35, wherein the selection
gene encodes a suppressor transfer RNA for a specific codon.
37. The extrachromosomal DNA molecule according to claim 36, wherein the suppressor
is a suppressor transfer tRNA for an amber codon.
38. The extrachromosomal DNA molecule according to claim 29, further comprising
a target region for a site-specific recombinase.
39. The extrachromosomal DNA molecule according to claim 38, wherein the target
region for the site-specific recombinase is a cer fragment from ColE1.
40. The extrachromosomal DNA molecule according to claim 29, wherein the heterologous
gene is under the control of a bacteriophage T7 promoter.
41. A prokaryotic recombinant host cell comprising a heterologous replication
initiating protein that activates a conditional origin of replication and an extrachromosomal
DNA molecule comprising a heterologous gene encoding acidic fibroblast growth factor
and a conditional origin of replication whose functionality in the prokaryotic
recombinant host cell requires a replication initiating protein specific for said
origin of replication and foreign to the host cell, wherein said DNA molecule does
not encode the replication initiating protein.
42. The prokaryotic recombinant host cell according to wherein the cell is
Escherichia
coli.
43. The prokaryotic recombinant host cell according to claim 41, wherein the
conditional origin of replication is from a bacterial plasmid or a bacteriophage.
44. The prokaryotic recombinant host cell according to claim 43, wherein the
conditional origin of replication is from a bacterial plasmid or a bacteriophage
selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010, P1,
P4, lambda, Phi82 and Phi80.
45. The prokaryotic recombinant host cell according to claim 43, wherein the
conditional origin of replication is from bacterial plasmid R6K.
46. The prokaryotic recombinant host cell according to claim 41, wherein the
host cell expresses a π initiator protein.
47. The prokaryotic recombinant host cell according to claim 46, wherein the
π initiator protein is encoded by gene pir-116.
48. The prokaryotic recombinant host cell according to claim 46, wherein the
π initiator protein is encoded by SEQ ID No.: 2.
49. The prokaryotic recombinant host cell according to claim 41, wherein the
host cell further comprises a gene comprising an amber mutation, wherein said gene
is essential under certain culture conditions.
50. A method for producing an extrachromosomal DNA molecule comprising a heterologous
gene encoding acidic fibroblast growth factor and a conditional origin of replication
whose functionality in a prokaryotic host cell requires a replication initiating
protein specific for said origin of replication and foreign to the host cell, wherein
said DNA molecule does not encode the replication initiating protein, comprising:
a) culturing a prokaryotic recombinant host cell according to claim 41; and
b) isolating the extrachromosomal DNA molecule produced by the host cell.
51. The method according to claim 50, wherein the prokaryotic recombinant host
cell is
Escherichia coli.
52. The method according to claim 50, wherein the conditional origin of replication
originates from a bacterial plasmid or a bacteriophage.
53. The method according to claim 52, wherein the bacterial plasmid or the bacteriophage
is selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010,
P1, P4, lambda, Phi82 and Phi80.
54. The method according to claim 53, wherein the bacterial plasmid is R6K.
55. The method according to claim 54, wherein the conditional origin of replication
comprises SEQ ID No. 1.
56. The method according to claim 50, wherein the prokaryotic recombinant host
cell expresses a π initiator protein.
57. The method according to claim 56, wherein the π initiator protein is
encoded by gene pir-116.
58. The method according to claim 56, wherein the π initiator protein is
encoded by SEQ ID No. 2.
59. The method according to claim 50, wherein the extrachromosomal DNA molecule
further comprises a selection gene encoding a suppressor transfer RNA for a specific codon.
60. The method according to claim 59, wherein the suppressor is a suppressor
transfer tRNA for an amber codon.
61. The method according to claim 60, wherein the selection gene is a suppressor
of an amber transfer RNA, and the host cell contains a gene essential under certain
culture conditions comprising an amber mutation.
62. The method according to claim 50, wherein the extrachromosomal DNA molecule
further comprises a target region for the site-specific recombinase.
63. The method according to claim 62, wherein the target region for the site-specific
recombinase is a cer fragment from ColE1.
64. An extrachromosomal DNA molecule comprising a heterologous gene encoding
a protein, a selection gene encoding a suppressor transfer RNA for a specific codon
a target for a site-specific recombinase and a conditional origin of replication
whose functionality in a prokaryotic host cell requires a replication initiating
protein specific for said origin of replication and foreign to the prokaryotic
host cell, wherein said DNA molecule does not encode the replication initiating protein.
65. The extrachromosomal DNA molecule according to claim 64, wherein the conditional
origin of replication originates from a bacterial plasmid or a bacteriophage.
66. The extrachromosomal DNA molecule according to claim 65, wherein the bacterial
plasmid or the bacteriophage is selected from the group consisting of RK2, R6K,
R1, pSC101, Rtsl, F, RSF1010, P1, P4, lambda, Phi82 and Phi80.
67. The extrachromosomal DNA molecule according to claim 66, wherein the bacterial
plasmid is R6K.
68. The extrachromosomal DNA molecule according to claim 67, wherein the conditional
origin of replication comprises the gamma origin of replication of plasmid R6K.
69. The extrachromosomal DNA molecule according to claim 68, wherein the conditional
origin of replication comprises SEQ ID No: 1.
70. The extrachromosomal DNA molecule according to claim 64, wherein the suppressor
is a suppressor transfer tRNA for an amber codon.
71. The extrachromosomal DNA molecule according to claim 64, wherein the target
region for the site-specific recombinase is a cer fragment from ColE1.
72. The extrachromosomal DNA molecule according to claim 64, wherein the heterologous
gene is under the control of a bacteriophage T7 promoter.
73. A prokaryotic recombinant host cell comprising a heterologous replication
initiating protein that activates a conditional origin of replication, a selection
gene encoding a suppressor transfer RNA for a specific codon, and an extrachromosomal
DNA molecule comprising a heterologous gene encoding a protein, a target region
for a site-specific recombinase, and a conditional origin of replication whose
functionality in the prokaryotic recombinant host cell requires a replication initiating
protein specific for said origin of replication and foreign to the host cell, wherein
said DNA molecule does not encode the replication initiating protein.
74. The prokaryotic recombinant host cell according to claim 73, wherein the
cell is
Escherichia coli.
75. The prokaryotic recombinant host cell according to claim 73, wherein the
conditional origin of replication is from a bacterial plasmid or a bacteriophage.
76. The prokaryotic recombinant host cell according to claim 75, wherein the
conditional origin of replication is from a bacterial plasmid or a bacteriophage
selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010, P1,
P4, lambda, Phi82 and Phi80.
77. The prokaryotic recombinant host cell according to claim 76, wherein the
conditional origin of replication is from bacterial plasmid R6K.
78. The prokaryotic recombinant host cell according to claim 73, wherein the
host cell expresses a π initiator protein.
79. A prokaryotic recombinant host cell comprising a heterologous replication
initiator protein encoded by gene pir-116, and an extrachromosomal DNA molecule
comprising a heterologous gene encoding a protein, a target region for a site-specific
recombinase, and a conditional origin of replication whose functionality in the
prokaryotic recombinant host cell requires the replication initiating protein encoded
by gene pir-116, wherein said DNA molecule does not comprise gene pir-116.
80. The prokaryotic recombinant host cell according to claim 79, wherein the
heterologous replication initiator protein is encoded by SEQ ID No.: 2.
81. A method for producing an extrachromosomal DNA molecule comprising a heterologous
gene encoding a protein, a target region for a site-specific recombinase, a selection
gene encoding a suppressor transfer RNA for a specific codon, and a conditional
origin of replication whose functionality in a prokaryotic host cell requires a
replication initiating protein specific for said origin of replication and foreign
to the host cell, wherein said DNA molecule does not encode the replication initiating, comprising:
a) culturing a prokaryotic recombinant host cell according to claim 73; and
b) isolating the extrachromosomal DNA molecule produced by the host cell.
82. The method according to claim 81, wherein the prokaryotic recombinant host
cell is
Escherichia coli.
83. The method according to claim 81, wherein the conditional origin of replication
originates from a bacterial plasmid or a bacteriophage.
84. The method according to claim 83, wherein the bacterial plasmid or the bacteriophage
is selected from the group consisting of RK2, R6K, R1, pSC101, Rtsl, F, RSF1010,
P1, P4, lambda, Phi82 and Phi80.
85. The method according to claim 84, wherein the bacterial plasmid is R6K.
86. The method according to claim 85, wherein the conditional origin of replication
comprises SEQ ID No. 1.
87. The method according to claim 81, wherein the prokaryotic recombinant host
cell expresses a π initiator protein.
88. The method according to claim 81, wherein the suppressor is a suppressor
transfer tRNA for an amber codon.
89. The method according to claim 88, wherein the selection gene is a suppressor
transfer RNA for an amber codon, and the host cell contains a gene essential under
certain culture conditions comprising an amber codon.
90. The method according to claim 81, wherein the target region for the site-specific
recombinase is a cer fragment from ColE1.
91. An extrachromosomal DNA molecule comprising a heterologous gene encoding
a protein, a cer fragment from ColE1, a selection gene, wherein the selection gene
does not impart resistance to an antibiotic, and a conditional origin of replication
whose functionality in a prokaryotic host cell requires a replication initiating
protein specific for said origin of replication and foreign to the prokaryotic
host cell, wherein said DNA molecule does not encode the replication initiating protein.
92. The extrachromosomal DNA molecule according to claim 91, wherein the conditional
origin of replication originates from a bacterial plasmid or a bacteriophage.
93. The extrachromosomal DNA molecule according to claim 92, wherein the bacterial
plasmid or the bacteriophage is selected from the group consisting of RK2, R6K,
R1, pSC101, Rtsl, F, RSF1010, P1, P4, lambda, Phi82 and Phi80.
94. The extrachromosomal DNA molecule according to claim 93, wherein the bacterial
plasmid is R6K.
95. The extrachromosomal DNA molecule according to claim 94, wherein the conditional
origin of replication comprises the gamma origin of replication of plasmid R6K.
96. The extrachromosomal DNA molecule according to claim 95, wherein the conditional
origin of replication comprises SEQ ID No: 1.
97. The extrachromosomal DNA molecule according to claim 91, wherein the selection
gene encodes a suppressor transfer RNA for a specific codon.
98. The extrachromosomal DNA molecule according to claim 97, wherein the suppressor
is a suppressor transfer tRNA for an amber codon.
99. The extrachromosomal DNA molecule according to claim 91, wherein the heterologous
gene encodes thymidine kinase (tk).
100. The extrachromosomal DNA molecule according to claim 91, wherein the heterologous
gene encodes acidic fibroblast growth factor.
101. A method for producing an extrachromosomal DNA molecule comprising a heterologous
gene encoding a protein, a target region for a site-specific recombinase, and a
conditional origin of replication whose functionality in a prokaryotic recombinant
host cell requires a replication initiating protein encoded by gene pir-116, wherein
said DNA molecule does not comprise gene pir-116, comprising:
a) culturing a prokaryotic recombinant host cell according to claim 79; and
b) isolating the extrachromosomal DNA molecule produced by the host cell.
102. The method of claim 101, wherein the replication initiator protein is encoded
by SEQ ID No.: 2.
Description
The present invention relates to a novel conditional replication DNA molecule
which can be used in gene therapy or for the production of recombinant proteins.
Gene therapy consists in correcting a deficiency or an anomaly by introducing
genetic information into the affected organ or cell. This information may be introduced
either in vitro into a cell extracted from the organ and then reinjected into the
body, or in vivo, directly into the target tissue. As a molecule of high molecular
weight and of negative charge, DNA has difficulty in spontaneously crossing phospholipid
cell membranes. Various vectors are thus used in order to enable gene transfer
to take place: viral vectors, on the one hand, and natural or synthetic chemical
and/or biochemical vectors, on the other hand.
Viral vectors (retroviruses, adenoviruses, adeno-associated viruses, etc.)
are very effective, in particular for crossing membranes, but present a certain
number of risks such as pathogenicity, recombination, replication, immunogenicity, etc.
Chemical and/or biochemical vectors allow these risks to be avoided (for
reviews, see Behr, 1993, Cotten and Wagner 1993). These are, for example, cations
(calcium phosphate, DEAE-dextran, etc.) which act by forming precipitates with
DNA, which may be "phagocytosed" by the cells. They may also be liposomes in which
the DNA is incorporated and which fuse with the plasma membrane. Synthetic gene-transfer
vectors are generally lipids or cationic polymers which complex the DNA and form
with it a particle bearing positive surface charges. As illustrations of vectors
of this type, mention may be made in particular of dioctadecylamidoglycylspermine
(DOGS, Transfectam™) or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
(DOTMA, Lipofectin™).
However, the use of chemical and/or biochemical vectors or naked DNA implies
the possibility of producing large amounts of DNA of pharmacological purity. The
reason for this is that in gene therapy techniques, the medicinal product consists
of the DNA itself and it is essential to be able to manufacture, in suitable amounts,
DNAs having properties which are appropriate for therapeutic use in man.
In the case of non-viral vectorology, the vectors used are plasmids of bacterial
origin. The plasmids generally used in gene therapy carry (i) an origin of replication,
(ii) a marker gene such as a gene for resistance to an antibiotic (kanamycin, ampicillin,
etc.) and (iii) one or more transgenes with sequences necessary for their expression
(enhancer(s), promoter(s), polyadenylation sequences, etc.).
However, the technology currently available is not entirely satisfactory.
On the one hand, the risk remains of dissemination in the body. Thus, a bacterium
which is present in the body can, at low frequency, receive this plasmid. There
is a greater likelihood of this taking place if it involves an in vivo gene therapy
treatment in which the DNA may be disseminated in the body of the patient and may
come into contact with bacteria which infect this patient or bacteria of the commensal
flora. If the bacterium receiving the plasmid is an enterobacterium, such as
E.
coli, this plasmid can be replicated. Such an event then leads to dissemination
of the therapeutic gene. Insofar as the therapeutic genes used in gene therapy
treatments can code, for example, for a lymphokine, a growth factor, an anti-oncogene
or a protein whose function is defective in the host and which thus makes it possible
to correct a genetic defect, the dissemination of some of these genes could have
unforeseeable and worrying effects (for example if a pathogenic bacterium acquired
a human growth factor gene).
On the other hand, the plasmids generally used in non-viral gene therapy also
possess a marker for resistance to an antibiotic (ampicillin, kanamycin, etc.).
The bacterium acquiring such a plasmid thus has an undeniable selective advantage
since any antibiotic therapy, using an antibiotic from the same family as that
which selects the plasmid resistance gene, will lead to selection of the plasmid
in question. In this respect, ampicillin belongs to the β-lactams, which
is the family of antibiotics which is most frequently used worldwide. The use in
bacteria of selection markers which are not antibiotic-resistance genes would thus
be particularly advantageous. This would avoid the selection of bacteria which
may have received a plasmid carrying such a marker.
It is thus particularly important to seek to limit the dissemination of therapeutic
genes and resistance genes as much as possible.
The subject of the present invention is specifically to propose novel DNA molecules
which can be used in gene therapy or for the production of recombinant proteins
in vitro and which replicate only in cells which can complement certain functions
of these non-viral vectors.
The invention also relates to a particularly effective method for preparing these
DNA molecules.
The DNA molecules claimed have the advantage of removing the risks associated
with dissemination of the plasmid, such as (1) replication and dissemination, which
can lead to uncontrolled overexpression of the therapeutic gene, (2) dissemination
and expression of resistance genes. The genetic information contained in the DNA
molecules according to the invention effectively comprises the therapeutic gene(s)
and the signals for regulating its (their) expression, a functional conditional
origin of replication which greatly limits the host cell spectrum of this plasmid,
a selection marker of reduced size which is preferably different from a gene which
imparts resistance to an antibiotic and, where appropriate, a DNA fragment which
allows the resolution of plasmid multimers. The probability of these molecules
(and thus the genetic information which they contain) being transferred to a microorganism,
and maintained stably, is very limited.
Lastly, the vectors according to the invention, also referred to as miniplasmids
on account of their circular structure, their reduced size and their supercoiled
form, have the following additional advantages: on account of their size which
is reduced in comparison with the ColE1-derived plasmids used conventionally, the
DNA molecules according to the invention potentially have better in vivo bioavailability.
In particular, they have improved capacities of cell penetration and distribution.
Thus, it is acknowledged that the diffusion coefficient in tissues is inversely
proportional to the molecular weight (Jain, 1987). Similarly, in the cell, high
molecular weight molecules have poorer permeability across the plasma membrane.
In addition, in order for the plasmid to pass into the nucleus, which is essential
for its expression, the high molecular weight is also a disadvantage, the nuclear
pores imposing a size limit for diffusion into the nucleus (Landford et al., 1986).
The reduction in size of the non-therapeutic parts of the DNA molecule (origin
of replication and selection gene in particular) according to the invention also
makes it possible to decrease the size of the DNA molecules. The part which allows
the replication and selection of this plasmid in the bacterium (1.1 kb) is decreased
by a factor of 3, counting, for example, 3 kb for the origin of replication and
the resistance marker vector part. This decrease (i) in molecular weight and (ii)
in negative charge imparts improved tissue, cellular and nuclear bioavailability
and diffusion to the molecules of the invention.
More precisely, the present invention relates to a circular DNA molecule, which
is useful in gene therapy, this molecule comprising at least one nucleic acid sequence
of interest, characterized in that the region which allows its replication comprises
an origin of replication whose functionality in a host cell requires the presence
of at least one specific protein which is foreign to the said host cell.
This DNA molecule may be in single- or double-stranded form and advantageously
possesses a supercoiled form.
For the purposes of the present invention, the host cells used can be of various
origins. They can be eukaryotic or prokaryotic cells. According to a preferred
embodiment of the invention, they are prokaryotic cells.
The replication of bacterial plasmids conventionally requires the presence of
at least one protein, which is coded for by the host cell, of the RNA polymerase,
Rnase, DNA polymerase, etc. type. For the reasons already explained above, it is
not possible to overcome entirely, with this type of replication, any possible
risks of dissemination in the treated organism. Advantageously, the functionality
of the origin of replication of the DNA molecule according to the invention requires
the presence of a specific protein which is foreign to the host cell. The significance
of this characteristic is to reduce the host spectrum of the claimed plasmid to
specific strains that express this initiator protein. The DNA molecule developed
within the context of the present invention thus advantageously possesses a so-called
conditional origin of replication.
The conditional origin of replication used according to the present invention
may originate from plasmids or bacteriophages which share the following characteristics:
they contain in their origin of replication repeat sequences, or iterons, and they
code for at least one replication-initiating protein (Rep) which is specific to
them. By way of example, mention may be made of the conditional replication systems
of the following plasmids and bacteriophages:
| |
|
| |
|
specific initiator |
| |
plasmid or bacteriophage |
protein |
| |
|
| |
RK2 (Stalker et al., 1981) |
TrfA |
| |
R1 (Ryder et al., 1981) |
RepA |
| |
pSC101 (Vocke and Bastia, 1983) |
RepA |
| |
F (Murotsu et al., 1981) |
protein E |
| |
Rts1 (Itoh et al., 1982, 1987) |
RepA |
| |
RSF1010 (Miao et al., 1995) |
RepC |
| |
P1 (Abeles et al., 1984) |
RepA |
| |
P4 (Flensburg and Calendar, 1987) |
alpha protein |
| |
lambda (Moore et al., 1981) |
protein O |
| |
phi 82 (Moore et al., 1981) |
protein O from phi 82 |
| |
phi 80 |
protein O from phi 80 |
| |
|
According to a preferred embodiment of the invention, the origin of replication
used in the DNA molecules claimed is derived from a natural
E. coli plasmid
referred to as R6K.
The replication functions of R6K are grouped together in a 5.5 kbp DNA fragment
(FIG. 1) comprising 3 origins of replication α, β and γ (γ
and β providing 90% of the replication) and an operon coding for the π
replication-initiator proteins and the protein Bis. The minimum amount of genetic
information required to maintain this plasmid at its characteristic number of copies
(15 copies per genome) is contained in two elements: the 400 bp of ori γ
and the gene pir, whose product is the π initiator protein.
Ori γ may be divided into two functional parts: the core part and the activator
element (FIG.
1). The core part, which is essential for replication, contains
the iterons (7 direct repeats of 22 bp) represented in SEQ ID No. 1 to which the
π protein becomes bound, and flanking segments, which are targets of the
host proteins (IHF, DnaA).
According to a preferred mode of the invention, the origin of replication
of the vector claimed consists entirely or partially of this γ origin of
replication of the plasmid R6K and more preferably, entirely or partially of SEQ
ID No. 1 or one of its derivatives.
For the purposes of the present invention, the term derivative denotes any sequence
which differs from the sequence considered on account of degeneracy of the genetic
code, obtained by one or more modifications of genetic and/or chemical nature,
as well as any sequence which hybridizes with these sequences or fragments thereof
and whose product possesses the activity indicated with regard to the replication-initiator
protein, π. The term modification of the genetic and/or chemical nature may
be understood to refer to any mutation, substitution, deletion, addition and/or
modification of one or more residues. The term derivative also comprises the sequences
homologous with the sequence considered, derived from other cellular sources and
in particular cells of human origin, or from other organisms, and possessing an
activity of the same type. Such homologous sequences may be obtained by hybridization
experiments. The hybridizations may be performed starting with nucleic acid libraries,
using the native sequence or a fragment thereof as probe, under conventional conditions
of stringency (Maniatis et al., cf. General techniques of molecular biology), or,
preferably, under conditions of high stringency.
The origin of replication described above, which has the advantage of being of
very limited size, is functional solely in the presence of a specific initiator
protein, protein Pi, produced by the gene pir (SEQ ID No. 2). Since this protein
can act in trans, it is possible to physically dissociate the ori gamma from the
pir gene, which may be introduced into the genome of the cell which is chosen as
the specific host for these plasmids. Mutations in π may alter its inhibitory
functions (Inuzuka and Wada, 1985) and lead to an increase in the number of copies
of the R6K derivatives, up to more than 10 times the initial number of copies.
These substitutions are all within a domain of 40 amino acids, which therefore
appears to be responsible for the control by π of the number of plasmid copies
(FIG.
2).
According to an advantageous embodiment of the present invention, the π
protein, expressed in the host cell, results from the expression of the gene represented
in SEQ ID No. 2 or one of its derivatives as defined above and more particularly
of the gene pir 116 which comprises a mutation when compared with the pir gene.
This mutation corresponds to the replacement of a proline by a leucine. In this
context, the number of copies of the R6K derivatives is about 250 copies per genome.
Besides a conditional origin of replication as defined above, the DNA molecules
claimed contain a region comprising one (or more) gene(s) which make it possible
to ensure selection of the DNA molecule in the chosen host.
This may be a conventional marker of gene type which imparts resistance to an
antibiotic, such as kanamycin, ampicillin, chloramphenicol, streptomycin, spectinomycin,
lividomycin or the like.
However, according to a preferred embodiment of the invention, this region
is different from a gene which imparts resistance to an antibiotic. It may thus
be a gene whose product is essential for the viability of the host envisaged, under
defined culturing conditions. It may be, for example:
- a gene coding for a suppressor tRNA, of natural or synthetic origin.
This is, more preferably, an amber codon tRNA (TAG)
- a gene whose product is necessary for metabolism of the cell, under
certain culturing conditions, namely a gene involved in the biosynthesis of a metabolite
(amino acid, vitamin, etc.), or a catabolism gene which makes it possible to assimilate
a substance present in the culture medium (specific nitrogen or carbon source), etc.
According to a preferred mode of the invention, this region contains an
expression cassette of a gene coding for a suppressor tRNA for specific codons.
This latter may be chosen, in particular, from those coding for phenylalanine,
cysteine, proline, alanine and histidine bases. It is more particularly a suppressor
tRNA for amber codons (TAG).
In this particular case, the system used to select, in the host cells, the DNA
molecules which are the subject of the present invention includes two elements:
1) on the DNA molecule, a gene coding for a suppressor transfer RNA for the amber
codon (TAG) which constitutes the selection marker, known as (sup) gene and 2)
a specific host, one of whose genes, which is essential under certain culture conditions,
contains an amber TAG codon. This cell may grow, under the culture conditions for
which the product of the gene containing the TAG codon is essential, only if the
plasmid allowing the expression of sup is present in the cell. The culture conditions
thus constitute the pressure for selection of the DNA molecule. The sup genes used
may be of natural origin (Glass et al., 1982) or may originate from a synthetic
construction (Normanly et al., 1986, Kleina et al., 1990).
Such a system offers great flexibility insofar as, depending on the gene containing
an amber mutation, it is possible to determine various selective media. In the
bacterium
Lactococcus lactis for example, the amber codon is located in
a purine biosynthesis gene. This allows the selection of the plasmid carrying the
gene coding for the suppressor tRNA when the bacteria multiply in milk. Such a
marker has the advantage of being very small and of containing no "foreign" sequences,
originating from phages or transposons.
According to a particular embodiment of the invention, the DNA molecule
also comprises a DNA fragment, the target for site-specific recombinases, which
allows the resolution of plasmid multimers.
Thus, such a fragment, introduced on to a DNA molecule which is circular and
whose origin of replication is, for example, ori gamma, allows the resolution of
multimers of such a plasmid. Such multimers are observed, in particular, when the
DNA molecule is prepared in a strain carrying a mutated allele of pir, such as
pir-116, which makes it possible to increase the number of copies of the R6K derivatives.
This recombination may be achieved by means of various systems which entail
site-specific recombination between sequences. More preferably, the site-specific
recombination of the invention is obtained by means of specific intramolecular
recombination sequences which are capable of recombining with each other in the
presence of specific proteins, generally referred to as recombinases. In this specific
case, these are the recombinases XerC and XerD. For this reason, the DNA molecules
according to the invention generally also comprise a sequence which allows this
site-specific recombination. The specific recombination system present in the genetic
constructions according to the invention (recombinases and specific recognition
site) may be of different origins. In particular, the specific sequences and the
recombinases used may belong to different structural classes, and in particular
to the transposon Tn3 resolvase family or to the bacteriophage lambda integrase
family. Among the recombinases belonging to the transposon Tn3 family, mention
may be made in particular of the resolvase of transposon Tn3 or of transposons
Tn21 and Tn522 (Stark et al., 1992); the Gin invertase of bacteriophage mu or alternatively
plasmid resolvases, such as that of the par fragment of RP4 (Abert et al., Mol.
Microbiol. 12 (1994) 131). Among the recombinases belonging to the bacteriophage
λ integrase family, mention may be made in particular of the integrase of
the phages lambda (Landy et al., Science 197 (1977) 1147), P22 and φ80 (Leong
et al., J. Biol. Chem. 260 (1985) 4468), HP1 of
Haemophilus influenzae (Hauser
et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase of phage P1, the integrase
of plasmid pSAM2 (EP 350 341) or alternatively the FLP recombinase of the 2μ
plasmid and the XerC and XerD recombinases from
E. coli.
Preferably, the DNA molecules which form the subject of the present invention
contain the fragment cer from the natural
E. coli plasmid ColE1. The cer
fragment used is a 382 bp HpaII fragment from ColE1 which has been shown to bring
about, in cis, the resolution of plasmid multimers (Summers et al., 1984; Leung
et al., 1985). It is also possible to use a HpaII-TaqI fragment of smaller size
(280 bp) or a smaller fragment (about 220 bp), contained in the HpaII fragment,
which fragments possess the same properties (Summers and Sherratt, 1988). This
resolution takes place by way of a specific intramolecular recombination, which
involves four proteins encoded by the genome of
E. coli: ArgR, PepA, XerC
and XerD (Stirling et al., 1988, 1989; Colloms et al., 1990, Blakely et al., 1993).
In this respect, it [lacuna] particularly advantageous to use all or part of
the
cer fragment of ColE1 or one of its derivatives as defined above.
According to an implementation variant, the DNA molecules of the invention
may also comprise a sequence capable of interacting specifically with a ligand.
Preferably, this is a sequence capable of forming, by hybridization, a triple helix
with a specific oligonucleotide. This sequence thus makes it possible to purify
the molecules of the invention by selective hybridization with a complementary
oligonucleotide immobilized on a support (see application WO 96/18744). The sequence
can be positioned at any site in the DNA molecule of the invention, provided that
it does not affect the functionality of the gene of interest and of the origin
of replication.
As a DNA molecule representative of the present invention, the plasmid pXL2774
and its derivatives may be claimed most particularly. For the purposes of the invention,
the term derivative is understood to refer to any construction derived from pXL2774
and containing one or more genes of interest other than the luciferase gene. Mention
may also be made of the plasmids pXL3029 and 3030 containing an expression cassette
of a therapeutic gene and a sequence capable of interacting specifically with a ligand.
The present invention also relates to the development of a process for the construction
of specific host cells, which are particularly effective for the production of
these therapeutic DNA molecules.
Another subject of the present invention relates to a process for the production
of a circular DNA molecule, characterized in that a host cell is cultured containing
at least one DNA molecule as defined above and a protein, which may or may not
be expressed in situ, which conditions the functionality of the origin of replication
of the said DNA molecule, which is specific and which is foreign to the said host
cell, under conditions which allow the selection of host cells transformed by the
said DNA molecules.
More preferably, the protein which conditions the functionality of the origin
of replication of the DNA molecule is expressed in situ from a corresponding gene.
The gene coding for the replication-initiating protein may be carried by a subsidiary
replicon, which is compatible with the derivatives of the conditional origin of
replication used or which may be introduced into the genome of the host cell by
recombination, by means of a transposon, a bacteriophage or any other vector. In
the particular case in which the gene expressing the protein is placed on a subsidiary
replicon, the latter also contains a promoter region for functional transcription
in the cell, as well as a region which is located at the 3′ end and which
specifies a transcription termination signal. As regards the promoter region, this
may be a promoter region which is naturally responsible for expressing the gene
under consideration when the latter is capable of functioning in the cell. It may
also be a case of regions of different origin (responsible for expressing other
proteins), or even of synthetic origin. In particular, it may be a case of promoter
sequences for prokaryotic or bacteriophage genes. For example, it may be a case
of promoter sequences obtained from the cell genome.
As genes coding for the replication-initiating protein, use may be made either
of wild-type genes or of mutated alleles which make it possible to obtain an increased
number of copies of the plasmids (or derivatives) specific for the initiator protein
which conditions the functionality of the origin of replication used in the DNA molecule.
Such mutants have been described in particular for the plasmids R6K (Inuzuka
and Wada, 1985; Greener et al., (1990), Rts1 (Terawaki and Itoh, 1985, Terawaki
et al., 1990; Zeng et al., 1990), F (Seelke et al., 1982; Helsberg et al., 1985;
Kawasaki et al., 1991), RK2 (Durland et al., 1990; Haugan et al., 1992, 1995),
pSC101 (Xia et al., 1991; Goebel et al., 1991; Fang et al., 1993).
In the particular case in which the DNA molecule used possesses an origin of
replication
derived from the plasmid R6K, the initiator protein is a derivative of the π
protein of this same plasmid. It is particularly advantageous to express a mutated
form of this protein which is capable of increasing the number of initial copies
appreciably. To do this, the gene incorporated into the host cell is preferably
represented by all or part of the sequence represented in SEQ ID No. 2 or one of
its derivatives and more preferably by the pir 116 gene. The associated mutation
corresponds to the replacement of a proline by a leucine. According to a particular
embodiment of the invention, this pir 116 gene is directly incorporated into the
host cell genome.
Advantageously, one of the genes of the specific host cell, which
is essential under the culture conditions chosen, contains a specific codon which
is recognizable by the selected suppressor tRNA in the DNA molecule. According
to a preferred mode of the invention, this is an amber TAG codon. In this particular
case, the cell may grow, under culture conditions for which the product of the
gene containing the TAG codon is essential, only if the plasmid allowing the expression
of sup is present in the host cell. The culture conditions thus constitute the
pressure for selection of the DNA molecule.
Preferably, the gene containing the amber codon is a gene involved in
the biosynthesis of an amino acid, arginine. This gene, argE, codes for an N-acetylornithinase
(Meinnel et al., 1992) and in this case contains a TAG codon corresponding to a
point mutation Gln-53 (CAG)->TAG; the pressure for selection of the plasmid
carrying the sup gene is then provided by culturing in minimal M9 medium (Maniatis
et al., 1989). However, this could also be, for example, a gene for biosynthesis
of a vitamin or a nucleic acid base, or alternatively a gene which allows a specific
nitrogen or carbon source to be used or any other gene whose functionality is essential
for cellular viability under the chosen culture conditions.
The host cell is preferably chosen from
E. coli strains and is more preferably
represented by the strain
E. coli XAC-1.
According to a specific embodiment of the invention, the host cell used
in the claimed process is a cell of the
E. coli strain XAC-1, containing
the pir 116 gene in its genome and transformed by the plasmid pXL2774 or one of
its derivatives.
According to an advantageous variant of the invention, the host cell used
in the process claimed is a prokaryotic cell in which the endA1 gene or a homologous
gene is inactivated. The endA gene codes for endonuclease I of
E. coli.
This periplasmic enzyme has a non-specific activity of cleaving double-stranded
DNA (Lehman, I. R., G. G. Roussos and E. A. Pratt (1962) J. Biol. Chem. 237: 819-828;
Wright M. (1971) J. Bacteriol. 107: 87-94). A study carried out on various strains
of
Escherichia coli (wild-type or endA) showed that the degradation of plasmid
DNA incubated in extracts of these bacterial strains existed in the endA+ strains
but not in the endA mutants. (Wnendt S. (1994) BioTechniques 17: 270-272). The
quality of the plasmid DNA isolated from endA+ strains or from endA mutants was
studied by the company Promega using their purification system (Shoenfeld, T.,
J. Mendez, D. Storts, E. Portman, B.†Patterson, J. Frederiksen and C. Smith.
1995. Effects of bacterial strains carrying the endA1 genotype on DNA quality isolated
with Wizard plasmid purification systems. Promega notes 53). Their conclusion is
as follows: the quality of the DNA prepared from endA mutants is, overall, better
than that of DNA prepared in the endA+ strains tested.
The quality of the plasmid DNA preparations is thus affected by any contamination
with this endonuclease (relatively long-term degradation of the DNA).
The deletion or mutation of the endA gene can be envisaged without difficulty
insofar as the mutants no longer having this endonuclease activity behave on the
whole like wild-type bacteria (Dürwald, H. and H. Hoffmann-Berling (1968)
J. Mol. Biol. 34: 331-346).
The endA1 gene can be inactivated by mutation, total or partial deletion, disru
