U.S. patent application number 09/734357 was filed with the patent office on 2002-08-01 for variants of thymidine kinase, related nucleic acids sequences and their use in genic therapy.
Invention is credited to Blanche, Francis, Cameron, Beatrice, Couder, Michel, Crouzet, Joel.
Application Number | 20020102247 09/734357 |
Document ID | / |
Family ID | 26232518 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102247 |
Kind Code |
A1 |
Crouzet, Joel ; et
al. |
August 1, 2002 |
Variants of thymidine kinase, related nucleic acids sequences and
their use in genic therapy
Abstract
The present invention relates to a nucleic acid sequence
characterized in that it is derived from the wild nucleic acid
sequence coding for a thymidine kinase, said nucleic acid sequence
having at least one mutation in the region corresponding to the ATP
binding site and conveniently a second mutation in the N-terminal
region and/or C-terminal region. It also relates to variants of the
wild thymidine kinase and their use in genic therapy.
Inventors: |
Crouzet, Joel; (Sceaux,
FR) ; Blanche, Francis; (Paris, FR) ; Couder,
Michel; (Sucy En Brie, FR) ; Cameron, Beatrice;
(Paris, FR) |
Correspondence
Address: |
BROBECK, PHLEGER & HARRISON, LLP
ATTN: INTELLECTUAL PROPERTY DEPARTMENT
1333 H STREET, N.W. SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
26232518 |
Appl. No.: |
09/734357 |
Filed: |
December 12, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09734357 |
Dec 12, 2000 |
|
|
|
09125099 |
Aug 6, 1998 |
|
|
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Current U.S.
Class: |
424/94.5 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 9/1211 20130101; A61K 38/00 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
424/94.5 ;
435/194; 536/23.2; 435/69.1; 435/320.1; 435/325 |
International
Class: |
A61K 038/52; C07H
021/04; C12N 009/12; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 1996 |
FR |
96/01603 |
Aug 1, 1996 |
FR |
96/09709 |
Claims
1. Nucleic acid sequence coding for a thymidine kinase,
characterized in that it possesses, relative to the wild-type
sequence, at least one mutation in the region corresponding to the
ATP-binding site combined with at least one mutation in the
N-terminal and/or C-terminal region.
2. Nucleic acid sequence, characterized in that it is derived from
the sequence coding for a wild-type thymidine kinase, the said
sequence possessing at least one mutation in the region
corresponding to the ATP-binding site and at least one mutation in
the N-terminal and/or C-terminal region.
3. Nucleic acid sequence according to claim 2, characterized in
that it is derived from the sequence coding for herpes simplex
virus type 1 thymidine kinase.
4. Nucleic acid sequence according to claim 3, characterized in
that the said sequence comprises at least one substitution of a
guanine at position 180 by an adenine (G180A).
5. Nucleic acid sequence according to claim 3 or 4, characterized
in that the said sequence comprises at least one substitution of a
guanine at position 180 by an adenine (G180A) and at least one
substitution of the guanine at position 16 by an adenine
(G16A).
6. Nucleic acid sequence according to claim 3 or 4, characterized
in that the said sequence comprises at least one substitution of a
guanine at position 180 by an adenine (G180A) and at least a double
substitution of the guanines at position 28 and 30 by adenines
(G28A and G30A).
7. Nucleic acid sequences according to one of claims 4 to 6,
characterized in that the said sequence comprises, in addition, at
least one mutation in its C-terminal region.
8. Nucleic acid sequence according to one of claims 4, 6 and 7,
characterized in that it possesses at least one substitution of a
guanine at position 180 by an adenine (G180A), at least a double
substitution of the guanines at position 28 and 30 by adenines
(G28A and G30A) a double substitution of the cystosines at position
591 and 892 by thymines (C591T and C892T) and a double substitution
of the guanines at position 1010 and 1011 by adenines (G1010A and
G1011A).
9. Nucleic acid sequence coding for a thymidine kinase variant,
characterized in that it is chosen from: (a) the sequence SEQ ID
No. 3 or a portion of the latter carrying the (G180A) mutation or
one of their complementary strand, (b) the sequences SEQ ID No. 6
and SEQ ID No. 7 or a portion of these sequences carrying the
(G180A) mutation and, respectively, the G16A mutation and the
(G28A; G30A) double mutation or one of their complementary strand,
(c) the sequence SEQ ID No. 8 or a portion of the latter carrying
the (G180A) mutation the (G28A: G30A) double mutation, and the
(C591T; C892T; G1010A; G1011A) quadruple mutation or one of their
complementary strand (d) any sequence hybridizing with the
sequences (a), (b) and/or (c) and coding for a thymidine kinase
variant and as defined in claim 1 or 2, (e) the variants of (a),
(b), (c) and (d) resulting from the degeneracy of the genetic
code.
10. Nucleic acid sequence coding for a variant of a wild-type
thymidine kinase, capable of being obtained by mutagenesis,
site-directed or otherwise, of a sequence according to one of
claims 1 to 9.
11. Nucleic acid sequence according to one of the preceding claims,
characterized in that it can be of eukaryotic, bacterial, viral,
synthetic or semi-synthetic origin.
12. Variant of a wild-type thymidine kinase, capable of being
expressed from a nucleic acid sequence according to any one of
claims 1 to 11.
13. Variant of a thymidine kinase, comprising at least one mutation
in the region corresponding to the ATP-binding site combined with
at least one mutation in its N-terminal and/or C-terminal
region.
14. Variant according to claim 13, characterized in that the region
involved in the region corresponding to the ATP-binding site is
represented by the consensus GXXXXGK(T/S).
15. Variant according to claim 14, characterized in that it is
preferably the motif GPHGMGKT.
16. Variant according to claim 15, characterized in that it
comprises at least one substitution at position 60 of a methionine
by an isoleucine.
17. Variant of a wild-type thymidine kinase, characterized in that
it is the mutant 1537:E4.
18. Variant according to one of claims 13 to 16, characterized in
that the mutation in the N-terminal region lies between amino acids
1 and 20 of the said region.
19. Variant according to claim 18, characterized in that this
mutation lies between amino acids 1 and 15 of the N-terminal
region.
20. Variant according to claim 19, characterized in that this
mutation lies between amino acids 1 and 10 of the N-terminal
region.
21. Variant according to claim 20, characterized in that this
mutation lies between amino acids 5 and 10 of the N-terminal
region.
22. Variant of herpes simplex virus type 1 thymidine kinase
comprising at least one substitution at position 60 of a methionine
by an isoleucine and a substitution at position 10 of an alanine by
a threonine.
23. Variant of herpes simplex virus type 1 thymidine kinase
comprising at least one substitution at position 60 of a methionine
by an isoleucine and a substitution at position 6 of a glycine by a
serine.
24. Variant of a wild-type thymidine kinase, characterized in that
it is the mutant 2-865:H12.
25. Variant of a wild-type thymidine kinase, characterized in that
it is the mutant 2-3361:D3.
26. Variant according to claims 14 to 23, comprising, in addition,
at least one mutation in the C-terminal region.
27. Variant according to claim 26, characterized in that this
mutation lies between amino acids 320 and 350 of the C-terminal
region.
28. Variant according to claim 27, characterized in that this
mutation lies between amino acids 325 and 345 of the C-terminal
region.
29. Variant according to claim 28, characterized in that this
mutation lies between amino acids 330 and 343 of the C-terminal
region.
30. Variant according to claim 29, characterized in that this
mutation lies between amino acids 335 and 340 of the C-terminal
region.
31. Variant of herpes simplex virus type 1 thymidine kinase
comprising at least one substitution at position 60 of a methionine
by an isoleucine, a substitution at position 10 of an alanine by a
threonine and a substitution at position 337 of an arginine by a
glutamine.
32. Variant of a wild-type thymidine kinase, characterized in that
it is the mutant 3-4216:H2.
33. Variant of a wild-type thymidine kinase, characterized in that
it displays the following kinetic properties: a substantial or even
complete decrease in the inhibition of the activity of
phosphorylation of ganciclovir contrary to the wild-type enzyme for
which the inhibition is very marked at and above 15 .mu.M; an
increase by at least a factor of 2 to 2.5 in the initial rate of
phosphorylation of GCV at and above 15-20 .mu.M, relative to the
wild-type enzyme and a Kcat/Km ratio for thymidine which is reduced
by a factor of 1 to 6 relative to that of the wild-type enzyme.
34. Variant of a thymidine kinase according to claims 13 to 25,
characterized in that it displays at least one of the following
kinetic performance features: a significant decrease in the
inhibition of the phosphorylation of ganciclovir or of the
nucleoside analogue at high concentrations of ganciclovir or of
nucleoside analogue; a rate of phosphorylation of ganciclovir or of
nucleoside analogue, which is at least tripled, and/or a Kcat/Km
ratio comes out unchanged either for thymidine reduced by a factor
greater than or equal to 5 relative to that of the wild-type
enzyme.
35. Variant of a thymidine kinase according to claims 26 to 32,
characterized in that it displays at least one of the following
kinetic performance features: an absence of inhibition of the
phosphorylation of ganciclovir or of the nucleoside analogue at
high concentrations of ganciclovir or of nucleoside analogue, an
increase by a factor greater than 3.5 in the initial rate of
phosphorylation of GCV at and above 15-20 .mu.M, relative to the
wild-type enzyme. a Kcat/Km ratio for thymidine which is decreased
by a factor of 4, relative to that of the wild-type enzyme.
36. Expression cassette comprising a nucleic acid according to one
of claims 1 to 11, a promoter permitting its expression and a
transcription termination signal.
37. Vector comprising a nucleic acid according to one of claims 1
to 11 or a cassette according to claim 36.
38. Vector according to according to claim 37, characterized in
that it is a viral vector.
39. Vector according to claim 38, characterized in that it is a
defective recombinant adenovirus.
40. Vector according to claim 38, characterized in that it is a
defective recombinant retrovirus.
41. Vector according to claim 38, characterized in that it is a
defective recombinant AAV.
42. Vector according to claim 38, characterized in that it is a
defective recombinant HSV.
43. Vector according to claim 37, characterized in that it is a
chemical or biochemical vector.
44. Pharmaceutical composition comprising a nucleic acid according
to any one of claims 1 to 11, and/or a vector according to one of
claims 37 to 43.
45. Pharmaceutical composition comprising a variant according to
one of claims 12 to 35.
46. Pharmaceutical composition according to either of claims 45 and
46, for the treatment of hyperproliferative disorders.
Description
[0001] The present invention relates to nucleic acid sequences
coding for enzymes derived from the wild-type thymidine kinase, TK,
enzyme, and possessing improved functions for the purpose of
therapeutic use. It relates more especially to new enzymes
possessing an improved substrate specificity and/or efficacy
relative to the wild-type thymidine kinase enzyme. It also relates
to vectors containing these nucleic acid sequences and to their
therapeutic uses, in particular in gene therapy.
[0002] The present invention relates more especially to the field
of gene therapy which employs suicide genes for the purpose of
inducing the cell death of specific cells such as cells infected
with a virus such as the HIV (human immunodeficiency virus), CMV
(cytomegalovirus) or RSV (respiratory syncytial virus) type virus.
This type of therapeutic treatment, consisting in causing a suicide
gene to be expressed within a cell, is also applied for the
treatment of cancers and of some cardiovascular diseases.
[0003] As suicide gene, it is preferable to use, in gene therapy,
genes whose expression product endows the cell with a sensitivity
to a therapeutic agent. More generally, the genes in question are
ones that code for non-mammalian and non-toxic enzymes which, when
they are expressed in mammalian cells, transform a prodrug which
initially has little or no toxicity to a highly toxic agent. Such a
mechanism of action of prodrugs is advantageous on several counts:
it makes it possible to optimize the therapeutic index by adjusting
the prodrug concentration or the expression of the enzyme, to
interrupt the toxicity by no longer administering the prodrug, and
to evaluate the mortality rate.
[0004] Numerous suicide genes are described in the literature, such
as, for example, the genes coding for cytosine deaminase, purine
nucleoside phosphorylase or a thymidine kinase such as, for
example, the chickenpox virus or the herpes simplex virus type 1
thymidine kinases. Among these genes, the gene coding for herpes
simplex virus type 1 thymidine kinase is most especially
advantageous from a therapeutic standpoint since, in contrast to
the other suicide genes, it generates an enzyme, thymidine kinase,
capable of specifically eliminating dividing cells. This enzyme has
a different substrate specificity from the cellular enzyme, and it
has been shown to be the target of guanosine analogues such as
acyclovir or ganciclovir (Moolten 1986 Cancer Res. 46, p.
5276).
[0005] In the particular case of the HSV1-TK/ganciclovir system,
the mechanism of action may be outlined as follows: mammalian cells
modified to express the HSV1-TK enzyme implement the first step of
phosphorylation of ganciclovir to yield ganciclovir monophosphate.
This step appears to be limiting. Subsequently, cellular kinases
enable this ganciclovir monophosphate to be metabolized
successively to diphosphate and then triphosphate. The ganciclovir
triphosphate thus generated then produces toxic effects by becoming
incorporated in the DNA, and partially inhibits the cellular DNA
polymerase alpha, thereby causing DNA synthesis to be stopped and
hence leading to cell death (Moolten 1986 Cancer Res. 46, p. 5276;
Mullen 1994 Pharmac. Ther. 63, p. 199).
[0006] Moreover, a propagated toxicity effect ("bystander" effect)
has been observed when TK is used. This effect manifests itself in
the destruction not only of the cells which have incorporated the
TK gene, but also the neighbouring cells. The mechanism of this
process may be explained in three ways: i) the formation of
apoptotic vesicles which contain thymidine kinase or phosphorylated
ganciclovir, originating from dead cells, followed by phagocytosis
of these vesicles by the neighbouring cells, ii) transfer of the
prodrug metabolized by thymidine kinase, by a process of metabolic
cooperation, from the cells containing the suicide gene to the
cells not containing it, and/or iii) an immune response linked to
regression of the tumour (Marini et al., 1995 Gene Therapy 2, p.
655).
[0007] For a person skilled in the art, the use of the suicide gene
coding for herpesvirus thymidine kinase is very amply documented.
In particular, the initial in vivo studies on rats having a glioma
show regression of tumours when the HSV1-TK gene is expressed and
when doses of 150 mg/kg of ganciclovir are injected (K. Culver et
al., 1992 Science 256, p. 1550). However, these doses are highly
toxic in mice (T. Osaki et al., 1994 Cancer Research 54, p. 5258)
and hence totally banned in gene therapy in man.
[0008] A number of therapeutic trials are also in progress in man,
in which the TK gene is delivered to the cells by means of
different vectors such as, in particular, retroviral or adenoviral
vectors. In clinical trials of gene therapy in man, the doses which
have to be administered are much smaller, of the order of 5 mg/kg,
and for a short treatment period (14 days) (E. Oldfield et al.,
1995 Human Gene Therapy 6, p. 55). With higher doses or treatments
over a longer period of time, adverse side effects are, in effect,
observed.
[0009] It will hence be especially advantageous to have at one's
disposal a suicide gene related to the gene coding for wild-type
thymidine kinase, capable of generating a variant of the wild-type
TK enzyme which would be more specific and/or more active in
phosphorylating ganciclovir. Advantageously, such a variant may
also be employed at a significantly reduced dose compared to the
dose of wild-type suicide gene and, in addition, enable the dose of
substrate which is traditionally combined with it to be
reduced.
[0010] The objective of the present invention is, specifically, to
provide a nucleic acid sequence coding for an enzyme of the
thymidine kinase type having more potent activating behaviour in
relation to ganciclovir or a nucleoside analogue.
[0011] The sequence of the gene coding for the herpes simplex virus
type 1 thymidine kinase enzyme has been described in the literature
(see, in particular, McKnight 1980 Nucl. Acids Res. 8, p. 5949).
Natural variants of it exist, leading to proteins having a
comparable enzyme activity with respect to thymidine, or
ganciclovir (M. Michael et al., 1995 Biochem. Biophys. Res. Commun
209, p. 966). Similarly, derivatives have been described which were
obtained by directed mutagenesis at the binding site of the enzyme
with the substrate. However, no precise biochemical
characterization has been carried out on the pure enzymes, and no
cellular test using these mutants has been published (Black et al.,
1993 Biochemistry 32, p. 11618). In addition, the inducible
expression of an HSV1-TK gene from which its first 45 codons have
been deleted has been carried out in eukaryotic cells, but the
doses of prodrug used remain comparable to those described in all
the trials in the literature (B. Salomon et al., 1995 Mol. Cell.
Biol. 15, p. 5322). Consequently, none of the variants described
hitherto displays improved activity in relation or with respect to
ganciclovir.
[0012] The present invention describes the construction of new
thymidine kinase variants possessing improved enzymatic properties.
The present application also describes the construction of nucleic
acid sequences coding for these variants, as well as vectors
containing the said sequences and permitting their administration
in vivo and the in vivo production of mutants.
[0013] Unexpectedly, the Applicant has, in effect, prepared,
isolated and characterized a series of particular nucleic acid
sequences coding for thymidine kinase variants possessing the
requisite activating behaviour, that is to say significantly
improved compared to that of wild-type thymidine kinase. The
Applicant has, in particular, demonstrated that new thymidine
kinase variants having improved enzyme properties could be
obtained, in particular, by modification of the region of the
protein responsible for the binding with ATP.
[0014] Thus, a first subject of the invention lies in a nucleic
acid sequence coding for a thymidine kinase, characterized in that
it possesses, in relation to the wild-type sequence, at least one
mutation in the region corresponding to the ATP-binding site
combined with at least one mutation in the N-terminal and/or
C-terminal region.
[0015] More specifically, the first subject of the present
invention is a nucleic acid sequence, characterized in that it is
derived from the nucleic acid sequence coding for a wild-type
thymidine kinase, the said nucleic acid sequence possessing at
least one mutation in the region corresponding to the ATP-binding
site and at least one mutation in the N-terminal and/or C-terminal
region.
[0016] For the purposes of the present invention, the term mutation
covers any substitution, deletion, addition and/or modification of
one or more residues of the nucleic acid sequence in question. It
is understood that the claimed nucleic acid sequence can comprise
other mutations, localized or otherwise, in the regions as defined
above.
[0017] According to a preferred embodiment of the invention, the
nucleic acid sequence is derived from the sequence coding for
herpes simplex virus type I TK. The mutation in the region
corresponding to the ATP-binding site is preferably represented
therein by at least one substitution of a guanine at position 180
by an adenine (G180A).
[0018] As regards the mutation present in the N-terminal portion of
the TK, it may be a substitution of the guanine at position 16 by
an adenine (G16A) or a double substitution of the guanines at
position 28 and 30 by adenines (G28A and G30A).
[0019] According to a preferred embodiment of the invention, the
claimed nucleic sequences carry, in addition to a mutation in the
region corresponding to the ATP-binding site, at least one mutation
in the C-terminal portion and more particularly localized between
positions 990 and 1030. According to another embodiment of the
invention, this mutation, which is present in the C-terminal
portion, is in addition combined with at least one mutation
localized in the N-terminal portion of the TK as defined above.
[0020] As representatives of such a sequence, there may be
mentioned the nucleic acid sequences comprising at least one
substitution of a guanine at position 180 by an adenine (G180A), at
least one double substitution of the guanines at position 28 and 30
by adenines (G28A and G30A) a double substitution of the cystosines
at position 591 and 892 by thymines (C591T and C892T) and a double
substitution of the guanines at position 1010 and 1011 by adenines
(G1010A and G1011A).
[0021] The nucleic acid sequence coding for a thymidine kinase
variant is advantageously chosen from:
[0022] (a) the sequence SEQ ID No. 3 or a portion of the latter
carrying the (G180A) mutation or one of their complementary
strand,
[0023] (b) the sequences SEQ ID No. 6 and SEQ ID No. 7 or a portion
of these sequences carrying the (G180A) mutation and, respectively,
the G16A mutation and the (G28A; G30A) double mutation or one of
their complementary strand,
[0024] (c) the sequence SEQ ID No. 8 or a portion of the latter
carrying the (G180A) mutation the (G28A ; G30A) double mutation,
and the (C591T; C892T; G1010A; G1011A) quadruple mutation or one of
their complementary strand
[0025] (d) any sequence hybridizing with the sequences (a), (b)
and/or (c) coding for a thymidine kinase variant and in accordance
with the present invention,
[0026] (e) the variants of (a), (b), (c) and (d) resulting from the
degeneracy of the genetic code.
[0027] The nucleic acid sequence according to the invention can be
of eukaryotic, bacterial, viral, synthetic or semi-synthetic
origin.
[0028] Generally, speaking, the nucleic acid sequences of the
invention may be prepared according to any technique known to a
person skilled in the art. By way of illustration of these
techniques, there may be mentioned, in particular:
[0029] chemical synthesis, using the sequences presented in the
application and, for example, a nucleic acid synthesizer,
[0030] the screening of libraries by means of specific probes, in
particular such as are described in the application, or
alternatively
[0031] mixed techniques including chemical modification
(elongation, deletion, substitution, and the like) of sequences
screened from libraries.
[0032] The nucleic acid sequences according to the invention may
also be obtained by mutagenesis, site-directed or otherwise, of a
natural or already mutated nucleic acid sequence coding,
respectively, for a wild-type thymidine kinase or one of its
variants. Numerous methods enabling site-directed or random
mutagenesis to be carried out are known to a person skilled in the
art, and there may be mentioned site-directed mutagenesis using PCR
or oligonucleotides, and random mutagenesis in vitro by chemical
agents such as, for example, hydroylamine or in vivo in mutator
strains of E. coli (Miller "A short course in bacterial genetics",
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1992).
[0033] The present invention thus extends to any nucleic acid
sequence coding for a variant of a wild-type thymidine kinase and
capable of being obtained from a nucleic acid sequence as claimed
by employing one of the modification techniques mentioned above,
and more preferably mutagenesis, site-directed or otherwise.
[0034] Advantageously, the products of the claimed sequences
according to the present invention prove more potent than the
natural enzyme from which they are derived by structural
modification(s). When expressed in target cells, they display an
improved enzyme activity relative to the natural enzyme with
respect to ganciclovir or a nucleoside analogue. The behaviour
terms "more activating" or "improved enzyme activity" of the
variants according to the invention is assessed by comparison to
that of the wild-type enzyme, according to protocols described in
detail in the examples below.
[0035] For the purposes of the present invention, nucleoside
analogue is understood to cover compounds of the acyclovir,
trifluorothymidine,
1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil, ara-A,
araT, 1-beta-D-arabinofuranosylthymidine, 5-ethyl-2'-deoxyuridine,
iodouridine, AZT, AIU, dideoxycytidine and AraC. By way of a
preferred analogue in the context of the present invention, BVDU,
ganciclovir and penciclovir may be mentioned more especially.
[0036] The subject of the present invention is also wild-type
thymidine kinase variants capable of being expressed from a claimed
nucleic acid sequence.
[0037] More especially, the invention extends to any variant of a
thymidine kinase, characterized in that it comprises at least one
mutation in its ATP-binding site combined with at least one
mutation in the N-terminal and/or C-terminal region.
[0038] The localization of this ATP-binding site within the
thymidine kinase peptide sequence varies according to the viral
origin of the enzyme. Thus, depending on whether the thymidine
kinase in question is from the chickenpox virus or from the herpes
simplex virus, type 1 or otherwise, this region is positioned in
different regions. However, generally speaking, it is present
therein in the consensus GXXXXGK(T/S) (SEQ ID No. 1) with X
representing any amino acid, and, in the particular case of herpes
simplex virus type 1 thymidine kinase, in the following specific
sequence: GPHGMGKT (SEQ ID No. 2).
[0039] Consequently, the present invention relates to any variant
of a wild-type thymidine kinase in accordance with the invention
and comprising at least one mutation in its following peptide
region: GXXXXGK(T/S).
[0040] According to a preferred embodiment of the present
invention, the sequence possessing at least one mutation is that
represented by GPHGMGKT (SEQ ID No. 2). In this particular case,
the mutation is more preferably represented therein by at least one
substitution at position 60 of a methionine by an isoleucine.
[0041] As a representative of this type of mutant, the mutant
1537:E4 described in the examples below may be mentioned more
especially.
[0042] As regards more especially the mutation in the N-terminal
region. It is preferably localized in the amino acids numbered from
1 to 20. More preferably, the said mutation is localized in the
amino acids numbered from 1 to 15. Still more preferably, the said
mutation is localized in the amino acids numbered from 1 to 10.
According to a preferred embodiment, the said mutation is localized
in the amino acids numbered from 5 to 10.
[0043] According to a preferred embodiment, the present invention
relates to a variant of herpes simplex virus type I thymidine
kinase comprising at least one mutation represented by a
substitution at position 60 of a methionine by an isoleucine and by
a substitution at position 10 of an alanine by a threonine.
[0044] As a representative of this type of mutant, the mutant
2-865:H12 described in the examples below may be mentioned more
especially.
[0045] According to another most especially preferred embodiment of
the present invention, it is a variant of herpes simplex virus type
I thymidine kinase comprising at least one mutation represented by
a substitution at position 60 of a methionine an isoleucine and
substitution at position 6 of a glycine by a serine.
[0046] As a representative of this type of mutant, the mutant
2-3361:D3 described in the examples below may be mentioned more
especially.
[0047] As regards the mutation in the C-terminal region, it is
preferably localized in the amino acids numbered from 320 to 350.
More preferably, the said mutation is localized in the amino acids
numbered from 325 to 345. Still more preferably, the said mutation
is localized in the amino acids numbered from 330 to 343. According
to a preferred embodiment, the said mutation is localized in the
amino acids numbered from 335 to 340.
[0048] According to a most especially preferred embodiment of the
present invention, it is a variant of herpes simplex virus type I
thymidine kinase comprising at least one substitution at position
60 of a methionine by an isoleucine, a substitution at position 10
of an alanine by a threonine and a substitution at position 337 of
an arginine by a glutamine.
[0049] As a representative of this type of mutant, the mutant
3-4216:H2 described in the examples below may be mentioned more
especially.
[0050] Advantageously, the variants according to the invention
display improved performance features in one or more of the
following enzymatic characteristics:
[0051] inhibition by the substrate: inhibition by ganciclovir at
high concentration is generally observed with the wild-type enzyme;
it is decreased or even eliminated with the variants according to
the invention.
[0052] Rate of phosphorylation of ganciclovir or of another
nucleoside analogue: the variants according to the invention
advantageously possess a higher rate of phosphorylation of
ganciclovir or of another analogue;
[0053] Rate of phosphorylation of thymidine: it is preferably
unchanged or decreased with the variants of the invention which
confers on them greater selectivity in relation to ganciclovir or
of the nucleoside analogue. This rate of phosphorylation of
thymidine will be defined in the text which follows through its
specificity constant Kcat/Km. This is a second-order apparent rate
constant which is familiar to persons skilled in the art. It makes
it possible to describe the properties and the reactions of the
free enzyme and of the free substrate. For substrates in
competition, it determines the specificity of the enzyme towards
these substrates. (A. Fersht, Enzyme Structure and Mechanism 1985,
W.H. Freeman, London).
[0054] Preferably, the variants according to the invention and in
particular the variant 1537:E4 advantageously manifest the
following kinetic properties:
[0055] a substantial or even complete decrease in the inhibition of
the activity of phosphorylation of ganciclovir contrary to the
wild-type enzyme for which the inhibition is very marked at and
above 15 .mu.M;
[0056] an increase by at least a factor of 2 to 2.5 in the initial
rate of phosphorylation of GCV at and above 15-20 .mu.M, relative
to the wild-type enzyme,
[0057] and a Kcat/Km ratio for thymidine which is reduced by at
least a factor of 1 to 6 relative to that of the wild-type enzyme,
and preferably by at least 2.
[0058] Preferably, the variants according to the invention, and
more particularly the variants 2-865:H12 and 2-3361:D3 display at
least one of the following performance features:
[0059] a significant decrease in the inhibition of the
phosphorylation of ganciclovir or of the nucleoside analogue at
high concentrations of ganciclovir or of nucleoside analogue,
[0060] a rate of phosphorylation of ganciclovir or of the
nucleoside analogue which is at least tripled and/or
[0061] a Kcat/Km ratio for thymidine which is either unchanged as
for the mutant 2-865:H12 or reduced by a factor at least equal to 5
as for the mutant 2-3361:D3 relative to that of the wild-type
enzyme.
[0062] More preferably, the variant 3-4216:H2 according to the
invention displays at least one of the following kinetic
performance features:
[0063] an absence of inhibition of the phosphorylation of
ganciclovir or of the nucleoside analogue at high concentrations of
ganciclovir or of nucleoside analogue,
[0064] an increase by a factor greater than 3.5 in the initial rate
of phosphorylation of GCV at and above 15-20 .mu.M, relative to the
wild-type enzyme and/or
[0065] a Kcat/Km ratio for thymidine which is decreased by a factor
of 4, relative to that of the wild-type enzyme.
[0066] From these data, it is apparent that the variant 3-4216:H2
manifests a particularly advantageous behaviour according to the
invention.
[0067] Such qualities are especially advantageous from a
therapeutic standpoint, since they make it possible to envisage a
significant reduction in the doses at which enzymes and/or
nucleoside analogue is/are used for an at least equivalent or even
greater efficacy. Safety is enhanced without this having any
detrimental effect on efficacy.
[0068] For the purposes of the invention, the term variant is also
understood, according to the present invention, to denote any
enzyme obtained by modification, using genetic engineering
techniques, of the nucleic acid sequence coding for a wild-type
thymidine kinase and possessing the behaviour defined above in
relation to the ganciclovir and/or a nucleoside analogue.
(Modification should be understood to mean any mutation,
substitution, deletion, addition or modification of a genetic
and/or chemical nature).
[0069] Naturally, these derivatives according to the invention,
capable of inducing the destruction of the said cells via the
activation of ganciclovir or one of its analogues, may
advantageously be expressed in vivo directly from the claimed
nucleic acid sequences.
[0070] To this end, the present invention also relates to any
expression cassette comprising a nucleic acid sequence as defined
above, a promoter permitting its expression and a transcription
termination signal. The promoter is advantageously chosen from
promoters which are functional in mammalian, preferably human,
cells. More preferably, the promoter in question is one that
permits the expression of a nucleic acid sequence in a
hyperproliferative cell (cancer cell, restenosis, and the like). In
this connection, different promoters may be used. A possible
promoter is, for example, the one actually belonging to the herpes
simplex type I TK gene. Sequences of different origin (responsible
for the expression of other genes, or even synthetic sequences) are
a further possibility. Thus, it is possible to use any promoter or
derived sequence that stimulates or represses the transcription of
a gene, specifically or otherwise, inducibly or otherwise, strongly
or weakly. The promoter sequences of eukaryotic or viral genes may
be mentioned in particular. Possible promoter sequences are, for
example, ones originating from the target cell. Among eukaryotic
promoters, it is possible to use, in particular, ubiquitous
promoters (promoter of the HPRT, PGK, alpha-actin, tubulin, DHFR,
and the like, genes), promoters of intermediate filaments (promoter
of the GFAP, desmin, vimentin, neurofilament, keratin, and the
like, genes), promoters of therapeutic genes (for example the
promoter of the MDR, CFTR, factor VIII, ApoAI, and the like,
genes), tissue-specific promoters (promoter of the pyruvate kinase,
villin, intestinal fatty acid binding protein, smooth muscle
alpha-actin, and the like, gene), promoters of specific cells of
the dividing cell type, such as cancer cells, or alternatively
promoters that respond to a stimulus (steroid hormone receptor,
retinoic acid receptor, glucocorticoid receptor, and the like) or
so-called inducible promoters. Similarly, the promoter sequences
may be ones originating from the genome of a virus, such as, for
example, the promoters of the adenovirus E1A and MLP genes, the CMV
early promoter or alternatively the RSV LTR promoter, and the like.
In addition, these promoter regions may be modified by adding
activating or regulatory sequences, or sequences permitting a
tissue-specific or -preponderant expression.
[0071] The present invention now provides new therapeutic agents
that make it possible to interfere with numerous types of cell
dysfunction. To this end, the nucleic acids or cassettes according
to the invention may be injected as they are at the site to be
treated, or incubated directly with the cells to be destroyed or
treated. It has, in effect, been reported that naked nucleic acid
sequences could enter cells without a particular vector.
Nevertheless, it is preferable in the context of the present
invention to use an administration vector enabling (i) the efficacy
of entry into the cell, (ii) targeting and (iii) extra- and
intracellular stability to be improved.
[0072] In an especially preferred embodiment of the present
invention, the nucleic acid sequence or cassette is incorporated in
a vector. The vector used may be of chemical, biochemical or viral
origin.
[0073] Chemical vector is understood to cover, for the purposes of
the invention, any non-viral agent capable of promoting the
transfer of nucleic acid sequences to eukaryotic cells and their
expression therein. These synthetic or natural, chemical or
biochemical vectors represent an advantageous alternative to
natural viruses, especially for reasons of convenience and safety
and also on account of the absence of theoretical limit regarding
the size of the DNA to be transfected. These synthetic vectors have
two main functions, to compact the nucleic acid which is to be
transfected and to promote its binding to the cell as well as its
passage through the plasma membrane and, where appropriate, both
nuclear membranes. To compensate for the polyanionic nature of
nucleic acids, non-viral vectors all possess polycationic charges.
As representatives of this type of non-viral transfection
techniques which are currently developed for the introduction of
genetic information, there may thus be mentioned those involving
complexes of DNA and DEAE-dextran (Pagano et al., J. Virol.1 (1967)
891), of DNA and nuclear proteins (Kaneda et al., Science 243
(1989) 375) and of DNA and lipids (Felgner et al., PNAS 84 (1987)
7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255
(1980) 10431), and the like.
[0074] More recently, the use of viruses as vectors for gene
transfer has been seen to be a promising alternative to these
physical transfection techniques. In this connection, different
viruses have been tested for their capacity to infect certain cell
populations. This applies especially to retroviruses (RSV, HMS,
MMS, and the like), the HSV virus, adeno-associated viruses and
adenoviruses.
[0075] The nucleic acid sequence or vector used in the present
invention may be formulated for the purpose of topical, oral,
parenteral, intranasal, intravenous, intramuscular, subcutaneous,
intraocular, transdermal, and the like, administration. Preferably,
the nucleic acid sequence or vector is used in an injectable form.
It may hence be mixed with any pharmaceutically acceptable vehicle
for an injectable formulation, in particular for direct injection
at the site to be treated. Possible formulations include, in
particular, sterile isotonic solutions, and dry, in particular
lyophilized compositions which, on addition of sterilized water or
of physiological saline as appropriate, enable injectable solutions
to be made up. A direct injection of the nucleic acid sequence into
the patient's tumour is advantageous, since it enables the
therapeutic effect to be concentrated in the affected tissues. The
doses of nucleic acid sequences used may be adapted in accordance
with various parameters, and in particular in accordance with the
vector, the mode of administration used, the pathology in question
or the desired treatment period.
[0076] The invention also relates to any pharmaceutical composition
comprising at least one nucleic acid sequence as defined above.
[0077] It also relates to any pharmaceutical composition comprising
at least one vector as defined above.
[0078] It also relates to any pharmaceutical composition comprising
at least one thymidine kinase variant as defined above.
[0079] As a result of their antiproliferative properties, the
pharmaceutical compositions according to the invention are most
especially well suited to the treatment of hyperproliferative
disorders such as, in particular, cancer and restenosis. The
present invention thus provides an especially effective method for
the destruction of cells, in particular hyperproliferative cells.
It is thus applicable to the destruction of tumour cells or
vascular wall smooth muscle cells (restenosis). It is most
especially suitable for the treatment of cancer. As an example,
there may be mentioned adenocarcinoma of the colon, thyroid cancer,
carcinoma of the lung, myeloid leukaemia, colorectal cancer, breast
cancer, lung cancer, stomach cancer, cancer of the oesophagus, B
lymphoma, ovarian cancer, bladder cancer, glioblastoma,
hepatocarcinoma, bone cancer, skin cancer, cancer of the pancreas
or kidney and prostate cancer, cancer of the larynx, cancer of the
head and neck, HPV-positive anogenital cancer, EBV-positive cancer
of the nasopharynx, and the like.
[0080] It may be used in vitro or ex vivo. Ex vivo, it consists
essentially in incubating the cells in the presence of a nucleic
acid sequence (or of a vector or cassette or of the derivative
directly). In vivo, it consists in administering to the body an
active amount of a vector (or of a cassette) according to the
invention, preferably directly at the site to be treated (tumour in
particular), prior to, simultaneously with and/or after injection
of the prodrug in question, that is to say ganciclovir or a
nucleoside analogue. In this connection, the subject of the
invention is also a method of destruction of hyperproliferative
cells, comprising the bringing of the said cells or of a portion of
them into contact with a nucleic acid sequence or a thymidine
kinase variant as are defined above.
[0081] Consequently, the present invention provides a TK enzyme
mutated in such a way that the phosphorylation of ganciclovir or of
the nucleoside analogue employed is very significantly increased.
Advantageously, it is thus possible, according to the invention, to
use in cellular and clinical tests a mutated TK nucleic acid
sequence at doses of prodrug i) which are significantly lower, ii)
or are capable of causing a more pronounced "bystander" effect,
iii) or alternatively which do not lead to a cellular toxicity
which could occur when wild-type thymidine kinase is
overexpressed.
[0082] The present invention will be described more fully by means
of the examples and figures which follow, which are to be
considered to be illustrative and non-limiting.
LEGEND TO THE FIGURES
[0083] FIG. 1: Expression plasmid pXL2645
[0084] FIG. 2: Initial rates of phosphorylation (specific activity
in nmol/min/mg) as a function of the thymidine concentration
(.mu.M) for the wild-type and mutant 1537:E4, 2-865:H12, 2-3361:D3
and 3-4216:H2 HSV1-TK enzymes
[0085] FIG. 3: The curves represent the rates of phosphorylation
(specific activity in nmol/min/mg) as a function of the ganciclovir
concentration in .mu.M for the wild-type and mutant (1537:E4,
2-865:H12, 2-3361:D3 and 3-4216:H2) HSV1-TK enzymes.
[0086] FIG. 4: Diagrammatic representation of plasmids pcDNA3-TK,
pXL 3022 and pXL3037. Table I below summarizes the kinetic
constants of these enzymes with respect to ganciclovir and
thymidine.
[0087]
1TABLE 1 Thymidine Ganciclovir Thymidine V.sub.max' Kcat/Km Vmax
obs Acyclovir kinase K.sub.m (.mu.M) nmol/min/mg
s.sup.-1/.mu.M.sup.-1 S.sub.0.5 (.mu.M) nmol/min/mg S.sub.0.5
(.mu.M) Vmax obs Wild-type 0.12 .+-. 0.02 1020 .+-. 105 3540 4.13
400 51 220 1537:E4 0.07 .+-. 0.01 305 .+-. 12 1815 6.4 550 66 250
2-865:H12 0.12 .+-. 0.04 880 .+-. 77 3055 6.15 1080 62 280
2-3361:D3 0.47 .+-. 0.12 718 .+-. 85 637 5.77 730 45 220 3-4216:H2
0.71 .+-. 0.05 1486 .+-. 37 870 14.3 2100 123 590 Km Vmax 255 .+-.
16 910 .+-. 28 24.3 .+-. 3.8 2800 .+-. 130
[0088] Given that GCV does not display conventional Michaelis
kinetic constants (except with the mutant 3-4216:H2), the values
are expressed with the aid of S0.5 (i.e. substrate concentration
leading to half the maximum speed observed).
[0089] Materials and Methods
[0090] Abbreviations
[0091] ACV: acyclovir
[0092] GCV: ganciclovir
[0093] HSV1-TK: herpes simplex virus type 1 thymidine kinase
[0094] General Techniques of Molecular Biology
[0095] The methods traditionally used in molecular biology, such as
preparative extractions of plasmid DNA, centrifugation of plasmid
DNA in a caesium chloride gradient, agarose or acrylamide gel
electrophoresis, purification of DNA fragments by electroelution,
phenol or phenol/chloroform extraction of proteins, ethanol or
isopropanol precipitation of DNA in a saline medium and
transformation in Escherichia coli, are well known to a person
skilled in the art and are amply described in the literature
(Sambrook et al., "Molecular Cloning, a Laboratory Manual", Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989; Ausubel et
al., "Current Protocols in Molecular Biology", John Wiley &
Sons, New York, 1987).
[0096] Plasmids of the pUC type and phages of the M13 series are of
commercial origin (Bethesda Research Laboratories); pBSK or pBKS
plasmids are obtained from Stratagen.
[0097] The enzymatic amplification of DNA fragments by the
so-called PCR (polymerase-catalyzed chain reaction) technique may
be performed using a "DNA thermal cycler" (Perkin Elmer Cetus)
according to the manufacturer's recommendations.
[0098] The electroporation of plasmid DNA into E. coli cells maybe
carried out using an electroporator (Bio-Rad) according to the
supplier's recommendations.
[0099] Verification of the nucleotide sequences may be performed by
the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA,
74 (1977) 5463-5467] using the kit distributed by Amersham or the
one distributed by Applied Biosystems.
EXAMPLE 1
Biochemical Screening of HSV1-TK Mutants
[0100] 1-1 Plasmid for the Prokaryotic Expression of the HSV1-TK
Gene
[0101] Several systems for the expression of the HSV1-TK gene in E.
coli are described in the literature (Colbere et al., 1979 Proc.
Natl. Acad. Sci. USA 76 p. 3755; Kit et al., 1981 Gene 16 p. 287;
Waldman et al., 1983 J. Biol. Chem. 258 p. 11571; Fetzer et al.,
1992 Pharm. Pharmacol. Lett. 2 p. 112; Brown et al., 1995 Nature
Structural Biology 2 p. 876). The one which is described below
permits a very well regulated and high production of the HSV1-TK
protein in its native (unfused, non-truncated) form.
[0102] The prokaryotic expression plasmid pXL2638 was constructed
from the plasmid pHSV-106 (Gibco-BRL) and the expression vector
pET11a (obtained from Novagen) in the following manner. After the
ends were blunted, the 1.5-kb BqlII-NcoI insert originating from
pHSV-106 and containing the HSV1-TK gene, the sequence of which is
published by McKnight 1980 Nucl. Acids Res. 8 p. 5949, was cloned
at the SmaI site of pBSK to form the plasmid pBTK1. An NdeI site
was introduced by site-directed mutagenesis starting from
position--3 of the coding sequence of the HSV1-TK gene. For this
purpose, a 500-bp fragment containing the 5' portion of the gene
was amplified by PCR using PBTK1 as template and the sense
oligonucleotide 5'(TTA TGA ATT CAT ATG GCT TCG TAC CCC GGC)3' SEQ
ID No. 4 and the antisense oligonucleotide 5'(TTA TTT CTA GAG GTC
GAA GAT GAG GGT)3' SEQ ID No. 5 as primers; this fragment was
cloned into M13mp19 and then sequenced. This fragment, digested
with EcoRI and SstI, generates a 460-bp insert which was cocloned
with the 1-kb SstI-XbaI insert from pBTK1 containing the 3' portion
of the HSV1-TK gene into plasmid pUC19 digested with EcoRI and
XbaI; this plasmid pUCTK contains the HSV1-TK gene in the form of
an NdeI-BamHI set, which was cloned between the NdeI and BamHI
sites of pET11a to create plasmid pXL2638. This plasmid enables the
HSV1-TK gene to be expressed under the control of the promoter of
gene 10 of bacteriophage T7; this promoter being induced when the
RNA polymerase of bacteriophage T7 is synthesized, as, for example,
in E. coli strain BL21, lambdaDE3 (Studier et al., 1990 Methods
Enzymol. 185 p. 89).
[0103] 1-2 Preparation of Acellular Extracts
[0104] Acellular extracts of E. coli strain overproducing the
HSV1-TK protein may be prepared in various ways, among which lysis
with lysozyme in the presence of EDTA, the use of Menton-Golin,
French Press or X-Press type grinding apparatuses or the action of
ultrasound may be mentioned. More especially, the acellular
extracts of E. coli strain BL21, lambdaDE3 (Novagen Inc) pXL2638
were prepared in the following manner:
[0105] E. coli strain BL21, lambdaDE3 pXL2638 is cultured in LB
(Luria-Bertani) medium+ampicillin (50 mg/l) at 37.degree. C. to an
absorbance at 600 nm of 0.7; production of the HSV1-TK protein is
induced by adding 1 mM IPTG (isopropyl beta-D-thiogalactoside), and
takes place on continuing the growth of the cells for 3 hours at
30.degree. C. After centrifugation (5000.times. g; 20 min), the
cells obtained from 1 1 of culture are resuspended in 10 ml of 50
mM Tris-HCl buffer pH 7.8 containing 5 mM DTT, 4 mM MgCl.sub.2 and
10% glycerol (v/v), and sonicated for 4 min at 4.degree. C. After
centrifugation (50,000.times.g; 1 h), the supernatant is injected
onto a column of Source 15Q (50 ml of gel, Pharmacia) equilibrated
in the above buffer. The proteins are eluted with a linear gradient
of 0 to 400 mM NaCl in buffer A. The fractions containing the TK
activity are pooled, taken to a final concentration of 1,1 M
ammonium sulphate and chromatographed on a column of
Phenyl-Superose HR 10/10 (Pharmacia) eluted with a linear gradient
decreasing from 1.1 to 0 M ammonium sulphate. The fractions
containing the TK activity are pooled. After this step, the
preparation displays a single band visible in SDS-PAGE after
visualization with Coomassie blue, and this band migrates with an
apparent molecular weight of approximately 41,000.
[0106] 1-3 Assay of TK Activity
[0107] The ATP-dependent activity of phosphorylation of nucleosides
may be detected by proceeding, for example, in the following
manner:
[0108] An enzyme extract containing approximately 0.1 unit of TK is
incubated for 15 min at 37.degree. C. in 100 .mu.l of 50 mM
Tris-HCl buffer pH 7.8 containing 1 mg/ml BSA (bovine serum
albumin), 5 mM ATP, 4 mM MgCl.sub.2, 12 mM KCl, 2 mM DTT, 600 .mu.M
EDTA and 100 .mu.M [8-.sup.3H]GCV (40 nCi/nmol). The reaction is
stopped by adding 10 .mu.l of 50 mM Tris-HCl buffer pH 7.8
containing 1 mM non-radioactive thymidine. The phosphorylated
species are bound to a column of DEAE-Sephadex (400 .mu.l of gel)
and then, after the column is washed, these species are eluted with
2 ml of 1 M HCl. The radioactivity in the sample is then counted by
liquid scintillation.
[0109] The assay of TK activity using thymidine as substrate is
performed in the same manner, employing 0.002 unit of TK and 1
.mu.M (methyl-.sup.14C)thymidine (56 nCi/nmol).
[0110] The unit of TK activity is defined as the amount of enzyme
required to phosphorylate 1 nmol of substrate per min under the
above conditions.
[0111] For the calculation of the kinetic constants, the amount of
TK introduced into the enzyme reaction is adjusted so as to convert
not more than 5% of the substrate introduced at the start, and the
specific activity of this substrate is increased accordingly. The
Michaelis curves are adjusted to the experimental points using the
Enzfitter software (Sigma).
[0112] Since GCV does not display conventional Michaelis kinetic
constants (except with the mutant 3-4216:H2), the values are
expressed with the aid of S0.5 (i.e. substrate concentration
leading to half the maximum speed observed)
[0113] 1-4 Plasmid PXL2645 Permitting a Biochemical Screening
[0114] The heterologous expression systems in E. coli are many and
well known to a person skilled in the art. Expression of the
HSV1-TK gene proved to be the best one for biochemical screening
when the gene is expressed using the tryptophan promoter pTryp at a
high copy number on plasmid pXL2645. The 1.4-kb NdeI-XbaI insert
from pUCTK is cocloned with the 120-bp NdeI-EcoRI and 3.1-kb
EcoRI-XbaRI inserts from pXL694 (Jung et al., 1988 Ann. Inst.
Pasteur/Microbiol. 139 p. 129) to generate the plasmid pXL2619. The
following inserts from pXL2619, namely the 1.5-kb EcoRI-XbaI insert
(containing the HSV1-TK gene and pTryp: the promoter/operator
region of the E. coli tryptophan operon followed by the RBS
(ribosome binding site) of the lambda cII gene) and 530-bp
XbaI-BamHI insert containing the T.sub.rrnB terminator region of
the E. coli ribosomal operon, are cocloned into the vector pBSK+ to
create plasmid pXL2645.
[0115] 1-5 Mutagenesis of the Plasmid
[0116] Numerous methods enabling site-directed or random
mutagenesis to be carried out on plasmids are known to a person
skilled in the art, and there may be mentioned site-directed
mutagenesis using PCR or oligonucleotides according to the
recommendations of the supplier, Amersham, and random mutagenesis
in vitro by chemical agents or in vivo in mutator strains of E.
coli (Miller "A short course in bacterial genetics", Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1992). Plasmid pXL2645
was mutagenized with hydroxylamine according to a protocol already
described and which leads to GC to AT transitions at random on the
plasmid (Humphreys et al., 1976 Mol. Gen. Genet. 145 p. 101). Five
.mu.g of plasmid DNA dissolved in 0.2 M phosphate buffer pH 6
containing 0.4 M hydroxylamine, are incubated at 80.degree. C. or
86.degree. C. for 30 min and then cooled to room temperature for 20
min; the solution is thereafter dialyzed and then precipitated. The
DNA is then redissolved in 50 Al of water. If the plasmid DNA is
pCH110 (obtained from Pharmacia and carrying the lacZ gene),
lacZ.sup.- mutants are obtained as a frequency of 2.4% (or 7.6%,
respectively) when the plasmid is heated to 80.degree. C. (or
86.degree. C., respectively) in the presence of hydroxylamine.
[0117] 1-6 Screening of Mutants Possessing a Modified TK
[0118] Plasmid pXL2645 mutagenized with hydroxylamine at 80.degree.
C. is introduced by electroporation into the E. coli tk.sup.-
strain ME8025 (obtained from the National Institute of Genetics,
Mishima, Shizuoka, Ken, Japan). The electroporants are inoculated
individually into the wells of a microtitration plate containing
100 .mu.l of M9 minimum medium supplemented with 0.4% of casamino
acids and 50 mg/l of ampicillin. The cultures are incubated at
37.degree. C. with agitation for 17 hours. Fifteen .mu.l of the
culture diluted to {fraction (1/25)} in 50 mM Tris-HCl buffer pH
7.8 are incubated for 20 min at 37.degree. C. in a volume of 250
.mu.l of 50 mM Tris-HCl buffer pH 7.8 containing 2 mg/ml of egg
white lysozyme, 5 mM ATP, 4 mM MgCl.sub.2, 12 mM KC.sub.1, 2 mM
DTT, 600 .mu.M EDTA, 16 .mu.M [8-.sup.3H]GCV (60 nCi/nmol) and 1
.mu.M [Methyl-.sup.14C]thymidine (56 nCi/nmol). The reaction is
stopped by adding 25 .mu.l of 50 mM Tris-HCl pH 7.8 buffer
containing 1 mM non-radioactive thymidine. The phosphorylated
species are bound to a column of DEAE-Sephadex (400 .mu.l of gel)
and then, after the column is washed, these species are eluted with
2 ml of 1 M HCl. The radioactivity (.sup.3H and .sup.14C) in the
sample is then counted by liquid scintillation using a
double-labelling programme, and the 3H/.sup.14C ratio is calculated
for each sample.
[0119] For each 96-well microtitration plate, the mean of the
.sup.3H/.sup.14C ratios of all of the clones (M) and the standard
deviation (.sigma.) of the distribution of the .sup.3H/.sup.14C
ratios are calculated. Furthermore, the amount of proteins present
in each of the wells of the 96-well plate is measured using
Bradford's reagent (Coomassie Plus Protein Assay Reagent, Pierce)
from an aliquot fraction of the {fraction (1/25)} dilution prepared
above. Any clone possessing a protein content less than one quarter
of the mean of the clones of the dish is permanently discarded.
[0120] The .sup.3H/14 ratio obtained for each clone of a 96-well
dish is compared with the mean M. Clones possessing a ratio higher
than the sum M+3.sigma. while displaying thymidine phosphorylation
activity greater than M'/2,M' being the mean of the thymidine
phosphorylation activities, are selected for confirmation and
study.
[0121] Table 2 summarizes the results obtained after screening of
4129 clones originating from hydroxylamine mutagenesis of pXL2645
at 80.degree. C. In this screening, for the wild-type enzyme, the
TK activity is designated 100% and the GCV/Thy ratio is 1.
[0122] At the end of this study, a mutant termed 1537:E4
manifesting more activating behaviour with respect to ganciclovir
is revealed.
2TABLE 2 Names of Frequency TK activity % < TK < % Number
mutants % High 233 < TK < 320 2 3841:D2 0.05 3841:F3 Low 5
< TK < 10 17 0.4 Zero TK < 5 99 2.4 Zero with respect to 1
2881:C8 0.02 GCV but unchanged with respect to thymidine High with
respect to GCV/Thy: 2 0.05 GCV but unchanged 1.76(.sigma.:0.11)
1537:E4 with respect to 1.70(.sigma.:0.19) 1921H:12 thymidine
EXAMPLE 2
Primary Structure and Biochemical Characterization of the Mutant
1537:E4
[0123] 2-1 Sequence of the HSV1-TK Gene of the Mutant 1537:E4
[0124] The HSV-TK gene expressed from the mutant 1537:E4 was
sequenced on both strands and a single G180A mutation was observed.
This mutation corresponds to a Met60Ile substitution. It should be
noted that this residue is located in the consensus region of the
ATP-binding site. A comparable study was carried out with the
mutant 1921:H12 and the same substitution (Met60Ile) was
observed.
[0125] 2-2 Cloning of the HSV1-TK Gene of the Mutant 1537:E4 into a
High Expression Prokaryotic Vector
[0126] The 1.4-kb NdeI-BamHI insert coding for the HSV1-TK gene of
the mutant 1537:E4 was cloned into the expression vector pET11a to
generate pET:E4. This plasmid pET:E4 is the one from which the
HSV1-TK enzyme of the mutant 1537:E4 was produced in E. coli BL21,
lambdaDE3 under the conditions described in 1-2.
[0127] 2-3 Biochemical Data
[0128] The kinetic constants for the mutant 1537:E4 TK and the
wild-type TK taken as reference are obtained under the enzyme assay
conditions described in Section 1-3.
[0129] The 2 TKs (wild-type and mutant 1537:E4) do not display a
Michaelis behaviour with ganciclovir and acyclovir (inhibition at
high concentration). This therefore prohibits the calculation of a
Km value with these 2 substrates. There maybe given only the
S.sub.0.5 concentration corresponding to the substrate
concentration giving an initial speed equal to half the maximum
speed observed. Vmax and Vmax obs are expressed therein in
nmol/min/mg of protein.
[0130] The curve in FIG. 2 shows the initial rates of
phosphorylation as a function of the GCV concentration for both
1537:E4 and wild-type enzymes. It brings out, in particular, the
almost complete absence of inhibition of the activity of
phosphorylation of GCV by the mutant 1537:E4 TK, contrary to the
wild-type enzyme for which inhibition is very marked at and above
15 .mu.M. This curve shows, furthermore, an increase by a factor of
2 to 2.5 in the initial rate of phosphorylation of GCV at and above
15-20 .mu.M with the mutant 1537:E4 enzyme, relative to the
wild-type enzyme. The mutant enzyme 1537:E4 displays therein a
Kcat/Km ratio for thymidine which is reduced by a factor of 2
relative to the wild-type enzyme.
EXAMPLE 3
Obtaining, Primary Structure and Biochemical Characterization of
the Mutants 2-865:H12 and 2-3361:D3
[0131] 3-1 Obtaining the Mutants 2-856:H12 and 2-3361:D3
[0132] The plasmid pXL2838 originating from the mutant 1537:E4
carries, under the control of the pTryp promoter, the HSV1-TK gene
whose TK protein differs from the wild-type protein by the mutation
M60I. This plasmid is mutagenized with hydroxylamine at 86.degree.
C. and then introduced by electroporation into the E. coli
tk.sup.31 strain ME8025. A total of 4992 electroporants are
analyzed for their capacity to phosphorylate ganciclovir and
thymidine as described in Example 1-6. The results of this
screening are summarized in Table 3, where the TK activity is
designated 100% and the GCV/Thy ratio is 1 for the wild-type
enzyme. Two mutants, 2-865:H12 and 2-3361:D3, display more
activating behaviour with respect to ganciclovir.
3TABLE 3 Names of Frequency TK activity % < TK < % Number
mutants % Zero TK < 5 227 4.5 Low 5 < TK < 10 55 1.1 High
with respect to GCV/Thy: 2 0.04 GCV but unchanged 4.95 +/- 0.66
2-865:H12 with respect to 3.99 +/- 0.53 2-3361:D3 thymidine
[0133] 3-2 Sequence of the HSV1-TK Gene of the Mutants 2-865:H12
and 2-3361:D3
[0134] The HSV1-TK gene expressed from the mutants 2-865:H12 and
2-3361:D3 was sequenced on both strands and the following mutations
were observed. With the mutant 2-865:H12, the mutations are G28A,
G30A and G180A, corresponding to the substitutions Ala10Thr and
Met60Ile. Whereas with the mutant 2-3361:D3, the mutations are
G16A, G180A, C306T and C308T, corresponding to the substitutions
Gly6Ser, Met60Ile and Thr103Ile.
[0135] 3-3 Importance of the Mutation Localized in the N-Terminal
Region of the TK
[0136] The enzymes of the mutants 2-865:H12 and 2-3361:D3 both
carry a mutation in the N-terminal portion of the thymidine kinase,
position 10 or 6. Since the enzyme corresponding to the mutant
2-3361:D3 also contains a mutation at position 103, plasmids
containing the mutations at positions 6 and 60 (pXL2964) or at
positions 60 and 103 (pXL2963) or at position 103 (pXL2965) or at
position 6 only (pXL2966) were constructed in the following manner:
the 400-bp SnaBI-BspEI insert from the plasmid pXL2840 (plasmid
extracted from the mutant 2-3361:D3) was cloned either into plasmid
pXL2645 digested with SnaBI-BspEI to generate pXL2965, or into
plasmid pXL2838 digested with SnaBI-BspEI to generate pXL2963.
Plasmid pXL2964 corresponds to the ligation of the 2.9-kb MluI-XmnI
insert from pXL2838 with the 2.1-kb XmnI-MluI fragment of pXL2840.
The plasmid pXL2966 corresponds to the ligation of the 2.9 kb
MluI-XmnI insert from pXL2645 with the 2.1 kb XmnI-MluI fragment of
pXL2840. These plasmids were transformed into E. coli strain
ME8025, and the thymidine kinase activity with respect to thymidine
and ganciclovir is determined as in Example 1-6; the GCV/Thy ratios
are seen in Table 4.
4TABLE 4 Plasmid (Mutant) Mutation of the TK enzyme GCV/Thy pXL2645
Wild-type TK 1.00 +/- 0.08 pXL2838 (1537:E4) M601 1.60 +/- 0.17
pXL2840 (2-3361:D3) G6S, M601, T1031 3.10 +/- 0.26 pXL2963 M601,
T1031 1.60/-0.18 pXL2964 G6S, M601 2.80 +/- 0.40 pXL2965 T1031 1.00
+/- 0.18 pXL2966 G6S 1.40 +/- 0.20
[0137] Only the GCV/Thy ratio obtained with plasmid pXL2964 is
comparable to that obtained with plasmid pXL2840. And the results
collectively show that the mutation at position 6, not the mutation
at position 103, is the one which leads to an improvement in the TK
activity with respect to GCV of the mutant 2-3361:D3 relative to
the mutant 1537:E4. Moreover, the mutation at position 6 alone
leads to an improvement in the TK activity. The effect of this
mutation is therefore additive to the effect of the mutation at
position 60 for improving the TK activity with respect to GCV.
Furthermore, the improvement obtained by combining these two
mutations Gly6Ser and Met60Ile can also be obtained by combining
with the mutation Met60Ile another mutation situated in the
N-terminal portion of the thymidine kinase, for example the
mutation Ala 10Thr.
[0138] 3-4 Cloning of the HSV1-TK Gene of the Mutants 2-865:H12 and
2-3361:D3 into a High Expression Prokaryotic Vector
[0139] The NdeI-BamHI insert coding for the HSV1-TK gene
originating from the mutant 2-865:H12 (or 2-3361:D3, respectively)
was cloned into the expression vector pET11a to form the plasmid
pXL2843 (or pXL2841, respectively). These plasmids were transformed
into E. coli BL21met.sup.-lambdaDE3 in order to produce the enzymes
of these two mutants.
[0140] 3-5 Biochemical Data
[0141] The TK enzymes originating from the E. coli
BL21met.sup.-lambdaDE3, pXL2843 and E. coli BL21met.sup.-
lambdaDE3, pXL2841 cultures were purified to homogeneity. The
kinetic constants for the TK of the mutants 2-865:H12 and 2-3361:D3
were determined and compared with those of the wild-type and the
mutant 1537:E4 TK. The set of values is recorded in FIG. 3. The
curves in FIG. 3 show the rates of phosphorylation as a function of
the GCV concentration for the four enzymes. They bring out a
partial lifting of the inhibition by GCV of the mutant 1537:E4,
2-865:H12 and 2-3361:D3 TKs, contrary to the wild-type enzyme for
which inhibition is marked above 30 .mu.M. These curves also show
an increase in the rate of phosphorylation of GCV by a factor of
1.6 to 2.5 at 16 .mu.M GCV and of 4.3 to 4.9 at 100 .mu.M GCV,
relative to the wild-type enzyme. Table 1 of FIG. 3 shows that the
enzyme of the mutant 2-865:H12 possesses a Kcat/Km ratio for
thymidine which is unchanged relative to that of the wild-type
enzyme, and that of the mutant 2-3361:D3 manifests a Kcat/Km ratio
which is reduced by a factor greater than or equal to 5 relative to
that of the wild-type enzyme.
EXAMPLE 4
Obtaining, Primary Structure of The Mutant and Biochemical
Characterization of the Mutant 3-4216:H2
[0142] 4.1 Obtaininq of the mutant 3-4216:H2 The plasmid pXL2842
originating from the mutant 2-865:H12 carries, under the control of
the pTryp promoter, the HSV1-TK gene whose TK protein differs from
the wild-type protein by the mutations Ala.sub.10Thr and Met60Ile.
This plasmid is mutagenized with hydroxylamine at 86.degree. C. and
then introduced by electroporation into the E.coli tk.sup.- strain
ME8025. A total of 3900 electroporants are analysed for their
capacity to phosphorylate ganciclovir and thymidine as described in
Example 1-6. The results of this screening are summarized in Table
5, where the TK activity is designated 100% and the GCV/Thy ratio
is 1 for the wild-type enzyme. A mutant 3-4216:H2 displays a more
activating behaviour with respect to ganciclovir.
5TABLE 5 Names of Frequency TK activity % < TK < % Number
mutants % Zero TK < 5 121 3.2 Low 5 < TK < 10 19 0.5 Zero
with respect 1 3-2293:C4 0.025 to GCV but unchanged with respect to
thymidine High with respect GCV/THy: 1 3-4216:H2 0.025 to GCV but
7.84 +/- 1.09 unchanged with respect to thymidine
[0143] 4.2 Sequence of the HSV1-TK Gene of the Mutant 3-4216:H2
[0144] The HSV1-TK gene expressed from the mutant 3-4216:H2 was
sequenced on both strands and the following mutations were observed
(G28A, G30A, G180A, C591T, C892T, G1010A and G1011A). These
mutations correspond to the substitutions Ala10Thr, Met60Ile and
Arg337Gln.
[0145] 4-3 Cloning of the HSVI-TK Gene of the Mutant 3-4216:H2 into
a High Expression Prokaryotic Vector.
[0146] The NdeI-BamHI insert coding for the HSV1-TK gene
originating from the mutant 3-4216:H2 was cloned into the
expression vector pET11a to form the plasmid pXL3129. This plasmid
was transformed into E.coli BL21met.sup.-lambdaDE3 in order to
produce the enzyme of this mutant.
[0147] 4-4 Biochemical Data.
[0148] The TK enzyme originating from the E.coli
BL21met.sup.-lambdaDE3, pXL3129 was purified to homogeneity. The
kinetic constants for the TK of the mutant 3-4216:H2 were
determined and compared with those of the wild-type and mutant
1537:E4, 2-865:H12 and 2-3361:D3 TK. The set of values is recorded
in FIG. 3 and Table 1. The enzyme of the mutant 3-4216:H2 is
remarkable by the complete lifting of the inhibition by the GCV
substrate and by the increase in the rate of phosphorylation of
GCV. The initial rate of phosphorylation of GCV with this mutant is
multiplied by a factor of 7, relative to the wild-type enzyme, and
a factor of 2.8 relative to the mutant 2-865:H12. Furthermore, the
mutant 3-4216:H2 possesses a lower catalytic activity for thymidine
phosphorylation than the wild-type enzyme and the other mutants
1537:E4, 2-865:H12 and 2-3361:D3, as indicated by the Kcat/Km
values in Table 1.
EXAMPLE 5
Construction of Vectors for the Expression of TK Variants
[0149] This example describes the construction of vectors which can
be used for the in vitro or in vivo transfer of the nucleic acid
sequences of the invention.
[0150] 5.1--Construction of Plasmid Vectors
[0151] To do this, commercial vectors such as the vectors pZeoSV,
pSV2pcDNA3 and the like can be used.
[0152] The vector pSV2, described in DNA Cloning, A practical
approach Vol. 2, D. M. Glover (Ed) IRL Press, Oxford, Washington
D.C., 1985. This vector is a eukaryotic expression vector. The
nucleic acids coding for the TK variants were inserted into this
vector at the HpaI-EcoRV sites. They are thus placed under the
control of the promoter of the SV40 virus enhancer.
[0153] The vector pCDNA3 (Invitrogen). This is also a eukaryotic
expression vector. The nucleic acid sequences coding for the TK
variants of the invention are thus placed in this vector under the
As regards the genes coding for wild-type HSVI-TK and control of
the CMV early promoter. All the constructions described in Example
1 were introduced into this vector between the HindIII/NotI sites
in order to be tested in the different in vivo evaluation
systems.
[0154] As regards the gene coding for the wild-type HSV1-TK and
that of the mutant 2-865:H12, constructs which were prepared with
the plasmid pXL2990. This vector originating from pBSK (Stratagen)
comprises the CMV promoter/enhancer amplified by PCR from the
plasmid pGCN (Tanaka et al., 1990, Cell 60, p. 375). The HSV1-TK
gene was amplified by PCR with the aid of the plasmid pBTK1 (see
Example 1.1) by creating NcoI and AvrII sites at the ATG and TGA
codons respectively. This gene was then cloned downstream of the
CMV promoter of pXL2990 and upstream of the SV40 late
polyadenylation sequence (amplified by PCR with the aid of the
plasmid pGL3basic (Promega)) in order to generate the plasmid
pXL3022. In a similar manner, the HSV1-TK gene derived from the
mutant 2-865:H12 was amplified by PCR with the aid of the plasmid
pXL2843 (see Example 3.4) and then cloned downstream of the CMV
promoter of pXL2990 and upstream of the SV40 late polyadenylation
sequence in order to generate the plasmid pXL3037, see FIG. 4.
[0155] 5.2--Construction of Viral Vectors
[0156] According to a particular embodiment, the invention lies in
the construction and use of viral vectors permitting the in vivo
transfer and expression of the nucleic acids as defined above.
[0157] As regards adenoviruses more especially, different
serotypes, the structure and properties of which vary somewhat,
have been characterized. Among these serotypes, it is preferable to
use, in the context of the present invention, human adenoviruses
type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see
Application WO 94/26914). Among adenoviruses of animal origin which
can be used in the context of the present invention, adenoviruses
of canine, bovine, murine (e.g.: Mav1, Beard et al., Virology 75
(1990) 81), ovine, porcine, avian or alternatively simian (e.g.:
SAV) may be mentioned. Preferably, the adenovirus of animal origin
is a canine adenovirus, and more preferably a CAV-2 adenovirus
[strain Manhattan or A26/61 (ATCC VR-800), for example]. It is
preferable to use adenoviruses of human or canine or mixed origin
in the context of the invention.
[0158] Preferably, the defective adenoviruses of the invention
comprise the ITRs, a sequence permitting encapsidation and a
nucleic acid according to the invention. Still more preferably, in
the genome of the adenoviruses of the invention, the E1 region at
least is non-functional. The viral gene in question may be rendered
non-functional by any technique known to a person skilled in the
art, and in particular by total elimination, substitution, partial
deletion or addition of one or more bases in the gene or genes in
question. Such modifications may be obtained in vitro (on the
isolated DNA) or in situ, for example by means of genetic
engineering techniques, or alternatively by treatment by means of
mutagenic agents. Other regions may also be modified, and in
particular the E3 (WO 95/02697), E2 (WO 94/28938), E4 (WO 94/28152,
WO 94/12649, WO 95/02697) and L5 (WO 95/02697) regions. According
to a preferred embodiment, the adenovirus according to the
invention comprises a deletion in the E1 and E4 regions. According
to another preferred embodiment, it comprises a deletion in the E1
region, into which are inserted the E4 region and the nucleic acid
sequence of the invention (see FR 94/13355). In the viruses of the
invention, the deletion in the E1 region preferably extends from
nucleotides 455 to 3329 on the Ad5 adenovirus sequence.
[0159] The defective recombinant adenoviruses according to the
invention may be prepared by any technique known to a person
skilled in the art (Levrero et al., Gene 101 (1991) 195, EP
185,573; Graham, EMBO J. 3 (1984) 2917). In particular, they may be
prepared by homologous recombination between an adenovirus and a
plasmid carrying, inter alia, the claimed nucleic acid sequence.
Homologous recombination takes place after cotransfection of the
said adenovirus and said plasmid into a suitable cell line. The
cell line used should preferably (i) be transformable by the said
elements, and (ii) contain the sequences capable of complementing
the portion of the genome of the defective adenovirus, preferably
in integrated form in order to avoid risks of recombination. As an
example of a line, there may be mentioned the human embryonic
kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which
contains, in particular, integrated in its genome, the left-hand
portion of the genome of an Ad5 adenovirus (12 %), or lines capable
of complementing the E1 and E4 functions as are described, in
particular, in Applications Nos. WO 94/26914 and WO 95/02697.
[0160] Thereafter, the adenoviruses which have multiplied are
recovered and purified according to standard techniques of
molecular biology, as illustrated in the examples.
[0161] More especially, the adenoviral constructs according to the
invention are obtained in accordance with the following
protocol:
[0162] They were constructed according to the technology described
in patent W096/25506 to which reference will be made for the
detailed description of the plasmids mentioned below. For that, the
shuttle plasmid pACK3 was used; it originates from ColE1 and
contains i) the gene which confers resistance to kanamycin, ii) the
B. subtilis sacB gene, iii) the ITR-Psi sequences (position 1-386
of the Ad5 adenovirus) separated from the sequences containing the
gene coding for the PIX protein (position 3447-4415 of the Ad4
adenovirus) by the EcoRV and SalI restriction sites. The eukaryotic
expression cassettes of the plasmids pXL3022 and pXL3037 were
cloned between the EcoRV and SalI sites of the shuttle plasmid
pACK3 in order to form the plasmids pXL3050 and 3051 respectively,
see FIG. 4. By double homologous recombination with the aid of the
adenoviral plasmid pXL2822 (described by J. Crouzet et al., 1997,
Proc. Natl. Acad. Sci. 94, in press), the adenoviral plasmids
pXL3075 and 3076 were generated, respectively. These plasmids were
digested with PacI and transfected into the human cell line 293 in
order to produce the viruses ADV3075 and ADV3076. These viruses are
deleted in the E1 and E3 regions and which contain respectively the
cassettes for expression of the HSV1-TK genes for the wild-type
enzyme and for the mutant 2-865:H12.
[0163] Adeno-associated viruses (AAV) are, for their part,
relatively small-sized DNA viruses which integrate stably and in a
site-specific manner in the genome of the cells they infect. They
are capable of infecting a broad range of cells without inducing an
effect on cell growth, morphology or differentiation. Moreover,
they do not appear to be implicated in pathologies in man. The AAV
genome has been cloned, sequenced and characterized. It comprises
approximately 4,700 bases, and contains at each end an inverted
repeat region (ITR) of approximately 145 bases, serving as origin
of replication for the virus. The remainder of the genome is
divided into 2 essential regions carrying the encapsidation
functions: the left-hand portion of the genome, which contains the
rep gene involved in the viral replication and expression of the
viral genes; and the right-hand portion of the genome, which
contains the cap gene coding for the capsid proteins of the
virus.
[0164] The use of vectors derived from AAV for the transfer of
genes in vitro and in vivo has been described in the literature
(see, in particular, WO 91/18088; WO 93/09239; U.S. Pat. No.
4,797,368, U.S. Pat. No. 5,139,941, EP 488,528). These applications
describe different constructions derived from AAV, in which the rep
and/or cap genes are deleted and replaced by a gene of interest,
and their use for transferring the said gene of interest in vitro
(to cells in culture) or in vivo (directly into a body). The
defective recombinant AAVs according to the invention may be
prepared by cotransfection, into a cell line infected with a human
helper virus (for example an adenovirus), of a plasmid containing a
nucleic acid sequence of the invention, of interest, flanked by two
inverted repeat regions (ITR) of AAV, and a plasmid carrying the
encapsidation genes (rep and cap genes) of AAV. A cell line which
can be used is, for example, the line 293. The recombinant AAVs
produced are then purified by standard techniques.
[0165] Regarding herpesviruses and retroviruses, the construction
of recombinant vectors has been amply described in the literature:
see, in particular, Breakfield et al., New Biologist 3 (1991) 203;
EP 453242, EP 178220, Bernstein et al., Genet. Eng. 7 (1985) 235;
McCormick, BioTechnology 3 (1985) 689, and the like. In particular,
retroviruses are integrative viruses which selectively infect
dividing cells. They hence constitute vectors of interest for
cancer applications. The retrovirus genome essentially comprises
two LTRs, an encapsidation sequence and three coding regions (gag,
pol and env). In the recombinant vectors derived from retroviruses,
the gag, pol and env genes are generally deleted wholly or
partially, and replaced by a heterologous nucleic acid sequence of
interest. These vectors may be produced from different types of
retrovirus such as, in particular, MoMuLV (Moloney murine leukaemia
virus; also designated MoMLV), MSV (Moloney murine sarcoma virus),
HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous
sarcoma virus) or alternatively Friend virus.
[0166] To construct recombinant retroviruses according to the
invention containing a nucleic acid sequence according to the
invention, a plasmid containing, in particular, the LTRs, the
encapsidation sequence and the said nucleic acid sequence is
constructed, and then used to transfect a so-called encapsidation
cell line capable of providing in trans the retroviral functions
which are deficient in the plasmid. Generally, the encapsidation
lines are hence capable of expressing the gag, pol and env genes.
Such encapsidation lines have been described in the prior art, and
in particular the line PA317 (U.S. Pat. No. 4,861,719), the line
PsiCRIP (WO 90/02806) and the line GP+envAm-12 (WO 89/07150).
Moreover, the recombinant retroviruses can contain modifications in
the LTRs to eliminate transcriptional activity, as well as extended
encapsidation sequences containing a portion of the gag gene
(Bender et al., J. Virol. 61 (1987) 1639). The recombinant
retroviruses produced are then purified by standard techniques.
[0167] To implement the present invention, it is most especially
advantageous to use a defective recombinant adenovirus or
retrovirus. These vectors possess, in effect, especially
advantageous properties for the transfer of suicide genes into
tumour cells.
[0168] 5.3--Chemical Vectors
[0169] Among the synthetic vectors developed, it is preferable to
use, in the context of the invention, cationic polymers of the
polylysine, (LKLK)n, (LKKL)n, polyethylenimine and DEAE-dextran
type, or alternatively lipofectants or cationic lipids. They
possess the property of condensing DNA and of promoting its
combination with the cell membrane. Among the latter compounds,
there may be mentioned lipopolyamines (lipofectamine, transfectam,
and the like), various cationic or neutral lipids (DOTMA, DOGS,
DOPE, and the like) and also peptides of nuclear origin. In
addition, the concept of receptor-mediated, targeted transfection
has been developed, which turns to good account the principle of
condensing DNA by means of the cationic polymer while directing the
binding of the complex to the membrane as a result of a chemical
coupling between the cationic polymer and the ligand for a membrane
receptor present at the surface of the cell type which it is
desired to graft. Targeting of the transferrin or insulin receptor
or of the asialoglycoprotein receptor of hepatocytes has thus been
described. The preparation of the composition according to the
invention using such a chemical vector is carried out according to
any technique known to a person skilled in the art, generally by
simply bringing the different components into contact.
SEQUENCE LISTING
[0170] (1) GENERAL INFORMATION:
[0171] (i) APPLICANT:
[0172] (A) NAME: RHONE POULENC RORER S. A.
[0173] (B) STREET: 20, Avenue Raymond Aron
[0174] (C) CITY: ANTONY
[0175] (E) COUNTRY: FRANCE
[0176] (F) POSTAL CODE: 92165
[0177] (G) TELEPHONE: 40.91.69.22
[0178] (H) TELEFAX: (1) 40.91.72.96
[0179] (ii) TITLE OF INVENTION: NEW THYMIDINE KINASE VARIANTS,
CORRESPONDING NUCLEIC ACID SEQUENCES AND THEIR USE IN GENE
THERAPY
[0180] (iii) NUMBER OF SEQUENCES: 8
[0181] (iv) COMPUTER READABLE FORM:
[0182] (A) MEDIUM TYPE: Tape
[0183] (B) COMPUTER: IBM PC compatible
[0184] (C) OPERATING SYSTEM: PC-DOS/MS-DOS
[0185] (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
[0186] (2) INFORMATION FOR SEQ ID No. 1:
[0187] (i) SEQUENCE CHARACTERISTICS:
[0188] (A) LENGTH: 8 amino acids
[0189] (B) TYPE: amino acid
[0190] (D) TOPOLOGY: linear
[0191] (ii) MOLECULE TYPE: peptide
[0192] (ix) FEATURE:
[0193] (D) OTHER INFORMATION: The first four Xaa's represent any
amino acid and the third Xaa a threonine or a serine.
[0194] (xi) SEQUENCE DESCRIPTION SEQ ID No. 1: Gly Xaa Xaa Xaa Xaa
Gly Lys Xaa
[0195] (2) INFORMATION FOR SEQ ID NO. 2:
[0196] (i) SEQUENCE CHARACTERISTICS:
[0197] (A) LENGTH: 8 amino acids
[0198] (B) TYPE: amino acids
[0199] (D) TOPOLOGY: linear
[0200] (ii) MOLECULE TYPE: peptide
[0201] (xi) SEQUENCE DESCRIPTION: SEQ ID No. 2: Gly Pro His Gly Met
Gly Lys Thr
[0202] (2) INFORMATION FOR SEQ ID No. 3:
[0203] (i) SEQUENCE CHARACTERISTICS:
[0204] (A) LENGTH: 1131 base pairs
[0205] (B) TYPE: nucleotide
[0206] (C) STRANDEDNESS: single
[0207] (D) TOPOLOGY: linear
[0208] (ii) MOLECULE TYPE: cDNA
[0209] (xi) SEQUENCE DESCRIPTION: SEQ ID No. 3:
Sequence CWU 0
0
* * * * *