U.S. patent application number 11/802679 was filed with the patent office on 2009-10-15 for recombinant viruses and their use for treatment of atherosclerosis and other forms of coronary artery disease and method, reagent, and kit for evaluating susceptibility to same.
This patent application is currently assigned to AVENTIS PHARMA S.A.. Invention is credited to Patrick Benoit, Patrice Denefle, Michael R. Hayden, M.E. Suzanne Lewis, Michel Perricaudet.
Application Number | 20090257981 11/802679 |
Document ID | / |
Family ID | 33312293 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090257981 |
Kind Code |
A1 |
Benoit; Patrick ; et
al. |
October 15, 2009 |
Recombinant viruses and their use for treatment of atherosclerosis
and other forms of coronary artery disease and method, reagent, and
kit for evaluating susceptibility to same
Abstract
Recombinant viruses comprising a heterologous DNA sequence
coding for a lipase involved in lipoprotein metabolism. The
invention also concerns the preparation and use in therapy of said
recombinant viruses, especially for the treatment or prevention of
dyslipoproteinemia-related pathologies.
Inventors: |
Benoit; Patrick; (Paris,
FR) ; Denefle; Patrice; (Saint Maur, FR) ;
Perricaudet; Michel; (Ecrosnes, FR) ; Lewis; M.E.
Suzanne; (West Vancouver, CA) ; Hayden; Michael
R.; (Vancouver, CA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
AVENTIS PHARMA S.A.
The University of British Columbia
|
Family ID: |
33312293 |
Appl. No.: |
11/802679 |
Filed: |
May 24, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10831268 |
Apr 26, 2004 |
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11802679 |
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09713268 |
Nov 16, 2000 |
6814962 |
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10831268 |
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08737954 |
Dec 16, 1996 |
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PCT/FR95/00669 |
May 22, 1995 |
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09713268 |
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08817192 |
Jul 10, 1997 |
6784162 |
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PCT/US95/13620 |
Oct 11, 1995 |
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08737954 |
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08320604 |
Oct 11, 1994 |
5658729 |
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08817192 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/325 |
Current CPC
Class: |
C12N 2740/13043
20130101; A61K 2039/51 20130101; C12N 9/20 20130101; C12Q 1/6858
20130101; C12Y 301/01034 20130101; C12N 2840/44 20130101; C12N
2840/20 20130101; C12N 15/86 20130101; A61K 48/00 20130101; C12N
2800/108 20130101; C12Q 2600/156 20130101; A61K 38/00 20130101;
A61P 9/00 20180101; C12Q 1/6883 20130101; C12N 2710/10343 20130101;
C12N 2830/42 20130101; C12Q 1/6858 20130101; C12Q 2535/131
20130101; C12Q 2525/131 20130101 |
Class at
Publication: |
424/93.2 ;
435/235.1; 435/325 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 7/01 20060101 C12N007/01; C12N 5/10 20060101
C12N005/10; A61P 9/00 20060101 A61P009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 1994 |
FR |
FR94/06759 |
Claims
1-15. (canceled)
16. A method for treating a patient with dyslipoproteinaemia
comprising administering to the patient via intravenous injection
an adeno-associated virus comprising a nucleic acid sequence coding
for a biologically active human lipoprotein lipase (LPL), wherein
the nucleic acid sequence is operably linked to a promoter, and
wherein the nucleic acid sequence is expressed so as to cause a
reduction of lipoprotein in the patient.
17. A method for treating a patient with hypertriglyceridaemia
comprising administering to the patient via intravenous injection
an adeno-associated virus comprising a nucleic acid sequence coding
for a biologically active human lipoprotein lipase (LPL) wherein
the nucleic acid sequence is operably linked to a promoter, and
wherein the nucleic acid sequence is expressed so as to cause a
reduction of triglyceride in the patient.
18. A method for treating a patient with hypercholesterolaemia
comprising administering to the patient via intravenous injection
an adeno-associated virus comprising a nucleic acid sequence coding
for a biologically active human lipoprotein lipase (LPL), wherein
the nucleic acid sequence is operably linked to a promoter, and
wherein the nucleic acid sequence is expressed so as to cause a
reduction of cholesterol in the patient.
19. A method for treating a patient with hyperlipidaemia comprising
administering to the patient via intravenous injection an
adeno-associated virus comprising a nucleic acid sequence coding
for a biologically active human lipoprotein lipase (LPL), wherein
the nucleic acid sequence is operably linked to a promoter, and
wherein the nucleic acid sequence is expressed so as to cause a
reduction of lipid in the patient.
20. A method for treating a patient with familial
hypertriglyceridaemia comprising administering to the patient via
intravenous injection an adeno-associated virus comprising a
nucleic acid sequence coding for a biologically active human
lipoprotein lipase (LPL) wherein the nucleic acid sequence is
operably linked to a promoter, and wherein the nucleic acid
sequence is expressed so as to cause a reduction of triglyceride in
the patient.
21. A method for treating a patient with combined familial
hyperlipidaemia and postprandial hyperlipidaemia comprising
administering to the patient via intravenous injection an
adeno-associated virus comprising a nucleic acid sequence coding
for a biologically active human lipoprotein lipase (LPL), wherein
the nucleic acid sequence is operably linked to a promoter, and
wherein the nucleic acid sequence is expressed so as to cause a
reduction of lipid in the patient.
22. The method of treatment according to claim 16, wherein the
adeno-associated virus is administered by direct injection into the
patient's portal vein, such that viral infection is targeted to the
liver.
23. The method according to one of claims 16-21, wherein the
promoter is a viral promoter.
24. The method according to claim 23, wherein the viral promoter is
selected from the group consisting of an E1A promoter, a MLP
promoter, a CMV promoter, and a RSV LTR promoter.
25. The method according to claim 24, wherein the viral promoter is
the RSV LTR promoter.
26. The method according to claim 22, wherein the promoter is a
viral promoter.
27. The method according to claim 22, wherein the viral promoter is
selected from the group consisting of an E1A promoter, a MLP
promoter, a CMV promoter, and a RSV LTR promoter.
28. The method according to claim 22, wherein the viral promoter is
the RSV LTR promoter.
29. A method for preventing or delaying the onset of coronary
artery disease in a human individual having lipoprotein lipase
enzyme in which a serine residue is present at amino acid 291 in
the enzyme, comprising administering to the individual an
adeno-associated virus comprising a nucleic acid sequence coding
for a replacement lipoprotein lipase enzyme, said replacement
lipoprotein lipase enzyme having an asparagine residue as amino
acid 291, wherein the replacement lipoprotein lipase enzyme is
produced in the individual to provide a functional lipoprotein
lipase enzyme.
30. The method according to claim 29, wherein the adeno-associated
virus further comprises a promoter effective to promote expression
of the nucleic acid sequence in human cells.
31. The method according to claim 29, wherein the adeno-associated
virus is administered by parenteral injection.
32. An adeno-associated virus comprising a nucleic acid sequence
coding for a biologically active lipoprotein lipase (LPL).
33. The adeno-associated virus according to claim 32, wherein the
nucleic acid sequence is placed under the control of a promoter
permitting its expression in an infected cell.
34. The adeno-associated virus according to claim 32, wherein the
nucleic acid sequence is a cDNA sequence.
35. The adeno-associated virus according to claim 32, wherein the
nucleic acid sequence codes for human LPL.
36. The adeno-associated virus according to claim 32, wherein the
promoter is selected from the group consisting of E1A, MLP, CMV,
and RSV LTR promoters.
37. An adeno-associated virus comprising a cDNA sequence coding for
a biologically active lipoprotein lipase (LPL) under the control of
an RSV LTR promoter.
38. The adeno-associated virus according to claim 32, wherein the
virus further comprises a nucleic acid sequence enabling the
lipoprotein lipase to be directed into a pathway of secretion in an
infected cell.
39. The adeno-associated virus according to claim 38, wherein the
secretion sequence is the native secretion sequence of lipoprotein
lipase.
40. The adeno-associated virus according to claim 32, wherein the
adeno-associated virus lacks the rep gene.
41. The adeno-associated virus according to claim 32, wherein the
adeno-associated virus lacks the cap gene.
42. The adeno-associated virus according to claim 32, wherein the
adeno-associated virus lacks the rep and cap genes.
43. The adeno-associated virus according to claim 32, further
comprising a gene coding for an apolipoprotein.
44. The adeno-associated virus according to claim 43, wherein the
apolipoprotein is selected from the group consisting of ApoA-I and
ApoA-IV.
45. An adeno-associated virus comprising a nucleic acid sequence
coding for hepatic lipase and further comprising a nucleic acid
sequence coding for apolipoprotein selected from the group
consisting of ApoA-I and ApoA-IV, wherein the nucleic acid
sequences are placed under the control of a promoter permitting
their expression in an infected cell.
46. A composition comprising the adeno-associated virus according
to claim 32 and a pharmaceutically acceptable vehicle.
47. The composition according to claim 46, wherein the composition
is in an injectable form.
48. A mammalian cell infected in vitro with the adeno-associated
virus according to claim 32, whereby a biologically active
lipoprotein lipase is expressed from the adeno-associated
virus.
49. The mammalian cell according to claim 48, wherein the cell is a
human cell.
50. The mammalian cell according to claim 49, wherein the cell is
selected from the group consisting of a fibroblast, myoblast,
hepatocyte, endothelial cell, glial cell, and keratinoctyte.
51. A composition comprising the infected mammalian cell according
to claim 48 and an extracellular matrix.
52. The composition according to claim 51, wherein the
extracellular matrix comprises a gelling compound selected from the
group consisting of collagen, gelatin, glycosaminoglycans,
fibronectin, and lectins.
53. The composition according to claim 52, wherein the
extracellular matrix comprises a support permitting anchorage of
the infected cell.
54. The composition according to claim 53, wherein the support
comprises polytetrafluoroethylene fibers.
55. The adeno-associated virus of claim 32, wherein the
adeno-associated virus is defective.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 08/737,954, filed Dec. 16, 1996, abandoned, which is a 371
of international application PCT/FR95/00669, filed May 22, 1995,
and a continuation-in-part of application Ser. No. 08/817,192,
filed Apr. 11, 1997, which is a 371 of international application
PCT/US95/13620, filed Apr. 18, 1996, which is a continuation of
application Ser. No. 08/320,604, filed Oct. 11, 1994, issued as
U.S. Pat. No. 5,658,729, each of which is incorporated by reference
herein.
[0002] The present invention relates to recombinant vectors of
viral origin, to their preparation and to their use, in particular
for the treatment and/or prevention of pathologies associated with
dyslipoproteinaemias. More particularly, it relates to recombinant
viruses containing a DNA sequence coding for a lipase involved in
lipoprotein metabolism. The invention also relates to the
preparation of these viral vectors, to pharmaceutical compositions
containing them and to their therapeutic use, in particular, in
gene therapy by which lipoprotein deficiencies can be treated. In
addition, the invention relates to a method, reagent and kit for
evaluating susceptibility to and causation of premature
atherosclerosis and other forms of coronary artery disease.
BACKGROUND OF THE INVENTION
[0003] Dyslipoproteinaemias are disorders of the metabolism of the
lipoproteins responsible for transport of lipids such as
cholesterol and triglycerides in the blood and the peripheral
fluids. They lead to major pathologies associated, respectively,
with hypercholesterolaemia or hypertriglyceridaemia, such as, for
example, coronary artery disease.
[0004] "Coronary artery disease" is a collective term for a variety
of symptomatic conditions including angina, myocardial infarction,
and non-specific chest, arm and face pain, which result from
atherosclerosis of the arteries that supply blood to the heart.
[0005] "Premature atherosclerosis" as used herein refers to the
clinical presentation of signs and symptoms of coronary artery
disease before the age of 65.
[0006] Atherosclerosis, commonly known as "hardening of the
arteries," is a complex disease of polygenic origin, which is
defined from a histological standpoint by deposits (lipid or
fibrolipid plaques) of lipids and of other blood derivatives within
the wall or endothelium of the large arteries (aorta, coronary
arteries, carotid). These plaques, which are more or less calcified
according to the degree of progression of the process, may be
coupled with lesions, and are associated with the accumulation of
fatty deposits in the arteries, consisting essentially of
cholesterol esters.
[0007] The plaques are accompanied by a thickening of the arterial
wall, with hypertrophy of the smooth muscle, appearance of foam
cells and accumulation of fibrous tissue. The atheromatous plaque
protrudes markedly from the wall, endowing it with a stenosing
character responsible for vascular occlusions by atheroma,
thrombosis or embolism which occur in those patients who are most
affected. Thus, the dyslipoproteinaemias can lead to very serious
cardiovascular pathologies such as infarction, sudden death,
cardiac decompensation, stroke, and the like.
[0008] Because of the significant relationship between coronary
artery disease and heart attacks, considerable effort has been
devoted to identifying the biochemical causes of atherosclerosis.
This research has shown that high levels of total cholesterol, low
density lipoprotein (LDL), very low density lipoprotein (VLDL) and
triglycerides are associated with increased risk of coronary artery
disease, while high levels of high density lipoproteins (HDL) are
associated with decreased risk of coronary artery disease. See,
Gordon et al., The Amer. J. Med. 62: 707-714 (1977). However, while
observation of lipoproteins, cholesterol and triglycerides can
provide a basis for identifying individuals at risk of coronary
artery disease, the levels of these substances are themselves
symptoms of an underlying biochemical defect which remains
unidentified. Thus, specific treatment of the ultimate cause rather
than an intermediate condition, and prediction of risk prior to the
onset of this intermediate condition is not possible through such
observation.
[0009] Studies directed towards the underlying cause of coronary
artery disease have identified a number of mutations in genes
coding for proteins involved in lipid transport and metabolism that
appear to be associated with an increased risk. Examples include a
large number of mutations in the low-density lipoprotein receptor
gene, Hobbs et al., Human Mutations 1: 445-466 (1992), and a single
mutation in the apolipoprotein-B (Apo-B) gene which underlies
familial defective Apo-B in many parts of the world. Soria et al.,
Proc. Nat'l Acad. Sci. USA 86: 587-91 (1989). In addition,
mutations in other genes which play a significant role in HDL
metabolism such as the cholesterol ester transferase protein (CETP)
gene, Brown et al., Nature 342: 448-451 (1989) and the gene for
Apo-A1, Rubin et al., Nature 353: 265-266 (1991), have also been
shown to be associated with either enhanced resistance or increased
susceptibility to atherosclerosis. However, these mutations are
uncommon and thus far no specific mutation in any gene has been
found in a significant number (i.e., >1%) of patients with
coronary artery disease or premature atherosclerosis. Accordingly,
these test results while interesting do not offer the opportunity
to provide evaluation or therapy to significant numbers of
patients
[0010] At the present time these pathologies, and especially the
hypercholesterolaemias, are treated essentially by means of
compounds which act either on cholesterol biosynthesis
(hydroxymethylglutarylcoenzyme A reductase inhibitors, statins), or
on the uptake and removal of biliary cholesterol (sequestering
agents or resins), or alternatively on lipolysis by a mode of
action which remains to be elucidated at molecular level
(fibrates). Consequently, all the major classes of medicinal
products which have been used in this indication (sequestering
agents, fibrates or statins) are directed only towards the
preventive aspect of atheromatous plaque formation and not, in
fact, towards the treatment of atheroma. Current treatments of
atheroma following coronary accident are merely palliative, since
they do not intervene in cholesterol homeostasis and are surgical
procedures (coronary bypass, angioplasty).
SUMMARY OF THE INVENTION
[0011] It has now been found that a single point mutation in the
human lipoprotein lipase gene which results in an A-G nucleotide
change at codon 291 (nucleotide 1127) of the lipoprotein lipase
gene, and a substitution of serine for the normal asparagine in the
lipoprotein lipase gene product is seen with increased frequency in
patients with coronary artery disease, and is associated with an
increased susceptibility to coronary artery disease, including in
particular premature atherosclerosis. This is expressed as a
diminished catalytic activity of lipoprotein lipase, lower
HDL-cholesterol levels and higher triglyceride levels. Thus, in
accordance with one embodiment of the present invention there is
provided a method for evaluating susceptibility of a human
individual to premature atherosclerosis and other forms of coronary
artery disease comprising the steps of: [0012] (a) obtaining a
sample of DNA from the individual; and [0013] (b) evaluating the
sample of DNA for the presence of nucleotides encoding a serine
residue as amino acid 291 of the lipoprotein lipase gene product.
The presence of a serine residue is indicative of increased
susceptibility in the patient.
[0014] The invention further provides a kit for performing the
method of the invention. Such a kit comprises a pair of primers
selected to amplify a region of a human lipoprotein lipase gene
spanning amino acid 291 of human lipoprotein lipase. Appropriate
additional reagents may also be included in the kit such as
polymerase enzymes, nucleoside stock solutions and the like.
[0015] A further aspect of the present invention is a method of
treating patients suffering from or likely to suffer from premature
atherosclerosis and other forms of coronary artery disease as a
result of a lipoprotein lipase deficiency using gene therapy. This,
for example, may be accomplished using adenovirus-mediated or
retrovirus-mediated gene therapy, and can be performed using either
an in vivo or an ex vivo approach.
[0016] Thus, the present invention also constitutes a novel
therapeutic approach to the treatment of pathologies associated
with dyslipoproteinaemias, which may be caused by, for example,
lipoprotein lipase deficiency. It proposes an advantageous solution
to the drawbacks of the prior art, by demonstrating the possibility
of treating pathologies associated with dyslipoproteinaemias by
gene therapy, by the transfer and expression in vivo of a gene
coding for a lipase involved in lipoprotein metabolism. The
invention thus affords a simple means permitting specific and
effective treatment of these pathologies.
[0017] Gene therapy consists in correcting a deficiency or an
abnormality (mutation, aberrant expression, and the like) or in
providing for the expression of a protein of therapeutic interest
by introducing genetic information into the affected cell or organ.
This genetic information may be introduced either ex vivo into a
cell extracted from the organ, the modified cell then being
reintroduced into the body, or directly in vivo into the
appropriate tissue. In this second case, different techniques
exist, including various techniques of transfection 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. 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.
[0018] The present invention constitutes a novel therapeutic
approach to the treatment of pathologies associated with
dyslipoproteinaemias, consisting in transferring and expressing in
vivo genes coding for lipases involved in lipoprotein metabolism.
It is especially advantageous that applicants have now shown that
it is possible to construct recombinant viruses containing a DNA
sequence coding for a lipase involved in lipoprotein metabolism,
and to administer these recombinant viruses in vivo, and that this
administration permits a stable and effective expression of a
biologically active lipase in vivo, and without a cytopathological
effect.
[0019] The present invention is also the outcome of the
demonstration that adenoviruses constitute especially effective
vectors for the transfer and expression of such genes. In
particular, the present invention shows that the use of recombinant
adenoviruses as vectors enables levels of expression of these genes
to be obtained which are sufficiently high to produce the desired
therapeutic effect.
[0020] The present invention thus affords a novel approach to the
treatment and prevention of cardiovascular and neurological
pathologies associated with dyslipoproteinaemias.
[0021] A subject of the invention lies in a defective recombinant
virus comprising a nucleic acid sequence coding for a lipase
involved in lipoprotein metabolism.
[0022] The subject of the invention is also the use of such a
defective recombinant virus for the preparation of a pharmaceutical
composition intended for the treatment and/or prevention of
cardiovascular diseases.
[0023] The present invention also relates to the use of cells
modified genetically in vivo or ex vivo with a virus as described
above, or of cells producing such viruses, implanted in the body,
permitting a sustained and effective in vivo release of a
biologically active lipase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1: Shows the structure of the vector pXL2418
[0025] FIG. 2: Shows the structure of the vector pXL2419
[0026] FIG. 3: Shows the structure of the vector pXL CMV-LPL
[0027] FIG. 4: Shows the structure of the vector pXL RSV-LPL
[0028] FIG. 5: Shows the structure of the vector pXL RSV-LPLc
[0029] FIG. 6 illustrates the use of strand displacement
amplification in a method in accordance with the present
invention;
[0030] FIG. 7 shows the sandwich formed when two oligonucleotide
probes are used to analyze for the presence of an Asn291Ser
mutation;
[0031] FIG. 8 illustrates the use of mismatch primers in accordance
with the invention to detect the Asn291Ser mutation; and
[0032] FIG. 9 shows a plasmid construct, PRc/CMV-hLPL useful in
accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Among lipases involved in lipoprotein metabolism for the
purposes of the invention, preferential mention may be made of
lipoprotein lipase (LPL).
[0034] Lipoprotein lipase (LPL) is an enzyme which permits the
hydrolysis of triglycerides contained in very low density
lipoproteins (VLDL) or chylomicrons. Apolipoprotein CII, which is
present at the surface of these particles, is used as cofactor in
this hydrolysis. Naturally, LPL is mainly synthesized by adipocytes
in the form of a 51-kDa monomeric precursor, which is then
glycosylated (58 kDa). In the blood, LPL is active in dimeric form.
Up to 80% of freshly synthesized LPL is degraded in the lysosomal
compartment before being able to be secreted. After its secretion,
LPL is taken up by the luminal face of the cells of the vascular
endothelium, to which it binds via glycosaminoglycans. It has a
very strong affinity for heparin, which enables LPL to be displaced
from its binding site at the surface of the endothelial cell.
Intravenous injection of heparin enables LPL concentration and
activity to be measured in patients. LPL is utilized in the
vascular cells and also in liver cells as an uptake agent for
lipoproteins, increasing their retention at the cell surface and
thereby promoting their uptake or their modification.
[0035] cDNA coding for human LPL has been cloned and sequenced
(Wion et al., Science 235 (1987) 1638-1641). Two forms of
messengers coexist, of 3350 and 3750 bases, mainly in adipose and
muscle tissue, and originate from the use of 2 polyadenylation
sites. They include a long untranslated 3' sequence and code for a
preprotein of 475 aa, from which a leader sequence of 27 aa is
cleaved to give rise to the mature monomeric protein of 448
residues. The LPL gene has also been cloned (Kirchgessner et al.,
Proc. Natl. Acad. Sci. USA, 1987. 262:9647-9651). The synthesis,
processing and secretion of LPL are regulated in a complex manner
during development and in response to hormonal stimuli. A sizeable
part of this regulation is accomplished at transcriptional level
(review in Auwerx et al., Critical Reviews in Clinical Laboratory
Sciences, 1992, 29:243-268).
[0036] The present invention shows that it is possible to
incorporate a DNA sequence coding for LPL in a viral vector, and
that these vectors enable a biologically active (dimeric) mature
form of LPL to be expressed and secreted effectively. More
especially, the invention shows that in vivo expression of active
LPL may be obtained by direct administration of an adenovirus or by
implantation of a cell which is productive or genetically modified
by an adenovirus or by a retrovirus incorporating such a DNA.
[0037] The vectors of the invention may be used, in particular, to
correct LPL deficiencies due to mutations in the LPL gene. Such
deficiencies are relatively common and can reach an incidence of
1:5,000-1:10,000 in some populations (S. Santamarina-Fojo, 1992,
Cur. Op. lipid., 3:186-195). These deficiencies can result from a
sizeable mutation in the gene, leading to the absence of LPL
synthesis or to the synthesis of a truncated or highly modified
protein. The existence has, in effect, been shown in some patients
of mutations of the insertion, deletion or nonsense mutation type
(review in S. Santamarina-Fojo, 1992, Cur. Op. lipid., 3:186-195).
They can also result from a defect at the catabolic site, which may
be due to mutations of the missense type in the gene. They can also
result from modification both at the heparin-binding site and at
the catalytic site. At the heterozygous stage, these deficiencies
can represent a considerable proportion of the commonest
hyperlipidaemias, including familial hypertriglycerinaemias,
combined familial hyperlipidaemias and postprandial
hyperlipidaemias.
[0038] The present invention is especially advantageous, since it
enables an expression of LPL which is controlled and without a
harmful effect to be induced in organs which are not normally
affected by the expression of this protein. In particular, an
altogether advantageous release is obtained by implantation of
cells which produce vectors of the invention, or are infected in
vivo or ex vivo with vectors of the invention.
[0039] The lipase activity produced in the context of the present
invention can be a human or animal lipase. The nucleic acid
sequence used in the context of the present invention can be a
cDNA, a genomic DNA (gDNA), an ARN (in the case of retroviruses) or
a hybrid construction consisting, for example, of a cDNA into which
one or more introns might be inserted. Other possible sequences are
synthetic or semi-synthetic sequences. It is especially
advantageous to use a cDNA or a gDNA. In particular, the use of a
gDNA permits better expression in human cells. To permit their
incorporation in a viral vector according to the invention, these
sequences are advantageously modified, for example by site-directed
mutagenesis, especially for the insertion of suitable restriction
sites. The sequences described in the prior art are not, in effect,
constructed for a use according to the invention, and prior
adaptations may prove necessary in order to obtain substantial
expressions. In the context of the present invention, it is
preferable to use a nucleic acid sequence coding for a human
lipase. Moreover, it is also possible to use a construction coding
for a derivative of these lipases, especially a derivative of human
LPL and HL. HL (hepatic lipase) is localized at the surface of
hepatic endothelial cells. It differs from LPL in its insensitivity
to the activating action of apoC-II. HL is involved in the
hydrolysis of IDL lipids, and also of HDL2 lipids, bringing about
their conversion to HDL3.
[0040] A derivative of these lipases comprises, for example, any
sequence obtained by mutation. deletion and/or addition relative to
the native sequence, and coding for a product retaining lipase
activity. These modifications may be carried out by techniques
known to a person skilled in the art (see general techniques of
molecular biology below). The biological activity of the
derivatives thereby obtained may then be readily determined, as
described, in particular, in Example 3. The derivatives for the
purposes of the invention may also be obtained by hybridization
from nucleic acid libraries, using the native sequence or a
fragment of the latter as probe.
[0041] These derivatives are, in particular, molecules having a
greater affinity for their binding sites, molecules displaying
greater resistance to proteases, molecules having greater
therapeutic efficacy or reduced side effects, or possibly novel
biological properties. The derivatives also include modified DNA
sequences permitting improved expression in vivo.
[0042] Among preferred derivatives, there may be mentioned, more
especially, natural variants, molecules in which some N- or
O-glycosylation sites have been modified or eliminated, molecules
in which one or more residues have been substituted or molecules in
which all the cysteine residues have been substituted (muteins).
There may also be mentioned derivatives obtained by deletion of
regions having little or no involvement in the interaction with the
binding sites of interest or expressing an undesirable activity,
and derivatives containing additional residues relative to the
native sequence, such as, for example, a secretion signal and/or a
junction peptide.
[0043] In a first embodiment, the present invention relates to a
defective recombinant virus comprising a cDNA sequence coding for a
lipase involved in lipoprotein metabolism. In another preferred
embodiment of the invention, the DNA sequence is a gDNA
sequence.
[0044] The vectors of the invention may be prepared from different
types of virus. Preferably, vectors derived from adenoviruses, from
adeno-associated viruses (AAV), from herpesviruses (HSV) or from
retroviruses are used. It is most especially advantageous to use an
adenovirus, for a direct administration or for the ex vivo
modification of cells intended to be implanted or a retrovirus, for
the implantation of productive cells.
[0045] The viruses according to the invention are defective, that
is to say they are incapable of replicating autonomously in the
target cell. Generally, the genome of the defective viruses used in
the context of the present invention hence lacks at least the
sequences needed for replication of the said virus in the infected
cell. These regions may be either removed (wholly or partially), or
rendered non-functional, or substituted by other sequences, and in
particular by the nucleic acid sequence coding for the lipase.
Preferably, the defective virus nevertheless retains the sequences
of its genome which are needed for encapsidation of the viral
particles.
[0046] 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
are useable in the context of the present invention, adenoviruses
of canine, bovine, murine (e.g.: Mavl, 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 CAV2 adenovirus
[Manhattan or A26/61 (ATCC VR-800) strain, for example]. It is
preferable to use adenoviruses of human or canine or mixed origin
in the context of the invention.
[0047] Preferably, the defective adenoviruses of the invention
comprise the ITRs, a sequence permitting encapsidation and the
sequence coding for the lipase. Advantageously, in the genome of
the adenoviruses of the invention, the E1 region at least is
rendered non-functional. Still more preferably, in the genome of
the adenoviruses of the invention, the E1 gene and at least one of
the genes E2, E4, L1-L5 are non-functional. The viral gene of
interest 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 of interest. 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, and
the sequence coding for LPL is inserted in the inactivated E1
region. According to another preferred embodiment, it comprises a
deletion in the E1 region, into which are inserted the E4 region
and the sequence coding for LPL (see FR 94/13355).
[0048] 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 DNA sequence coding for the
lipase. 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.
[0049] Thereafter, the adenoviruses which have multiplied are
recovered and purified according to standard techniques of
molecular biology, as illustrated in the examples.
[0050] Thereafter, the adenoviruses which have multiplied are
recovered and purified according to standard techniques of
molecular biology, as illustrated in the examples.
[0051] 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 growth, morphology or cell 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 4,7,00 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; the right-hand portion of the genome, which contains
the cap gene coding for the capsid proteins of the virus.
[0052] 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
(into cells in culture) or in vivo (directly into a body). However,
none of these documents describes or suggests the use of a
recombinant AAV for the transfer and expression of a lipase ex vivo
or in vivo, or the advantages of such a transfer. The defective
recombinant AAVs according to the invention may be prepared by
cotransfection, into a cell line infected with a human helper virus
(for encapsidation genes (rep and cap genes) of AAV. The
recombinant AAVs produced are then purified by standard
techniques.
[0053] Regarding herpes viruses 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.
[0054] In particular, retroviruses are integrative viruses which
infect dividing cells. 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.
[0055] To construct recombinant retroviruses containing a sequence
coding for LPL according to the invention, a plasmid containing, in
particular, the LTRs, the encapsidation sequence and the coding
sequence is generally constructed, and then used to transfect a
so-called encapsidation cell line capable of providing in trans the
retroviral functions that are deficient in the plasmid. Generally,
the encapsidation lines are 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.
[0056] To implement the present invention, it is most especially
advantageous to use a defective recombinant adenovirus. The results
given below demonstrate the especially advantageous properties of
adenoviruses for expressing in vivo a protein having lipase
activity. The adenoviral vectors according to the invention are
especially advantageous for a direct administration of a purified
suspension in vivo, or for the in vivo transformation of cells, in
particular autologous cells, for the purpose of their implantation.
Furthermore, the adenoviral vectors according to the invention
possess, in addition, considerable advantages such as, in
particular, their very high efficiency of infection, enabling
infection to be carried out using small volumes of viral
suspension.
[0057] In an especially preferred embodiment, an adenovirus
containing, in addition to the gene coding for the lipase, a gene
coding for an apolipoprotein is used according to the invention.
The lipase is preferably hepatic lipase and the apolipoprotein is
preferably selected from apoA-I and apoAIV. The two genes are
advantageously used in the form of a bicistronic construction which
is introduced into an adenoviral vector according to the protocol
described above for the construction of an adenovirus containing a
single gene. Advantageously, the invention relates to a recombinant
adenovirus containing a gene coding for HL and a gene coding for
ApoA-I, inserted into the E1 region. The adenovirus construction
containing a gene coding for an apo has been described in
Application PCT/FR94/00422, which is incorporated herein by
reference.
[0058] According to another especially advantageous embodiment of
the invention, a line is used which produces retroviral vectors
containing the sequence coding for the lipase, for implantation in
vivo. The lines which are usable for this purpose are, in
particular, PA317 (U.S. Pat. No. 4,861,719), PsiCrip (WO 90/02806)
and GP+envAm-12 (U.S. Pat. No. 5,278,056) cells, modified to permit
the production of a retrovirus containing a nucleic acid sequence
coding for a lipase according to the invention.
[0059] Advantageously, in the vectors of the invention. the
sequence coding for the lipase is placed under the control of
signals permitting its expression in infected cells. These signals
can be ones for homologous or heterologous expression, that is to
say signals different from the ones naturally responsible for the
expression of the lipase. They can, in particular, be sequences
responsible for the expression of other proteins, or synthetic
sequences. In particular, they can be sequences of eukaryotic or
viral genes or derived sequences, stimulating or repressing the
transcription of a gene, specifically or non-specifically and
inducibly or non-inducibly. As an example, they can be promoter
sequences originating from the genome of the cell which it is
desired to infect, or from the genome of a virus, and in particular
the promoters of the adenovirus E1A and MLP genes, the CMV, RSV LTR
promoter, and the like. Among eukaryotic promoters, there may also
be mentioned the ubiquitous promoters (HPRT, vimentin,
.alpha.-actin, tubulin, and the like), the promoters of
intermediate filaments (desmin, neurofilaments, keratin, GFAP, and
the like), the promoters of therapeutic genes (MDR, CFTR, factor
VIII type, and the like), tissue-specific promoters (pyruvate
kinase, villin, promoter of the fatty acid-binding intestinal
protein, .alpha.-actin promoter of smooth muscle cells, promoters
specific to the liver; Apo AI, Apo AII, human albumin, and the
like) or alternatively promoters responding to a stimulus (steroid
hormone receptor, retinoic acid receptor, and the like). In
addition, these expression sequences may be modified by adding
activation, regulatory, and the like, sequences. Moreover, when the
inserted gene does not contain expression sequences, it may be
inserted into the genome of the defective virus downstream of such
a sequence.
[0060] In a particular embodiment, the invention relates to a
defective recombinant virus comprising a nucleic acid sequence
coding for a lipase involved in lipoprotein metabolism, under the
control of a promoter chosen from RSV LTR or the CMV early
promoter.
[0061] More preferably, the nucleic acid sequence used also
comprises signals permitting secretion of the lipase by infected
cells. To this end, the nucleic acid sequence generally contains,
upstream of the coding sequence, a signal sequence directing the
lipase synthesized into the pathways of secretion of the infected
cell. This signal sequence can be the natural signal sequence of
the lipase synthesized, but it can also be any other signal
sequence which is functional in the infected cell, or an artificial
signal sequence.
[0062] As stated above, the present invention also relates to any
use of a virus as described above for the preparation of a
pharmaceutical composition intended for the treatment and/or
prevention of pathologies associated with dyslipoproteinaemias.
[0063] The present invention also relates to a pharmaceutical
composition comprising one or more defective recombinant viruses as
are described above. These pharmaceutical compositions may be
formulated with a view to topical, oral, parenteral, intranasal,
intravenous, intramuscular, subcutaneous, intraocular, transdermal,
and the like, administration. Preferably, the pharmaceutical
compositions of the invention contain a pharmaceutically acceptable
vehicle for an injectable formulation, in particular for an
intravenous injection, such as, for example, into the patient's
portal vein. The formulations can be, in particular, isotonic
steryl solutions, or dry, in particular lyophilized, compositions
which, on adding sterilized water or physiological saline, as the
case may be, enable injectable solutions to be produced. Direct
injection into the patient's portal vein is advantageous, since it
enables the infection to be targeted to the liver, and thus the
therapeutic effect to be concentrated in this organ.
[0064] The doses of defective recombinant virus used for the
injection may be adapted in accordance with different parameters,
and in particular in accordance with the viral vector, the mode of
administration used, the pathology in question or alternatively the
desired period of treatment. Generally speaking, the recombinant
adenoviruses according to the invention are formulated and
administered in the form of doses of between 10.sup.4 and 10.sup.14
pfu/ml, and preferably 10.sup.6 to 10.sup.10 pfu/ml. The term pfu
(plaque forming unit) corresponds to the infectious power of a
solution of virus, and is determined by infecting a suitable cell
culture and measuring, generally after 48 hours, the number of
plaques of infected cells. The techniques of determination of the
pfu titre of a viral solution are well documented in the
literature.
[0065] As regards retroviruses, the compositions according to the
invention can contain the productive cells directly, with a view to
their implantation.
[0066] In this connection, another subject of the invention relates
to any mammalian cells infected with one or more defective
recombinant viruses as are described above. More especially, the
invention relates to any human cell population infected with these
viruses. The cells in question can be, in particular, fibroblasts,
myoblasts, hepatocytes, keratinocytes, endothelial cells, glial
cells, and the like.
[0067] The cells according to the invention can originate from
primary cultures. They may be removed by any technique known to a
person skilled in the art and then set up in culture under
conditions permitting their proliferation. As regards fibroblasts,
more especially, the latter may be readily obtained from biopsies,
for example according to the technique described by Ham [Methods
Cell. Biol. 21a (1980) 255]. These cells may be used directly for
infection with the viruses, or stored, for example by freezing, to
establish autologous banks with a view to subsequent use. The cells
according to the invention can also be secondary cultures obtained,
for example, from pre-established banks (see, for example, EP
228458, EP 289034, EP 400047, EP 456640).
[0068] The cells in culture are then infected with the recombinant
viruses to endow them with the capacity to produce. a biologically
active lipase. Infection is carried out in vitro according to
techniques known to a person skilled in the art. In particular,
depending on the cell type used and the desired number of copies of
virus per cell, a person skilled in the art can adapt the
multiplicity of infection and, where appropriate, the number of
infection cycles carried out. It should be obvious that these steps
must be performed under suitable conditions of sterility when the
cells are intended for administration in vivo. The doses of
recombinant virus used for infecting the cells may be adapted by a
person skilled in the art in accordance with the desired objective.
The conditions described above for in vivo administration may be
applied to infection in vitro. For infection with retroviruses, it
is also possible to coculture the cells which it is desired to
infect with cells producing the recombinant retroviruses according
to the invention. This makes it possible to eliminate the need to
purify the retroviruses.
[0069] Another subject of the invention relates to an implant
comprising mammalian cells infected with one or more defective
recombinant viruses as are described above, or cells which produce
recombinant viruses, and an extracellular matrix. Preferably, the
implants according to the invention comprise 10.sup.5 to 10.sup.10
cells. More preferably, they comprise 10.sup.6 to 10.sup.8
cells.
[0070] More especially, in the implants of the invention, the
extracellular matrix comprises a gelling compound and, where
appropriate, a support permitting anchorage of the cells.
[0071] For the preparation of the implants according to the
invention, different types of gelling agents may be employed. The
gelling agents are used for inclusion of the cells in a matrix
having the constitution of a gel, and to promote anchorage of the
cells to the support, where appropriate. Different cellular
adhesion agents may hence be used as gelling agents, such as, in
particular, collagen, gelatin, glycosaminoglycans, fibronectin,
lectins, and the like. Preferably, collagen is used in the context
of the present invention. The collagen may be of human, bovine or
murine origin. More preferably, type I collagen is used.
[0072] As stated above, the compositions according to the invention
advantageously comprise a support permitting anchorage of the
cells. The term anchorage denotes any form of biological and/or
chemical and/or physical interaction bringing about adhesion and/or
binding of the cells to the support. Moreover, the cells can either
coat the support used or enter the interior of this support, or
both. It is preferable, in the context of the invention, to use a
non-toxic and/or biocompatible solid support. In particular,
polytetrafluoroethylene (PTFE) fibres or a support of biological
origin may be used.
[0073] The implants according to the invention may be implanted in
different sites of the body. In particular, the implantation may be
performed in the peritoneal cavity, in the subcutaneous tissue
(subpubic region, iliac or inguinal fossae, and the like), in an
organ, a muscle, a tumour or the central nervous system, or
alternatively under a mucosa. The implants according to the
invention are especially advantageous in that they enable the
release of the lipase in the body to be controlled: this is, in the
first place, determined by the multiplicity of infection and by the
number of cells implanted. The release can then be controlled
either by withdrawing the implant, which stops the treatment
permanently, or by the use of regulable expression systems enabling
the expression of the therapeutic genes to be induced or
repressed.
[0074] The present invention thus affords a very effective means
for the treatment or prevention of pathologies associated with
dyslipoproteinaemias, especially obesity, hypertriglyceridaemia or,
in the field of cardiovascular complaints, myocardial infarction,
angina, sudden death, cardiac decompensation and stroke.
[0075] In addition, this treatment can be applied equally well to
man and to any animal such as sheep, cattle, domestic animals
(dogs, cats, and the like), horses, fish, and the like.
[0076] The present invention also involves detecting a mutation in
the gene coding for the enzyme lipoprotein lipase in a sample of
DNA obtained from a patient.
[0077] The first step in the method in accordance with the
invention is obtaining an appropriate sample of DNA. A suitable
source of such a sample is from patient blood. Isolation of the DNA
from the blood can be performed by many different methods. For
example, the DNA may be isolated from the leukocytes using a
salt-chloroform extraction as described in Trends in Genetics 5:
391 (1989).
[0078] Once the sample of patient DNA is obtained, it may be
desirable to amplify a portion of the DNA including the region of
interest. One technique which can be used for amplification is
Polymerase Chain Reaction (PCR) amplification. This technique,
which is described in U.S. Pat. Nos. 4,683,202 and 4,683,195, which
are incorporated herein by reference, makes use of two
amplification primers each of which hybridizes to a different one
of the two strands of the DNA duplex at regions which do not
overlap the site of the mutation being tested for, in this case the
mutation in amino acid 291. Multiple cycles of primer extension,
and denaturation are used to produce additional copies of DNA to
which the primers can hybridize. This amplification can be
performed in a solution, or on a solid support (see, e.g. U.S. Pat.
No. 5,200,314 which is incorporated herein by reference).
[0079] The mutation site of interest is at a defined location
within exon 6 of the lipoprotein lipase gene, the sequence of which
is known in the art. Oka et al., Biochim. Biophys. Acta 1049: 21-26
(1990); Deeb et al., Biochemistry, 28: 4131-4135 (1989); Wion et
al., Science 235: 1638-1641,(1987). Amplification primers may be
used which bind to the intron regions on either side of exon 6, or
which bind to portions of exon 6 itself. Where amplification of the
mutation site is desired, the primers should not overlap the site
of the mutation of interest. Suitable primers include those
described for exon 6 in Monsalve et al., J. Clin. Invest. 86:
728-734 (1990).
[0080] Another amplification technique which may be used in
accordance with the present invention is known as Strand
Displacement Amplification (SDA). In this technique, which is
described in U.S. Pat. No. 5,270,184, incorporated herein by
reference, and EP 0 497 272, and which is exemplified in FIG. 6, a
gene fragment is used as the target, and a primer is used which
binds to the 3'-end of this fragment. The primer is selected to
include a restriction site near its 5'-end. This can be achieved by
using a primer which extends beyond the 3'-end of the target gene
fragment if there is no restriction site conveniently located
towards the 3'-end of the fragment from the site of interest. The
primer and the target fragment (if the primer extends beyond the
end of the fragment) are extended to form a duplex using modified
nucleoside feedstocks, e.g., .alpha.-thio nucleoside triphosphates,
at least in the region of the restriction cleavage site so that the
newly formed strand is not susceptible to cleavage by the
endonuclease. For subsequent amplification normal feedstocks are
used. A restriction endonuclease is introduced which nicks the
duplex at the restriction site. Extension then starts over at the
site of the nick, at the same time that the previously hybridized
oligonucleotide is displaced. In this way, multiple copies of one
or both strands of a gene or gene fragment can be amplified without
the use of temperature cycling. To use strand displacement
amplification to amplify the mutation site responsible for the
Asn291Ser mutation, primers flanking exon 6, such as those
described in Monsalve et al. could be used.
[0081] Once amplified, the DNA may be evaluated by any of a number
of methods to determine if the Asn291Ser mutation is present.
First, the amplified DNA can be sequenced (optionally after cloning
into a TA cloning vector. available from Invitrogen, Inc.) using
manual or automated sequencing of the amplified product. Since the
complete sequence of exon 6 of normal lipoprotein lipase is known,
targeted sequencing primers can be readily developed for this
purpose.
[0082] Another approach to the detection of Asn291Ser mutations,
generally used following amplification, is the use of sequence
specific oligonucleotide probes which bind to one of the mutant or
wildtype form, but not to the other. Such probes generally have a
length of 15 to 20 bases. Because the difference being evaluated is
a single base, the analysis is conducted under very stringent
hybridization conditions such that only perfect matches will form
stable hybrids.
[0083] The probe used in the invention is advantageously labeled to
permit its easy detection. Suitable labels include radioactive
labels, fluorescent labels, and reactive labels such as biotin. The
probe may also be labeled twice, for example with a radiolabel and
a reactive label, in which case the reactive label may be used to
the capture the DNA hybrid, for example through the reaction of
biotin with an avidin-coated support.
[0084] A preferred format for testing using sequence specific
probes involves the use of a sandwich assay in which the amplified
DNA is evaluated using two probes. The first oligonucleotide probe
is either selected to bind specifically to a gene encoding a mutant
human lipoprotein lipase having a serine residue as amino acid 291,
wherein said probe binds to a portion of the gene including the
bases coding for the serine residue or selected to bind
specifically to a gene encoding a normal human lipoprotein lipase
having an asparagine residue as amino acid 291, wherein said probe
binds to a portion of the gene including the bases coding for the
asparagine residue. The second oligonucleotide probe is selected to
bind to a different, non-overlapping portion of the human-LPL gene
which is the same in both mutant and non-mutant forms. One of the
two probes is labeled with a detectable label while the other is
labeled with a reactive label to facilitate immobilization. Only
when both probes are bound to a single piece of amplified DNA will
the detectable label be immobilized through the formation of a
sandwich of the structure shown in FIG. 2.
[0085] Various modifications of the amplification process may also
be used in accordance with the present invention to detect the
presence of an Asn291Ser mutation. If intentionally mismatched
primers are used during the amplification, the amplified nucleic
acids may also be evaluated for the presence of the Asn291Ser
mutation using a technique called restriction fragment length
polymorphism (RFLP). In order to make use of RFLP directly to
detect a point mutation (as opposed to an insertion or deletion
mutation), the mutation must result in the addition or loss of a
site cleaved by a restriction endonuclease. If this is the case,
the fragments produced upon restriction endonuclease digestion of
the normal and mutant gene differ in number, in size, or in both.
This difference can be detected by gel electrophoresis of the
restriction fragments.
[0086] In the case of the Asn291Ser mutation, the nucleotide
sequence of the coding strand changes from
TABLE-US-00001 5'------GAG ATC AAT AAA GTC ------3' SEQ. ID. NO: 6
to 5'------GAG ATC AGT AAA GTC ------3' SEQ. ID. NO: 7
These fragments lack the two-fold symmetry that is associated with
cleavage sites of restriction endonucleases, and thus one cannot
simply use an enzyme which will cleave one of the sequences, but
not the other. RFLP can be used, however, if a special mismatch
primer is used during the amplification process. This, primer,
shown below in Example 8, binds to the LPL gene at a site adjacent
to the mutation of interest, and introduces an intentional error
into the amplified DNA. Thus, as illustrated in FIG. 8, instead of
the expected sequence, the mismatch primer produces the duplex
region
TABLE-US-00002 5'---ATAC---3' coding strand 3'---TATG---5'
non-coding strand
when a wild-type gene is amplified, and the sequence
TABLE-US-00003 5'---GTAC---3' coding strand 3'---CATG---5'
non-coding strand
when a mutant gene is amplified, where the C/G pair in the fourth
position of the above fragments is the intentional mismatch.
Amplified mutant genes therefore contain a restriction site
(5'-GTAC-3') which is cleaved by the restriction endonuclease RsaI,
but amplified wild-type sequence (5'-ATAC-3') does not. Thus, a
polymorphism measurable through restriction fragment lengths is
artificially introduced into the amplified DNA using the mismatch
primers.
[0087] The amplification process may also be modified by using
labeled primers which facilitate detection and/or capture of the
amplified product. For example, as described in British Patent No.
2 202 328, using a biotin-labeled primer as one of the two primers
permits the recovery of the extended primers produced during the
amplification reaction, e.g., by binding the extended primers to a
support coated with (strept)avidin. If the primer used is in a
region flanking the mutation site, the presence of the mutation can
be detected by adding a labeled probe, which specifically binds to
the mutant or wild-type gene, to the biotinylated amplified DNA
either before or after capture of the amplified DNA on a support.
If the label becomes bound to the support, this indicates that the
probe was bound. Alternatively, the primer may be one which spans
the mutation site in which case amplification will occur using a
primer corresponding to the mutant sequence only when the mutation
is present (and vice versa). In this case, a labeled probe which
binds to a portion of the LPL gene away from the mutation site or
labeled nucleoside feedstocks may be used to introduce a label into
the amplified DNA.
[0088] The presence of the Asn291Ser mutation may also be detected
using a catalytic hybridization amplification system of the type
described in International Patent Publication No. WO89/09284, which
is incorporated herein by reference. Basically, in this technique,
the target nucleic acid acts as a cofactor for enzymatic cleavage
of probe oligonucleotides. Thus, a substantial excess of labeled
probe oligonucleotide (which binds specifically to either the
mutant or the wild-type gene) is combined with the target nucleic
acid under stringent hybridization conditions such that only
exactly complementary strands will hybridize to any measurable
extent. An enzyme is added which will cleave the probe when it is
part of a duplex, but not in single stranded form. The mixture is
then cycled through multiple cycles of annealing/enzyme digestion
and denaturation. If the probe binds to the target, the result is
the production of many small labeled probe-fragments, and the
concurrent reduction in the number of full-size labeled probes.
Either the increase in the number of fragments or the decrease in
the number of full-sized probes can be detected and provides an
amplified indication of the presence or absence of the target
sequence in the sample.
[0089] An example of an enzyme which can be used in the catalytic
hybridization amplification system is RNaseH which is used in
combination with RNA probes; which are selectively cleaved when
hybridized to a strand of target DNA. Restriction endonucleases
which do not cleave phosphorothioate-modified DNA may also be used,
provided that the target DNA is first copied to produce a
phosphorothioate-modified target. Because this method combines both
amplification and detection, prior amplification of the genomic DNA
from the sample is generally not necessary.
[0090] Another technique useful in the present invention which
combines amplification and detection relies on the autocatalytic
replication of certain RNA's as described in U.S. Pat. No.
4,957,858, which is incorporated herein by reference. Briefly, in
this technique a replicative RNA segment is ligated to a sequence
specific oligonucleotide probe which binds to either the mutant or
the wild-type form of the Asn291Ser mutation site in exon 6 of the
LPL gene. This ligated probe is then combined with the genomic DNA
in such a manner that the probe will bind if the matching sequence
is present in the genomic DNA, and so that unbound probe can be
separated from bound probe. For example, the genomic DNA may be
immobilized on a solid support to facilitate washing out of unbound
probe molecules. Thereafter, the RNA portion of the ligated probe
is amplified, for example using the enzyme Q-beta replicase.
[0091] Yet another form of combination amplification/detection
technique which is useful in the present invention is described in
U.S. Pat. No. 5,124,246 which is incorporated herein by reference.
In this technique, a total of five types of oligonucleotide probes
are used. The first type of probe is a multimer oligonucleotide
having a "star" type configuration with many generally identical
arms. The second type of probe is a labeling probe. The labeling
probe is complementary to the sequence of one of the arms of the
multimer probe and includes a detectable label. The third type of
probe is an immobilized probe. A plurality of this third type of
probe is affixed to a solid support. The specific sequences used in
these first three types of probes are independent of the nature of
DNA being analyzed, except that they should not hybridize with this
DNA directly.
[0092] The fourth type of probe is referred to as an amplifier
probe. These probes are synthesized in two parts, one which is
complementary to a portion of the normal sequence of exon 6 of the
LPL gene away from the Asn291Ser mutation site, and one which is
complementary to an arm of the multimer probe. A plurality of
different types of amplifier probes is formed. These various types
of probes are complementary to different, non-overlapping portions
of the sequence. The fifth type of probe is a capture probe. The
capture probe is also formed in two parts: one which is
complementary to the site of the Asn291Ser mutation and one which
is complementary to the immobilized probe.
[0093] The assay is performed by combining denatured genomic DNA
with the plurality of amplifier probes and capture probes under
conditions permitting hybridization. The result is the binding of
numerous amplifier probes to exon 6 of the LPL gene. The capture
probe will only bind, however, if the corresponding mutant (or
non-mutant, depending on the sequence of the probe) is present.
Thereafter, the solid support having the third probe immobilized
thereon is introduced. A solid support-immobilized probe-capture
probe-genomic DNA-amplifier probe sandwich will form if DNA
complementary to the capture probe is present. The support is then
washed to remove unbound material, and the multimer probe is added.
The multimer probe binds to the support via the amplification probe
only if the sandwich was formed in the first place. The support is
then washed and a labeling probe is added. The labeling probe will
bind to all of the available arms of the multimer probe on the
solid support, thus providing numerous detectable labels for each
actual mutation site in the DNA sample.
[0094] In the foregoing discussion of amplification and detection
techniques, there is frequent mention of labeled probes or labeled
primers. For purposes of this application, the label applied to the
primer may take any form, including but not limited to radiolabels;
fluorescent or fluorogenic labels; colored or chromogenic labels;
chemically reactive labels such as biotin; enzyme-labels, for
example phosphatase, galactosidase or glucosidase enzymes which can
produce colored or fluorescent reaction product in combination with
substrates such as p-nitrophenyl phosphate (colored reaction
product) or 4-methyl umbelliferyl phosphate (fluorescent cleavage
product); and chemiluminescent labels.
[0095] A further aspect of the present invention is the particular
oligonucleotide probes which may be used in one or several of the
techniques as discussed above for detection of the Asn291Ser
mutation. Thus, for use in the case of mismatch primer
amplification followed by RFLP analysis there is provided an
oligonucleotide primer which binds specifically to a gene encoding
for human lipoprotein lipase in a region adjacent to, but not
overlapping the second base in the codon corresponding to residue
291 in human lipoprotein lipase, and which includes a mismatched
base which does not correspond to the normal sequence of human
lipoprotein lipase, whereby upon extension of the primer, using a
target human lipoprotein lipase gene as a template, an extension
product is produced which contains a restriction site which can be
cleaved by a restriction endonuclease when the lipoprotein lipase
product made by the target gene has a serine residue as amino acid
291, and does not contain such a restriction site when the
lipoprotein lipase product made by the target gene has an
asparagine residue as amino acid 291. A preferred primer which
binds to the coding strand is one in which a base complementary to
base number 1130 is changed from the normal thymine to guanine. For
the non-coding strand, the change is from adenine to cytosine. A
particularly preferred mismatch primer for binding to the coding
strand has the sequence
TABLE-US-00004 CTGCTTCTTT TGGCTCTGAC TGTA. SEQ. ID NO: 8
[0096] For several of the detection methods discussed above, an
oligonucleotide probe is utilized which binds to a site which
includes the site of the specific mutation of interest. Thus, the
present invention encompasses two types of oligonucleotide probes:
(1) an oligonucleotide probe selected to bind specifically to a
gene encoding a mutant human lipoprotein lipase having a serine
residue as amino acid 291, wherein said probe binds to a portion of
the gene including the bases coding for the serine residue; and (2)
an oligonucleotide probe selected to bind specifically to a gene
encoding a normal human lipoprotein lipase having a asparagine
residue as amino acid 291, wherein said probe binds to a portion of
the gene including the bases coding for the asparagine residue.
These probes are preferably from 15 to 20 bases in length, and may
be selected to bind to either the coding or the non-coding strand
of the genomic DNA. Further, the probes will advantageously include
a detectable label.
[0097] A further aspect of the present invention is a kit which may
be used to detect the presence of the Asn291Ser mutation. The
specific components of the kit will depend on the nature of the
evaluation being conducted. In general, however, the kit will
include a pair of primers selected to amplify a region of a human
lipoprotein lipase gene encoding for amino acid 291 of human
lipoprotein lipase. These primers may be primers for PCR, primers
adapted for strand displacement amplification, or a normal primer
and a mismatch primer. In addition, the kit may include
oligonucleotide probes for use in the detection of the Asn291Ser
mutation.
[0098] The discovery of the significance of the Asn291Ser mutation
opens the door to the possibility of providing gene therapy to
individuals having this mutation and thus to prevent or delay the
onset of coronary artery disease and particularly premature
atherosclerosis. In addition, since gene therapy to correct this
defect would provide a patient with a fully functional lipoprotein
lipase enzyme, therapeutic agents and methods used for this purpose
may also be used effectively for other conditions associated with
LPL mutations. Such conditions include infantile failure to thrive,
hepatosplenomegaly, eruptive xanthomas, chronic and/or episodic
abdominal pain, pancreatitis and lactescent plasma due to an
accumulation of chylomicrons and very low density lipoproteins or
their remnants in the plasma.
[0099] Gene therapy to introduce functional LPL may reduce the
clinical manifestations stemming from hypertriglyceridemia in both
LPL deficient homozygotes and heterozygotes. This gene transfer can
be accomplished, as previously described, using
adenovirus-DNA-polylysine conjugates; adenovirus constructs in
which the normal LPL gene is inserted into the viral genome; or
retroviral constructs in which the normal LPL gene is inserted into
the viral genome. The vector may be introduced directly, for
example by parenteral injection into the patient, or may be
introduced via an implanted pseudo-organ.
[0100] FIG. 9 shows a plasmid construct useful in accordance with
the present invention. As shown, the plasmid pRc/CMV-hLPL is 7.90
Kbases in size. The preparation of this particular plasmid is
described below in Example 9. It will be appreciated by persons
skilled in the art, however, that variations in this technique, or
the precise structure of the plasmid may be made without departing
from the present invention provided that the plasmid contains a
functional h-LPL gene and an appropriate promoter. For example,
tissue-specific promoters, particularly adipose tissue specific or
muscle specific promoters might be used in place of the CMV
promoter. Furthermore, while the SV40 promoter and the antibiotic
resistance markers are convenient for research purposes, they are
not necessary for therapeutic purposes.
[0101] To prepare a plasmid for transfection into mammalian, and
particularly human cells, the plasmid is complexed with an
adenovirus-polylysine conjugate. In general this process involves
the harvesting and purification of a suitable adenovirus, for
example a virus which is incompetent as a result of an E1A or an E3
deletion mutation. The purified virus is then conjugated with a
polycationic material for associating with DNA such as polylysine,
polyarginine or protamine, for example using a bifunctional reagent
such as ethyl-3,3-dimethyl aminopropyl carbodiimide (EDC) as a
crosslinking agent. When the resulting adenovirus-polylysine
conjugate is combined with a plasmid containing a normal LPL gene,
an adenovirus-DNA-polylysine complex forms spontaneously. This
complex transfects mammalian cells of various types when placed in
media with the cells with relatively high efficiency, and the
transfected cells produce functional LPL.
[0102] Mammalian cells may also be transduced (or transfected)
using an adenovirus into which a gene encoding for normal LPL has
been inserted. Preferred adenoviruses are those with an E1 or an E3
deletion mutation rendering the virus incompetent. The h-LPL gene
can be conveniently inserted into the virus at the site of the
deletion.
[0103] Specific modified adenoviruses useful in the present
technique are based on the RSV .beta.-Gal adenovector described by
Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-630 (1990).
This adenovector is based on adenovirus Ad5. Human LPL cDNA is
introduced into the vector by homologous recombination using a
modified form of Strafford-Perricaudet's pLTR.beta.GalpIX plasmid.
The plasmid contains an RSV LTR promoter or a CMV plus intron
promoter, human LPL cDNA, a poly A site plus small intron from SV40
derived from a pSV2 vector. Mulligan et al., Science 209: 1422-1427
(1980) which are inserted between nucleotides 455 to 3329 of an Ad5
DNA which is also deleted in the E3 region. This results in the
deletion of E1A and part of E1B, but, leaves pIX intact. The
resulting adenoviruses are non-replicating but can be propagated in
293 cells which transcomplement the E1A activity.
[0104] A third type of vector which may be used to transduce (or
transfect) mammalian cells is a retroviral vector. Suitable vectors
include myeloproliferative sarcoma virus (MPSV)-based retroviral
vectors into which human LPL cDNA is inserted under the
transcriptional control of the constitutive enhancer-promoter
regulatory elements of the MPSV long terminal repeat (LTR).
[0105] Gene transfer vectors can be introduced into a human subject
either in vivo or ex vivo. In the case of an in vivo treatment, the
gene transfer vector may be simply injected into the patient, for
example parenterally, and allowed to find suitable target cells. In
the case of ex vivo treatment, cells are grown in vitro and
transduced or transfected with the virus, embedded in a carrier
such as a collagen matrix, which is then implanted in the patient,
for example as a sub-cutaneous implant. Preferred cells for use in
ex vivo applications are fibroblast cells taken from the patient
who will receive the implant.
[0106] General Techniques of Molecular Biology
[0107] 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, transformation
in Escherichia coli, and the like, are well known to a person
skilled in the art and are amply described in the literature
[Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel
F. M. et al. (eds), "Current Protocols in Molecular Biology", John
Wiley & Sons, New York, 1987].
[0108] Plasmids of the pBR322 and pUC type and phages of the M13
series are of commercial origin (Bethesda Research
Laboratories).
[0109] To carry out ligation, the DNA fragments may be separated
according to their size by agarose or acrylamide gel
electrophoresis, extracted with phenol or with a phenol/chloroform
mixture, precipitated with ethanol and then incubated in the
presence of phage T4 DNA ligase (Biolabs) according to the
supplier's recommendations.
[0110] The filling in of 5' protruding ends may be performed with
the Klenow fragment of E. coli DNA polymerase I (Biolabs) according
to the supplier's specifications. The destruction of 3' protruding
ends is performed in the presence of phage T4 DNA polymerase
(Biolabs) used according to the manufacturer's recommendations. The
destruction of 5' protruding ends is performed by a controlled
treatment with S1 nuclease.
[0111] Mutagenesis directed in vitro by synthetic
oligodeoxynucleotides may be performed according to the method
developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749-8764]
using the kit distributed by Amersham.
[0112] The enzymatic amplification of DNA fragments by the
so-called PCR [Polymerase-catalysed Chain Reaction, Saiki R. K. et
al., Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A.,
Meth. Enzym. 155 (1987) 335-350] technique may be performed using a
DNA thermal cycler (Perkin Elmer Cetus) according to the
manufacturer's specifications.
[0113] The 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.
[0114] The present invention will be described more completely by
means of the examples that follow, which one should consider as
illustrative and non-limiting.
Example 1
[0115] Construction of the Vector pXL2418 Carrying the Gene Coding
for LPL Under the Control of the Cytomegalovirus (CMV) Early
Promoter (FIG. 1)
[0116] This example describes the construction of a vector
comprising a cDNA sequence coding for LPL, under the control of a
promoter consisting of the cytomegalovirus (CMV) early promoter, as
well as a region of the Ad5 adenovirus genome permitting homologous
recombination. This vector was constructed as described below.
[0117] 1.1. Construction of the Vector pXL2375
[0118] The vector pXL2375 contains, in particular, a region of the
Ad5 adenovirus genome and a DNA sequence coding for apolipoprotein
AI under the control of the CMV promoter. More especially, the CMV
promoter used extends as far as the donor 5' splicing site linked
to the 107 bp nearest the 3' end of the synthetic intron described
by O'Gorman et al. (Science 251 (1991) 1351). The construction of
this vector has been described in detail in copending Application
FR 93/05125. It is understood that similar constructions may be
carried out by a person skilled in the art.
[0119] 1.2. Construction of the cDNA Sequence Coding for LPL
[0120] Plasmid pHLPL 26-1 described by Wion et al. (Science 235
(1987) 1638-1641) contains an incomplete sequence of LPL cDNA.
Thus, this plasmid contains bases 272 to 1623 of LPL cDNA flanked
by two EcoRI sites. cloned to the EcoRI site of a plasmid pGEM1
(Promega).
[0121] The EcoRI fragment of pHLPL 26-1 containing the partial LPL
cDNA was recloned into the EcoRI site of a plasmid pMTL22 (Chambers
et al., Gene, 1988, 68:138-149), in the orientation placing the 5'
bases of the cDNA on the same side as the BglII site of pMTL22. The
resulting plasmid was called pXL2402.
[0122] The RNA of human adipose tissues was then extracted
according to the technique of Chromczynski and Sacchin (Anal.
Biochem. 162 (1987) 156-159). From this RNA preparation, an
amplification was carried out by RT-PCR so as to isolate the
missing portion of the LPL cDNA. To this end, the following primers
were used:
TABLE-US-00005 Sq4541: (SEQ ID NO: 1) TTA GAT CTA TCG ATA GAT GGA
GAG CAA AGC CCC TG
This primer makes it possible to introduce a BglII site as well as
a ClaI site upstream of the ATG.
TABLE-US-00006 (SEQ ID NO: 2) Sq3810: TAC ATT CCT GTT ACC GTC CAG
CCA TGG ATC
[0123] The 260-bp PCR fragment obtained after 25 amplification
cycles was then cloned into plasmid pCR-II (Invitrogen) and
sequenced for verification. It was then introduced via the BglII
and NcoI sites into the vector pXL2402, which reconstitutes a
complete cDNA preceded by a ClaI site and followed by a SalI site.
The resulting plasmid was called pXL2417.
[0124] 1.3. Construction of the Vector pXL2418.
[0125] Lastly, the LPL cDNA was inserted into plasmid pXL2375
between the SalI and ClaI sites, following excision of the Apo A1
cDNA with these same two enzymes. The plasmid obtained was
designated pXL2418 (FIG. 1).
Example 2
[0126] Construction of the Vector pXL2419 Carrying the Gene Coding
for LPL Under the Control of the Promoter of the Rous Sarcoma Virus
LTR (RSV LTR) (FIG. 2)
[0127] This example describes the construction of a vector
comprising a cDNA sequence coding for LPL, under the control of a
promoter consisting of the Rous sarcoma virus LTR (RSV LTR), as
well as a region of the Ad5 adenovirus genome permitting homologous
recombination. This vector was constructed as described below.
[0128] 2.1. Construction of the Vector pXL2244.
[0129] The vector pXL2244 contains, in particular, a region of the
Ad5 adenovirus genome and a DNA sequence coding for apolipoprotein
AI under the control of the RSV LTR promoter.
[0130] 2.2. Construction of a cDNA Sequence Coding for LPL.
[0131] The cDNA sequence coding for LPL used in this example is
that described in Example 1.2.
[0132] 2.3. Construction of the Vector pXL2419.
[0133] The LPL cDNA was inserted into plasmid pXL2244 between the
SalI and ClaI sites, following excision of the Apo A1 cDNA with
these same two enzymes. The plasmid obtained was designated pXL2419
(FIG. 2).
Example 3
Construction of the Vectors pXL RSV-LPL and pXL CMV-LPL
[0134] The vector pRC-CMV LPL contains a fragment of LPL cDNA
extending from bases 1 to 2388 of the sequence published by Wion et
al., cloned at the HindIII and XbaI sites of the expression vector
pRC-CMV (Invitrogen). The HindIII site was modified to a ClaI site
by inserting the oligonucleotide AGC TAC ATC GAT GT (SEQ ID NO: 3).
The LPL cDNA and the polyadenylation site of bovine growth hormone
(initially contained in pRC-CMV) are finally extracted from the
pRCMV-LPL by SphI cleavage, treatment with T4 polymerase and ClaI
cleavage. The fragment thereby obtained was cloned into the vectors
pXL2418 (Example 1) and pXL2419 (Example 2) cut with ClaI and
EcoRV, generating the vectors pXL CMV-LPL (FIG. 3) and pXL RSV-LPL
(FIG. 4), respectively.
Example 4
Construction of the Vector pXL RSV-LPLc
[0135] This example describes the construction of a vector which is
usable to generate recombinant viruses containing a short cDNA
coding for LPL.
[0136] A shorter cDNA (bases 146 to 1635 of the sequence of Wion et
al.) was cloned from the RNA of human adipose tissue. The primers
ATC GGA TCC ATC GAT GCA GCT CCT CCA GAG GGA CGC (SEQ ID NO: 4) and
ATC TCT AGA GTC GAC ATG CCG TTC TTT GTT CTG TAG (SEQ ID NO: 5),
which create, respectively, a BamHI site and a CalI site at the 5'
end of the cDNA, as well as an XbaI site and a SalI site at the 3'
end of the LPL cDNA, were used.
[0137] This PCR fragment was cloned into PCR II, and its sequence
verified in its entirety. The LPL cDNA was then released via the
BamHI and XbaI sites and cloned into an expression vector pcDNA3
(Invitrogen) for verification of the expression, generating plasmid
pcDNA3-LPLc.
[0138] The ClaI-SalI fragment containing the LPL cDNA was finally
cloned at the same sites into plasmid pXL RSV-LPL (Example 3) to
generate the shuttle plasmid pXL RSV-LPLc (FIG. 5).
Example 5
Functionality of the Vectors of the Invention: Demonstration of an
LPL Activity
[0139] The capacity of the vectors of the invention to express a
biologically active form of LPL in a cell culture was demonstrated
by transient transfection of 293 CosI cells. To this end, cells
(2.times.10.sup.6 cells per dish 10 cm in diameter) were
transfected (8 .mu.g of vector) in the presence of Transfectam. The
expression of the sequence coding for LPL and production of a
biologically active protein were demonstrated either in terms of
mass using an immunoenzymatic test (5.1.), or in terms of lipase
activity (5.2.).
[0140] 5.1. Measurement of LPL in Terms of Mass.
[0141] An Immulon I ELISA plate (Dynatech) was coated with
anti-bovine LPL monoclonal antibodies cross-reacting with human LPL
(20 .mu.g/ml in PBS, 50 .mu.l/well). The potential sites remaining
in the wells were then blocked (saturated) by incubation in the
presence of 1% gelatin for 1 hour at room temperature. The samples
to be measured were then incubated for 1 hour at 37.degree. C.
[0142] Visualization was carried out with an anti-LPL serum diluted
to 10 .mu.g/ml, 100 .mu.l/well, followed by a peroxidase-labelled
antiserum. Peroxidase activity was detected using a TMB substrate
(Kirkegaard and Perry Laboratories Inc. kit) and reading of the
absorbance at 490 nm.
[0143] 5.2. Measurement of LPL Activity.
[0144] Total lipase activity was measured on a substrate composed
of an emulsion of 0.3 mg of triolein (Sigma), 75 nCi of
tri(1-.sup.14C)oleoylglycerol (55 mCi/mmol, Amersham), 18 mg of BSA
(Fraction V, Sigma) and 25 .mu.l of normal human plasma as a source
of ApoCII, all these constituents in a final volume of 500 .mu.l of
0.223M Tris pH 8.5. Generally speaking, activity was measured on
100 .mu.l of supernatant of transfected cells or 50 .mu.l of
post-heparin plasma.
[0145] After 1 hour of incubation at 37.degree. C., the reaction
was stopped by adding 3.25 ml of extraction buffer
(chloroform/methanol/heptane, 10:9:7 v/v/v) and 0.75 ml of
carbonate/borate buffer pH 10.5, and the organic phase counted to
determine the amount of fatty acids liberated.
[0146] To determine the activity specifically associated with LPL,
the measurement of hepatic lipase activity was carried out in the
presence of a 1M concentration of NaCl (which inhibits LPL), and
then subtracted from the total activity. It was also possible to
inhibit lipoprotein lipase activity with a specific monoclonal
antibody (Babirak et al., Atheriosclerosis, 1989, 9:326-334).
[0147] Plasmids pXL RSV-LPL and pXL CMV-LPL were tested by
transfection into CosI cells by comparison with plasmid pRC
CMV-LPL. The results are presented in Table 1.
TABLE-US-00007 TABLE 1 Activity in the supernatant Expression
vector Day 1 pRC-CMV-LPL 24.5 pXL RSV-LPL 15.1 pXL CMV-LPL 22.9
[0148] Plasmid pcDNA-LPLc was tested by transfection into 293 cells
by comparison with an expression vector pcDNA3 containing the same
cDNA as the vector pRC-CMV-LPL. The results are presented in Table
2.
TABLE-US-00008 TABLE 2 Activity in the Activity in the Expression
supernatant supernatant vector Day 1 Day 2 pcDNA3-LPL 106.4 mU/ml
106.7 mU/ml pcDNA3-LPLc 114 mU/ml 109.6 mU/ml
Example 6
Construction of Recombinant Adenovirus Ad-CMV.LPL Containing a
Sequence Coding for LPL Lipase
[0149] The plasmids prepared in Examples 1 to 4 were linearized and
cotransfected for recombination with the deficient adenoviral
vector, in helper cells (line 293) providing in trans the functions
encoded by the E1 (E1A and E11B) regions of adenovirus.
[0150] More especially, the adenovirus Ad.CMV.LPL was obtained by
homologous recombination in vivo between the adenovirus
Ad.RSV.beta.gal (Stratford-Perricaudet et al., J. Clin. Invest 90
(1992) 626) and plasmid pXL2418 or pXL CMV-LPL according to the
following protocol: the linearized plasmid pXL2418 or pXL CMV-LPL
and the adenovirus labelled Ad.RSV.beta.gal linearized with ClaI
were cotransfected into line 293 in the presence of calcium
phosphate to permit homologous recombination. The recombinant
adenoviruses thus generated were selected by plaque purification.
After isolation, the recombinant adenovirus was amplified in the
cell line 293, yielding a culture supernatant containing the
unpurified recombinant defective adenovirus having a titre of
approximately 10.sup.10 pfu/ml.
[0151] The viral particles were then purified by centrifugation on
a caesium chloride gradient according to known techniques (see, in
particular, Graham et al., Virology 52 (1973) 456). The adenovirus
Ad-CMV.LPL were stored at -80.degree. C. in 20% glycerol.
[0152] The same protocol was reproduced with plasmid pXL2419 or pXL
RSV-LPL or pXL RSV-LPLc, yielding the recombinant adenovirus
Ad.RSV.LPL or Ad.RSV.LPLc.
Example 7
In Vivo Transfer of the LPL Gene by a Recombinant Adenovirus
[0153] This example describes the transfer of the LPL gene in vivo
by means of an adenoviral vector according to the invention.
[0154] The adenoviruses injected were the adenoviruses Ad-CMV.LPL
and Ad.LTR.LPL prepared in Example 5, used in purified form
(3.5.times.10.sup.6 pfU/.mu.l, in saline phosphate solution (PBS).
These viruses were injected into C57B1/6 mice intravenously using
the tail vein, the retro-orbital sinus or the portal vein. The
expression of an active form of LPL was demonstrated under the
conditions described in Example 5.
Example 8
[0155] The significance of the mutation resulting in a serine in
place of an asparagine as amino acid 291 in human lipoprotein
lipase ("Asn291Ser mutation") was discovered as a result of a case
controlled study of a large homogeneous sample of patients
undergoing diagnostic coronary angiography. A total of 807 men, all
of whom were of Dutch descent and had angiographically proven
atherosclerosis with more than 50% stenosis of at least one major
coronary vessel were included in the study. All of the patients
were less than 70 years of age, and had total cholesterol levels
between 4 and 8 mmol/l and triglyceride levels which did not exceed
4 mmol/l. The control group for the study included 157 persons who
did not have any history of angina or premature atherosclerosis,
and who exhibited no signs of vascular disease upon physical
examination. The controls were all less than 60 years of age and
had baseline HDL levels greater than 0.95 mmol/l and triglyceride
levels of less than 2.3 mmol/l.
[0156] DNA was extracted from leukocytes using a salt-chloroform
extraction method as described in Trends in Genetics 5: 391 (1989).
Exon 6 of the LPL gene was amplified with a 5'-PCR primer located
in intron 5 near the 5' boundary of exon 6 having the sequence
TABLE-US-00009 GCCGAGATAC AATCTTGGTG SEQ. ID. NO: 9
and a 3' mismatch primer which was located in exon 6 near the
Asn291Ser mutation. The mismatch primer had the sequence
TABLE-US-00010 CTGCTTCTTT TGGCTCTGAC TGTA SEQ. ID. NO: 8
[0157] PCR amplification reactions were performed using 0.5 .mu.g
of genomic DNA in BRL PCR buffer containing 1.5 mM MgCl.sub.2, 200
.mu.M dNTPs, 1 .mu.M each primer and 2.5 units Taq polymerase
(BRL). The reaction mixture was denatured at 95.degree. C. for 1
minute, annealed at 51.degree. C. for 1 minute and extended at
72.degree. C. for 45 seconds for a total of 35 cycles. Twenty .mu.l
of the PCR product was then digested with 10 units RsaI enzyme, 3.5
.mu.l of 10.times. reaction buffer 1 (BRL), and 9.5 .mu.l of water
at 37.degree. C. for 2 hours. The digested fragments were then
separated on 2% agarose gel.
[0158] Because the combination of the mismatch primer and the
Asn291Ser mutation produces an RsaI restriction site which is
absent when the mismatch primer is used to amplify the wild-type
gene, the restriction fragments observed on the agarose gel were
different when the mutation was present. Using this difference as a
diagnostic indicator, it was determined that the Asn291Ser mutation
was seen in 41 of the 807 or 5.09% of the patients in the test
group, but in only 3 out of 157 or 1.9% of the patients in the
control group. When a subgroup of the 494 patients in the test
group with hypoalphalipoproteinemia was considered, it was found
that a higher percentage of these patients, i.e., 6.9% (34 out of
494) had the Asn291Ser mutation. When a further subgroup of the
test group was considered by selecting those individuals with low
HDL-C levels (<1.0%), and excluding those individuals who had
blood glucose >6.8 mmol/l (suggestive of diabetes) and those on
.beta.-blocker therapy, 11.3% (12 out of 106 patients) had the
mutation. This proportion further increased when those with still
lower HDL-C levels were considered separately. Thus, among persons
with HDL-C levels less than 0.9 mmol/l, 8 out of 68 or 12.5% had
the Asn291Ser mutation, while among those with HDL-C levels less
than 0.8 mmol/l, 5 out of 32 or 15.6% had the Asn291Ser
mutation.
Example 9
[0159] pRc/CMV vector (Invitrogen) was linearized using XbaI and
Hind III. An XbaI/HindIII fragment containing h-LPL cDNA having a
length of about 2.4 kb was inserted into the vector. DH5-alpha was
transformed with the construct. Transformed cells were selected
from agar plates based upon ampicillin resistance, and grown in LB
medium. The plasmid construct, pRc/CMV-hLPL which is shown in FIG.
9, was isolated from the cultures by alkaline lysis and CsCl
centrifugation.
Example 10
[0160] A purified preparation of an incompetent adenovirus (E1A
deletion mutant) was prepared by growing 293 cells in 2 liter
spinner flasks to a cell density of 4.5.times.10.sup.6/ml and
infecting the cells with DL312 adenovirus stock at MOI
(multiplicity of infection) 20-50 for 1 hour. Forty hours post
infection, the cells were harvested by centrifugation. A lysate was
prepared by subjecting the harvested cells to 3 freeze/thaw cycles.
This lysate was centrifuged in a two-layer CsCl gradient (d=1.25,
d=1.4) in a Beckman SW41 swing rotor at 35,000 rpm and 18.degree.
C. for 90 minutes. After the ultracentrifugation, the virus was
recovered from the interface between the two CsCl layers using a
syringe and a long needle. The recovered virus was then placed onto
a CsCl solution (d=1.34) and centrifuged for 16 hours at 35,000 rpm
and 18.degree. C. After this centrifugation, the virus was again
recovered from the interface and was then dialyzed three times (1
hour per cycle) against a sterile buffer (Tris 10 mM, MgCl.sub.21
mM, NaCl 0.135 M). In the third dialysis cycle, the buffer included
10% glycerol to enhance storage stability. The purified virus was
kept frozen at -80.degree. C. until ready to use.
Example 11
[0161] Virus prepared as described in Example 10 was mixed with
polylysine (10 mM) and EDC (2 mM) for 4 hours at 4.degree. C. in
HBS/buffered saline to form adenovirus-polylysine conjugates. The
conjugates were re-isolated by CsCl gradient centrifugation using
the same protocol as the final centrifugation in Example 3.
[0162] The re-isolated conjugates (5.times.10.sup.9/ml) were
incubated with 60-70% confluent Chinese Hamster Ovary cells (CHO
K-1) in 2% FBS medium (1 ml) and 6 .mu.g of the plasmid
pRc/CMV-hLPL. As a control to assess the extent to which
transfection occurred, a second set of samples was prepared in the
same manner using the plasmid pRc/CMV-B-gal which includes a gene
encoding .beta.-galactosidase in place of h-LPL. After two hours,
the medium containing the conjugates was aspirated out, and new
medium (10% FBS) was added to the cells.
[0163] By incubating the control cells infected with pRc/CMV-B-gal
in the presence of X-gal, and counting the number of cells which
evidenced the characteristic blue color which result from cleavage
of X-gal by .beta.-galactosidase, it was determined that the
transfection efficiency in this system varied from 2% when the
virus solution was diluted 2000.times. to 50% when the virus
solution was diluted 125.times.. Thus, 50% transfection efficiency
could be achieved in vitro at titers of 0.5-1.times.10.sup.8, which
is at least 10-fold less than the titers which would normally be
used in vivo.
[0164] To determine the expression of LPL in cells transfected with
pRc/CMV-LPL, the activity of LPL was determined and compared to the
activity observed for control cells transfected with pRc/CMV-B-gal.
For the control cells, the activity measured was 12 mU/ml. For the
cells transfected with pRc/CMV-LPL, the activity measured was 20
mU/ml.
Example 12
[0165] The experiments described in Example 11 were repeated,
except that the cells used were LPL-deficient cat fibroblast cells
or HepG-2 liver cells. Table 3 shows the infection efficiencies at
various virus dilutions which were determined for these cell types
as well as the CHO K-1 cells.
TABLE-US-00011 TABLE 3 DILUTION VIRUS 2000.times. 1000.times.
500.times. 250.times. 125.times. CHO K-1 2 5 15 30 50 Cat
Fibroblast 10 20 50 100 100 HepG-2 20 50 100 100 100
[0166] Table 4 shows the LPL activity measured for Cat fibroblast
cells, and the LPL mass measured for cat fibroblast cells and
HepG-2 cells. In addition, Table 4 shows positive control results
for COS EV101 cells which are over producers of LPL. It can be seen
from this data that there is a substantial increase in the plasmid
activity and also in the amount of the active dimer form of the
enzyme.
TABLE-US-00012 TABLE 4 LPL Activity LPL MASS (ng/ml) Cell Type
plasmid (mU/ml) total monomer dimer CHO K-1 control 12 n.d. n.d.
n.d. pRc/CMV-LPL 20 n.d. n.d. n.d. Cat Fibroblasts control 0.15 26
24 2 pRc/CMV-LPL 1.5 128 88 34 HepG-2 control n.d. 33 28 6
pRc/CMV-LPL n.d. 164 113 51.5 COS EV101 50 530 87 443
Example 13
[0167] Vectors for introducing human LDL cDNA into mammalian cells
were made using the murine leukemia retroviral backbones M3neo,
M5neo and JZen1 which contain long terminal repeat (LTR) regulatory
sequences for the myeloproliferative sarcoma virus. To generate the
vectors M3neoLPL and M5neoLPL, a 1.56 kb DraI-EcoRI fragment
encompassing the entire LPL amino acid coding region was subcloned
into a unique BamHI site located 3' or 5' to the neomycin
phosphotransferase (neo.sup.f), respectively. Expression of both
genes is LTR driven in these vectors; in M3neoLPL, functional LPL
message would derive from the spliced proviral transcripts whereas
for M5neoLPL, LPL message would derive from the full length
unspliced proviral transcript. To construct JZenLPLtkneo, a 1092 bp
Xho I/SalI fragment for neo.sup.r was isolated from pMCIneo and
inserted into the SalI site of the plasmid pTZ19R, containing the
herpes simplex virus thymidine kinase (tk) promoter. The
SmaI/HindIII tkneo fragment from the pTZ19R was inserted into the
Hpa I/Hind III site of JZen1. A 1.56 kb human LPL cDNA sub-fragment
was then cloned in the BamHI site of JZentkneo. Human LPL cDNA was
also subcloned directly into JZen1 to construct JZenLPL.
[0168] Virus producer cells lines were then made for each of the
viral constructs using the amphotropic retroviral packaging cell
line GP-Am12 and the ecotropic packaging line GP-E86. Both cell
lines were cultured in HXM medium, which is Dulbecco's modified
Eagle's medium (DME) supplemented with 10% heat-inactivated
(55.degree. C. for 20 minutes) newborn calf serum (Gibco-BRL),
hypoxanthine (15 .mu.g/ml), xanthine (250 .mu.g/ml) and
mycophenolic acid (25 .mu.g/ml). For GP-AM12 cells, hygromycin B
(200 .mu.g/ml) was also added to the HXM medium. All cells were
cultured at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2.
Example 14
[0169] A variety of hematopoietic cell lines were tested using the
neomycin resistance marker incorporated in the vector to determine
whether transduction occurred as a result of coincubation with
M3neoLPL in vitro. K562 erythroid cells, HL60 myeloid cells, and
U937 and THP-1 monocytic cells obtained from the American Type
Culture Collection were grown in RPMI 1640 medium containing 10%
fetal bovine serum. The cells were then infected by cocultivation
(24-48 hours) with irradiated (15 Gy x-ray) near confluent producer
cells with polybrene 4 .mu.g/ml added to the co-cultivation medium
(RPMI/10% fetal bovine serum). After the infection period, the
hematopoietic target cells were maintained in suspension culture
for 24 hours before selection in 1 mg/ml G418. The gene transfer
efficiencies observed are summarized in Table 5.
[0170] The mass of LPL produced was determined for each of the
transduced hematopoietic cells lines using two ELISAs. The
antibodies used were MAb 5D2 which binds to the bioactive dimeric
form of LPL and MAb 5F9 which binds to both the bioactive dimer and
the inactive monomeric form of LPL. The results are summarized in
Table 5. Finally media supernatants were measured for LPL
bioactivity. The results of this study are also reported in Table
5.
TABLE-US-00013 TABLE 5 Gene Transfer Increase in Increase in Cell
Line Efficiency Bioactivity LPL Dimer K562 57% 11-fold 5-fold HL60
47% 9-fold 3-fold U937 45% 14-fold 54-fold THP-1 41% 4-fold
2-fold
These results demonstrate that for each cell type, good
transduction efficiencies were achieved, and production of
functional LPL resulted.
[0171] Transduced HL60 and THP-01 cells were differentiated in
macrophages by exposing the cells to 10 ng/ml of phorbal ester,
PdBU (Phorbol 12,13-dibutyrate) for 5 days. For HL60 cells, the LPL
bioactivity increased a further 1.8-fold, while the amount of LPL
dimer increased another 1.8-fold. No further increase was observed
upon differentiation of THP-1 cells.
Example 15
[0172] NIH 3T3 murine fibroblasts were grown in DME medium
containing 10% (vol/vol) fetal bovine serum. The medium on near
confluent 60 mm tissue culture plates of viral producer cells 24
hours prior to the planned infection with 10 ml DME/10% newborn
calf serum. This medium was removed at the time of infection,
concentrated 10-fold to a 1.0 ml final volume by filter
centrifugation in Centriprep-30 tubes (Amicon) and diluted 1:4 with
DME/10% fetal bovine serum with 4 .mu.g/ml polybrene added.
Fibroblasts were added to this preparation and incubated for 24-48
hours at 37.degree. C. 24 hours after viral exposure, cells were
subjected to selection in 1.0 mg/ml G418 and grown to confluence.
Testing for LPL production revealed a 16-fold increase in total LPL
production above constitutive levels which consisted almost
entirely of dimeric protein, and a 10-fold increase in secreted LPL
bioactivity.
Example 16
[0173] The experiment of Example 15 was repeated using primary
human fibroblast cells, FC 1898 and FC 1901 from diagnostic skin
biopsies. No measurable levels of endogenous LPL protein mass or
bioactivity could be detected prior to retroviral-mediated LPL gene
delivery. Post transduction levels of total LPL mass were massively
elevated at least 400 times above normal. However, at least 82% of
this exogenous LPL protein was of the inactive monomeric form. At
least a 52-fold (74.8.+-.22/9) increase in dimeric LPL production
was seen with significantly elevated secretion of bioactive LPL,
approximately 24 times higher (26.9.+-.3.0) than background LPL
levels.
Sequence CWU 1
1
9135DNAHomo sapiens 1ttagatctat cgatagatgg agagcaaagc ccctg
35230DNAHomo sapiens 2tacattcctg ttaccgtcca gccatggatc 30314DNAHomo
sapiens 3agctacatcg atgt 14436DNAHomo sapiens 4atcggatcca
tcgatgcagc tcctccagag ggacgc 36536DNAHomo sapiens 5atctctagag
tcgacatgcc gttctttgtt ctgtag 36615DNAHomo sapiens 6gagatcaata aagtc
15715DNAHomo sapiens 7gagatcagta aagtc 15824DNAHomo sapiens
8ctgcttcttt tggctctgac tgta 24924DNAHomo sapiens 9ctgcttcttt
tggctctgac tgta 24
* * * * *