U.S. patent application number 09/962459 was filed with the patent office on 2002-03-14 for process for the preparation of endotoxin-free or endotoxin-depleted nucleic acids and/or oligonucleotides for gene therapy.
This patent application is currently assigned to QIAGEN GMBH. Invention is credited to Colpan, Metin, Moritz, Peter, Schorr, Joachim.
Application Number | 20020032324 09/962459 |
Document ID | / |
Family ID | 27435911 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020032324 |
Kind Code |
A1 |
Colpan, Metin ; et
al. |
March 14, 2002 |
Process for the preparation of endotoxin-free or endotoxin-depleted
nucleic acids and/or oligonucleotides for gene therapy
Abstract
A process for the isolation and purification of nucleic acids
and/or oligonucleotides for use in gene therapy wherein said
nucleic acids and/or oligonucleotides are isolated or purified from
an essentially biological source, characterized in that said
essentially biological sources are lysed, the fractions obtained
are optionally freed or depleted from the remainder of said
biological sources by per se known mechanical methods, such as
centrifugation, filtration; the fractions thus treated are
subsequently treated with affinity chromatographic material or with
inorganic chromatographic material for the removal of endotoxins;
followed by isolation of said nucleic acids and/or oligonucleotides
on an anion exchanger which is designed such that DNA begins to
desorb from the anion exchanger only at an ionic strength
corresponding to a sodium chloride solution of a concentration
higher by at least 100 mM than one corresponding to the ionic
strength at which RNA begins to desorb from the anion exchanger
material.
Inventors: |
Colpan, Metin; (Essen,
DE) ; Schorr, Joachim; (Dusseldorf, DE) ;
Moritz, Peter; (Kerpen, DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
QIAGEN GMBH
|
Family ID: |
27435911 |
Appl. No.: |
09/962459 |
Filed: |
September 26, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09962459 |
Sep 26, 2001 |
|
|
|
09253702 |
Feb 22, 1999 |
|
|
|
6297371 |
|
|
|
|
09253702 |
Feb 22, 1999 |
|
|
|
08687529 |
Oct 18, 1996 |
|
|
|
5990301 |
|
|
|
|
08687529 |
Oct 18, 1996 |
|
|
|
PCT/EP95/00389 |
Feb 3, 1995 |
|
|
|
Current U.S.
Class: |
536/25.3 ;
435/270 |
Current CPC
Class: |
C12N 15/87 20130101;
B01D 15/3804 20130101; Y10S 435/81 20130101; C12N 15/101 20130101;
Y10S 435/82 20130101; B01D 15/3804 20130101; A61P 43/00 20180101;
B01D 15/363 20130101; A61P 35/00 20180101; Y10S 436/808 20130101;
G01N 30/461 20130101; A61P 21/04 20180101; B01D 15/363 20130101;
Y10T 436/10 20150115 |
Class at
Publication: |
536/25.3 ;
435/270 |
International
Class: |
C07H 021/04; C12N
001/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 1994 |
DE |
P 44 03 692.2 |
Jun 25, 1994 |
DE |
P 44 22 291.2 |
Sep 1, 1994 |
DE |
P 44 31 125.7 |
Sep 14, 1994 |
DE |
P 44 32 654.8 |
Claims
1. A process for the isolation and purification of nucleic acids
and/or oligonucleotides for use in gene therapy wherein said
nucleic acids and/or oligonucleotides are isolated or purified from
an essentially biological source, characterized in that said
essentially biological sources are lysed, the fractions obtained
are optionally freed or depleted from the remainder of said
biological sources by per se known mechanical methods, such as
centrifugation, filtration; the fractions thus treated are
subsequently treated with affinity chromatographic material or with
inorganic chromatographic material for the removal of endotoxins;
followed by isolation of said nucleic acids and/or oligonucleotides
on an anion exchanger which is designed such that DNA begins to
desorb from the anion exchanger only at an ionic strength
corresponding to a sodium chloride solution of a concentration
higher by at least 100 mM than one corresponding to the ionic
strength at which RNA begins to desorb from the anion exchanger
material.
2. The process according to claim 1, characterized in that porous
or non-porous inorganic and/or organic support materials modified
with anion exchanging groups are used as the support material of
the inorganic chromatographic material.
3. The process according to any of claims 1 and/or 2, characterized
in that silica gel, diatomaceous earth, glass, aluminium oxides,
titanium oxides, zirconium oxides, hydroxyapatite are used as
inorganic support materials, and dextrane, agarose, acrylic amide,
polystyrene resins, or copolymers of the materials mentioned are
used as organic support materials.
4. The process according to at least one of claims 1 to 3, wherein
said modified support material is obtained by reacting one of the
support materials mentioned in claim 3 in a first step with a
silanizing reagent of the general formula I,
R.sup.1R.sup.2R.sup.3SiR.sup.4 (I) wherein R.sup.1 is an alkoxy
residue of from 1 to 10 carbon atoms, especially --OCH.sub.3,
--OC.sub.2H.sub.5 or --OC.sub.3H.sub.7, or a halogen atom,
especially --Cl, or a dialkylamino group with identical or
different alkyl residues of from 1 to 6 carbon atoms; R.sup.2 and
R.sup.3 are independently a hydrocarbon residue of from 1 to 10
carbon atoms, especially --CH.sub.3, --C.sub.2H.sub.5 or
--C.sub.3H.sub.7, or an alkoxy residue of from 1 to 10 carbon
atoms, especially --OCH.sub.3, --OC.sub.2H.sub.5 or
--OC.sub.3H.sub.7, or a halogen atom, or an alkyl residue of from 4
to 20 carbon atoms which is interrupted by at least one oxa or
amino group wherein said residue may also be substituted with one
or more of halogen, cyano, nitro, amino, monoalkylamino,
dialkylamino, hydroxy or aryl; R.sup.4 is a hydrocarbon chain of
from 1 to 20 carbon atoms, or an alkyl residue which is interrupted
by at least one oxa or amino group wherein said residue may also be
substituted with one or more of halogen, cyano, nitro, amino,
monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl, and/or epoxy,
especially 4followed by a second step wherein the support which has
been modified in the first step is reacted with a reagent of the
general formula II: X-R-Y (II) wherein X is an amino, hydroxy,
epoxy group or a halogen atom; R is a hydrocarbon chain of from 2
to 20 carbon atoms, or an alkyl residue which is interrupted by at
least one oxa or amino group wherein said residue may also be
substituted with one or more of halogen, cyano, nitro, amino,
monoalkylamino, dialkylamino, alkoxy, hydroxy, aryl, and/or epoxy;
Y is a hydrocarbon residue having functional groups which form an
anion exchange material and having from 1 to 10 carbon atoms which
may be substituted with one or more of amino, monoalkylamino,
dialkylamino, quarternary alkylamino.
5. The process according to at least one of claims 1 to 4, wherein
diethylaminoethyl (DEAE) groups or dimethylaminoethyl (DMAE) groups
are arranged on the surface of the support either directly or
through so-called spacers.
6. The process according to at least one of claims 1 to 5, wherein
DNA, such as plasmids, cosmids, DNA isolated from viruses, also in
enzymatically or chemically modified form, and/or RNA in any form
and of any origin, or ribozymes are isolated as said nucleic
acids.
7. The process according to any of claims 1 to 6, wherein a
treatment of said fractions containing nucleic acids with non-ionic
detergents, such as Triton X 100 or a treatment with affinity
chromatographic supports, such as nickel/NTA, nickel/IDA,
polymyxin, DNA ETOX, is performed for the removal of endotoxins
from the nucleic acid fractions.
8. The process according to any of claims 1 to 7, wherein the
salts, which are necessary for eluting the nucleic acids under
conditions of high ionic strength, are removed by treating with a
mineral support material, such as one essentially consisting of
glass, the fractions containing nucleic acids and having the high
salt concentrations, wherein the nucleic acid adsorbs to the
surface of said mineral supports, and subsequent desorption of the
nucleic acids with water or buffer solutions of low ionic
strength.
9. The process according to any of claims 1 to 7, wherein cell
debris of the cell lysate are separated off by filtration, in
particular using a filter the pore size of which decreases in the
direction of flow of the sample to be filtrated and/or a filter
having a filter layer consisting of glass, silica gel, alumina or
packed diatomaceous earth or interlaced or bonded non-wovens made
of fiber glass and silica gel as well as cellulose, paper, pressed
paper, non-wovens made of paper.
10. The process according to at least one of claims 1 to 9, wherein
a preliminary purification of a sample of a cell lysate containing
nucleic acids is performed on a layer of unmodified diatomaceous
earth.
11. Use of the anion exchange materials mentioned in claims 1 to 6
for the separation, purification and isolation of nucleic acids for
the preparation of an agent containing nucleic acids for gene
therapy.
12. The use according to claim 11 for in vivo and ex vivo gene
therapy.
13. The use according to any of claims 11 or 12 for the preparation
of an agent for the treatment of genetically caused diseases, such
as cystic fibrosis, muscular dystrophy.
14. Use of the anion exchange materials mentioned in claims 1 to 6
for the purification of oligonucleotides for in vivo/ex vivo gene
therapy by the antisense/sense strategy.
15. Use of the anion exchange materials mentioned in claims 1 to 6
for the purification of virus particles, also intact virus
particles, for in vivo/ex vivo gene therapy.
16. Use of isopropanol as a reagent for the purification of nucleic
acids in a process according to any of claims 1 to 10.
17. A kit containing the components necessary for performing the
process according to any of claims 1 to 10, in particular,
reagents, also in concentrated form for final mixing by the user,
chromatographic materials for the separation of the nucleic acids,
aqueous solutions (buffers, optionally also in concentrated form
for final adjusting by the user), further auxiliaries, and
substances for the removal of endotoxins.
Description
[0001] The present invention pertains to a process for the
isolation and purification of nucleic acids and/or oligonucleotides
for use in gene therapy wherein said nucleic acids and/or
oligonucleotides are purified from an essentially biological
source, the use of anion exchange materials for the separation,
purification and isolation of nucleic acids for the preparation of
an agent containing nucleic acids for gene therapy, and a kit
containing components for performing the process according to the
invention.
[0002] A new form of therapy for genetically caused diseases, such
as cystic fibrosis or muscular dystrophy, is based on the discovery
that such diseases are caused by particular genetic defects. A
therapy for the genetic defect appears to be possible if the
healthy gene is supplied to the afflicted organism in a sufficient
amount. Gene therapy not only enables the treatment of genetically
caused diseases, but is also suitable for the treatment of tumors,
and is suited as a new form of inoculation against infectious
diseases, such as hepatitis, influenza, and HIV, to give but a few
examples (TIBTECH, Special Issue: Gene Therapy Therapeutic Strategy
and Commercial Prospects, May 1993, Vol. 11, No. 5 (112)).
[0003] A central problem of gene therapy is to administer the
therapeutic DNA in such a manner that it will reach the scene of
action. To date, part of the cells to be treated, in which the
defect gene is expressed, such as blood cells, has been withdrawn
from the patients. These cells have been cultured in culture dishs
(in vitro). In order to introduce the therapeutically active
foreign DNA into the cells, gene segments of a retrovirus, e.g.,
have been used which were linked to the DNA to be introduced. The
genetically altered cells have been retransferred into the organism
(Anderson, W. F. (1992), Human Gene Therapy, Science 256:
808-813).
[0004] Currently, a number of clinical studies are already being
performed with this so-called ex vivo approach. This has lately
involved the use of plasmid DNA, oligonucleotides, mRNA, genomic
DNA, YACs (yeast artificial chromosomes), in addition to the
retroviruses mentioned above, for the transfection of cell
cultures. However, the ex vivo method involves a high expenditure
of work and is not suited for the treatment of all diseases. There
may be mentioned, for example, muscular dystrophy or cystic
fibrosis. Thus, it is desirable to provide simpler procedures to
administer therapeutically useful DNA to an organism. It has been
found in this context that it is possible to administer plasmid DNA
directly into the tissue of an organ. Part of the DNA will be
transported to the nucleus. The genetic information administered
via the DNA is translated there into the therapeutically active
protein. The treatment within the organisms is a direct one and is
called in viva treatment.
[0005] For in vivo treatment, the DNA or RNA may also be mixed with
liposomes or other substances, resulting in a better intake of the
nucleic acids into the cell. However, the nucleic acid may also be
directly injected into the organ to be treated, for example, a
muscle or a tumor (Plautz, G. E. et al., 1993, PNAS, Vol. 90,
4645-4649). The advantage is that the DNA entering the organism
does not cause any immunological reactions in the organism if it is
free of accompanying immunogenic contaminations. Therefore, in viva
gene therapy makes high demands on the quality of the nucleic acids
to be administered. The DNA must be free of toxic substances which
might result in pathogenic effects in the organism to be
treated.
[0006] Clinical phase I studies on humans using this technology
have resulted in rather detailed and strict requirements for the
nucleic acids used therein. According to the requirements of the
FDA in the U.S.A., the nucleic acids employed for therapeutical
uses have to pass the following quality controls:
1 Examination of the nucleic acid for: requirement/limit Endotoxins
<300 I.U./mg of DNA E. coli genomic DNA <50 .mu.g/mg of DNA
Protein <100 .mu.g/mg of DNA Supercoiled DNA >90% 1.75-1.85
Residual salt scan from A.sub.220 to A.sub.320 RNA <1% Sterility
no colonies after 14 days of tryptose culture
[0007] In addition to the quality of the purified nucleic acid, the
scale on which the nucleic acid can be purified is also of crucial
importance. Thus, a future technology must enable to purify nucleic
acids on a scale of from 1 mg to 100 kg which in turn requires
culture volumes of from 1 l to 100 m.sup.3.
[0008] A general problem in the purification of nucleic acids from
bacterial cultures is at first the lysis of the microorganisms. In
addition to the alkaline lysis described by Birnborn and Dohly
(Nucl. Acids Res. 7, pages 1513-1522 (1979)) which is preferred
herein, this may also involve the rupture of the bacterial cells by
high pressure (French Press), lysis in the presence of detergents,
or the application of heat (boiling lysis).
[0009] Subsequently, the nucleic acid can be separated more or less
effectively from the other components of the bacterial cell, such
as proteins or genomic DNA and metabolites, by various methods. The
most simple, but also not very efficient, possibility is the
separation by the addition of salts, such as LiCl, causing
precipitation of the cellular proteins. The nucleic acid can
subsequently be precipitated with alcohol. A drawback of this
method is that contaminations of RNA, ssDNA and proteins cannot be
separated off quantitatively. As an additional purification step,
phenol extraction is frequently performed to remove any protein
contaminations. The drawback of this method, desingated as "salting
out", is that endotoxin contaminations as well as RNA and ssDNA
which may be present cannot be removed. In addition, phenol
extraction involves the risk of contaminating the nucleic acid with
phenol. Further, phenol treatment of nucleic acids usually results
in an increased content of so-called "nicked" nucleic acid, i.e.
break of the nucleic acid strand at many sites, which in turn
highly affects its stability.
[0010] CsCl gradient centrifugation has been an established method
for the purification of nucleic acids for nearly 30 years. This
makes use of the different sedimentation behaviors of differently
sized nucleic acid molecules (RNA, plasmid DNA, genomic DNA) in a
CsCl concentration gradient in the presence of intercalating
agents, such as ethidium bromide, for the separation of nucleic
acids. This type of separation can only be used with large
quantities and requires the use of ultracentrifuges. In addition to
the high financial expenditure of about DM 60,000.--per
ultracentrifuge, another drawback is the considerable expenditure
of time of at least 48 h for such a purification. This method
achieves a yield of only 5 mg of nucleic acid at most per
centrifugal run.
[0011] The purification of nucleic acids by chromatographic methods
is also known per se. There are generally two types of distinct
methods.
[0012] Purification by anion exchange chromatography is described
in EP 0 268 946 B1. The bacterial cells are preferably lysed by
alkaline lysis. The cellular proteins and genomic DNA are separated
by means of detergents and subsequent centrifugation. The
supernatant thus obtained which contains the plasmid DNA is called
the "cleared lysate". The cleared lysate is further purified over
an anion exchange column (QIAGEN.RTM.), wherein RNA and ssDNA are
quantitatively separated off. Removal of endotoxins does not take
place.
[0013] Gillespie and Vogelstein, Proc. Natl. Acad. Sci., USA, 76,
p. 615-619, state that nucleic acids may be further purified by
binding to silica gel or diatomaceous earth in the presence of
chaotropic salts, such as GuHCl, NaCl etc. In contrast to anion
exchange chromatography, binding of the DNA is here performed in
the presence of high salt concentrations whereas elution is
performed at low salt concentrations. The mechanism is not
understood in all details, but it is considered that the nucleic
acid is precipitated by dehydration on the surface of the silica
gel particles. Since this involves binding and elution according to
an "all-or-none" principle, a quantitative separation of RNA, ssDNA
and proteins is not possible. Therefore, unfortunately, such DNA
preparations are unsuited for obtaining nucleic acids for use in
gene therapy due to RNA, protein and ssDNA contaminations. In
addition, 1000 times higher endotoxin values can be found in such
preparations.
[0014] The nucleic acids obtained should also be suited for use in
gene therapy according to the "antisense" or "sense" strategy.
"Anti-sense" strategy makes use of the tendency of, for example,
mRNA to form hybrids with complementary nucleic acids. The hybrids
are inactive. Thus, the "antisense" nucleic acid inactivates the
mRNA. The "antisense" RNA obtained according to the invention may
be administered to the subject to be treated continuously from
outside or may be generated inside the subject himself by
correspondingly transformed cells. The "sense" strategy involves a
supplementation or assistance of, e.g., mRNA which serves important
functions. The RNA required is administered to the subject to be
treated. The nucleic acid to be obtained should also be suited for
use in so-called genetic vaccination methods.
[0015] The quality requirements stated above cannot be satisfied by
the DNA preparation methods described using cesium chloride
gradient centrifugation or by the isolation of DNA in the presence
of chaotropic salts alone since in this method the DNA to be
isolated gets in contact with various toxic or cancerogenic
substances, such as phenol, chloroform, guanidinium chloride, or
ethidium bromide. Thus, it can be shown by electron microscopic
analysis that ethidium bromide incorporated in the double helix
cannot be completely removed any more (Schleef and Heinemann (Bio
Techniques Vol. 14, No. 4, 1993). DNA molecules which are
contaminated, e.g., with ethidium bromide in the course of the
preparation may induce allergic reactions in the body due to the
intercalated ethidium bromide so that any therapeutical approach
with DNA thus prepared cannot be justified.
[0016] High purity DNA can be prepared with anion exchange
chromatography without the use of toxic substances. However, even
when chromatography is used, endotoxins can be conveyed into the
nucleic acid and/or oligonucleotid fractions to a not unrisky
extent.
[0017] The object of the invention is to provide a one-step process
for the purification, isolation and preparation of nucleic acids
which can meet the high quality requirements for nucleic acids
and/or oligonucleotides for gene therapy. A drastic reduction of
endotoxin levels should already take place, if possible, in sample
preparation.
[0018] Surprisingly, the object of the invention is achieved by a
process in which the anion exchange chromatographic material
mentioned in claim 1 is employed as follows.
[0019] In the manner described above, a cleared lysate can be
obtained by various methods. In a preferred embodiment, the
centrifugation step after the lysis for the separation of genomic
DNA and SDS/protein complex can be dispensed with by using a
filtration device as described in P 44 32 654.8. At the same time,
such filtration enables a considerable reduction of the endotoxin
contaminations of the nucleic acid solution.
[0020] By using the buffer proposed in P 44 31 125.7 in connection
with anion exchange chromatography, gel filtration or binding to
silica gel or diatomaceous earth in the presence of chaotropic
salts in a one-step process, the nucleic acid can be purified to
meet all quality requirements stated above.
[0021] The anion exchange material employed in the process
according to the invention enables a neat separation of RNA and DNA
due to the elution points differing by at least 100 mM NaCl.
[0022] The anion exchange material which can be used in the process
according to the invention is also suited for the purification of
virus particles, especially also intact virus particles, for in
vivo/ex vivo gene therapy.
[0023] However, endotoxins may also be depleted or removed
according to the method proposed in P 44 31 125.7. Endotoxins are
depleted or removed therein by treatment with chromatographic
material. After the lysis of the natural sources from which the
nucleic acids and/or oligonucleotides are to be obtained, the
fractions obtained are treated with metal-chelating chromatographic
methods. This method may be employed in addition to or in
combination with the incubation of the fractions obtained with
aqueous salt solutions and detergents wherein the detergent
treatment is followed by anion exchange chromatography. The
metal-chelating chromatographic materials include chelating agents,
IDA (iminodiacetate) or NTA (nitrilotriacetate), which are bound to
supports, such as silica gel, diatomaceous earth, glass, aluminium
oxides, titanium oxides, zirconium oxides, hydroxyapatite,
dextrane, agarose, acrylic amide, polystyrene resins, or copolymers
of the monomeric building blocks of the polymers mentioned. Other
materials which may be used include polymyxin or DNA ETOX. On this
affinity support, nickel ions, for example, can be complexed which
may interact with side-chain nitrogen containing amino acid
residues in proteins through additional coordination sites. The
lysed biological sources which have been freed from cell debris may
be incubated, in particular, with Ni/NTA chromatographic material
based on silica gel. The chromatographic material may be
centrifuged off, for instance, after the incubation is completed,
if batch-mode was used, and the supernatant may then be further
processed according to the invention. In addition to batch mode,
the affinity chromatography may also be performed in columns if the
sample condition allows.
[0024] The nucleic acid to be isolated may be derived directly from
cells which have been lysed. The process according to the invention
ensures the separation of contaminants and yields nucleic acids
having the purity required for gene therapy. Surprisingly, the
commercial material QIAGEN.RTM. of the firm Qiagen, in particular,
proves to be suitable for use in the process according to the
invention.
[0025] This material enables a very efficient separation of the DNA
from RNA. DNA elutes at a salt concentration corresponding to about
480 mM sodium chloride, whereas the double-stranded plasmid DNA
elutes only at about 1260 mM sodium chloride. The difference
between these two elution points is about 420 mM with the
QIAGEN.RTM. material whereas the difference between the elution
points of RNA and plasmid DNA is about 80 mM of sodium chloride
concentration at most with all known anion exchange materials. Such
a low difference in elution points involves a high risk of
coelution of DNA and RNA, in particular with single-stranded
DNA.
[0026] The material which is commercially available under the
designation of QIAGEN.RTM. is particularly suitable for the
purification of plasmid DNA for gene therapy. This chromatographic
support material is a modified porous inorganic material. As
inorganic support materials, there may be used materials such as
silica gel, diatomaceous earth, glass, aluminium oxides, titanium
oxides, zirconium oxides, hydroxyapatite, and as organic support
materials, such as dextrane, agarose, acrylic amide, polystyrene
resins, or copolymers of the monomeric building blocks of the
polymers mentioned.
[0027] The anion exchanger which is preferably used may be
obtained, for instance, by the reaction of one of the
above-mentioned support materials in a first step with a silanizing
reagent of the general formula I,
R.sup.1R.sup.2R.sup.3SiR.sup.4 (I)
[0028] wherein R.sup.1 is -an alkoxy residue of from 1 to 10 carbon
atoms, especially --OCH.sub.3, --OC.sub.2H.sub.5 or
--OC.sub.3H.sub.7, or a halogen atom, especially --Cl, or a
dialkylamino group with identical or different alkyl residues of
from 1 to 6 carbon atoms;
[0029] R.sup.2 and R.sup.3 are independently a hydrocarbon residue
of from 1 to 10 carbon atoms, especially --CH.sub.3,
--C.sub.2H.sub.5 or --C.sub.3H.sub.7, or an alkoxy residue of from
1 to 10 carbon atoms, especially --OCH.sub.3, --OC.sub.2H.sub.5 or
--OC.sub.3H.sub.7, or a halogen atom, or an alkyl residue of from 4
to 20 carbon atoms which is interrupted by at least one oxa or
amino group wherein said residue may also be substituted with one
or more of halogen, cyano, nitro, amino, monoalkylamino,
dialkylamino, hydroxy or aryl;
[0030] R.sup.4 is a hydrocarbon chain of from 1 to 20 carbon atoms,
or an alkyl residue which is interrupted by at least one oxa or
amino group wherein said residue may also be substituted with one
or more of halogen, cyano, nitro, amino, monoalkylamino,
dialkylamino, alkoxy, hydroxy, aryl, and/or epoxy, especially 1
[0031] followed by a second step wherein the support which has been
modified in the first step is reacted with a reagent of the general
formula II:
X-R-Y (II)
[0032] wherein X is an amino, hydroxy, epoxy group or a halogen
atom;
[0033] R is a hydrocarbon chain of from 2 to 20 carbon atoms, or an
alkyl residue which is interrupted by at least one oxa or amino
group wherein said residue may also be substituted with one or more
of halogen, cyano, nitro, amino, monoalkylamino, dialkylamino,
alkoxy, hydroxy, aryl, and/or epoxy;
[0034] Y is a hydrocarbon residue having functional groups which
form an anion exchange material and having from 1 to 10 carbon
atoms which may be substituted with one or more of amino,
monoalkylamino, dialkylamino, quarternary alkylamino.
[0035] In particular, support materials may be used made of silica
gel and having diethylaminoethyl (DEAE) or diethylaminopropyl
groups or dimethylaminoethyl (DMAE) or dimethylaminopropyl groups
arranged on the surface thereof either directly or through
so-called spacers.
[0036] In the process according to the invention, an anion
exchanger is used, in particular, having the formula
[0037] support material 2
[0038] The ethyl groups of the amine may also be replaced by methyl
groups.
[0039] The nucleic acids may also be purified by anion exchange
materials based on polystyrene/DVB, such as Poros 20 for medium
pressure chromatography, Poros 50 HQ, of the firm of BioPerseptive,
Cambridge, U.S.A., or over DEAE sepharose, Q sepharose, DEAE
Sephadex of the firm of Pharmacia, Sweden; DEAE Spherodex LS, DEAE
Spherosil, of the firm of Biosepra, France.
[0040] Also, silica materials, such as kieselguhr, siloid,
diatomaceous earth, glass, silica gel, alumina, titania,
hydroxyapatite, in the presence of chaotropic salts, such as sodium
iodide, guanidinium chloride, and/or alcohols, are useful for the
preparation of the nucleic acids according to the invention.
[0041] In the preparation of cell contents, especially nucleic
acids, the problem frequently arises, to separate the lysed natural
sources from which these contents are derived from the dissolved
materials. The separation of the cells or cell debris is performed
by centrifugation wherein the larger cell debris or cells will
deposit as a pellet in the centrifuge tube. The cell contents are
then found in the supernatant and may be pipetted. Filtration
methods which are simpler per se could not prevail, in particular,
in the preparation of nucleic acids since either the lysed cells or
their fragments will pass through the too large-pored filters,
resulting in turbidity and contaminations in the filtrate, or, when
filters having appropriately small pores are used, obstruction of
the filters will necessarily occur so that a reasonable preparation
of the cell contents is no longer possible.
[0042] Usually, the samples are centrifuged in 50 to 500 ml vessels
at about 20,000 rpm (about 30,000.times. g) for 5 to 60 min in
order to remove cell debris.
[0043] Such centrifugation is time-consuming and with larger cell
lysate volumes of 2 1 and more can hardly be performed in an
economical way. Although flow-through centrifuges exist, they are
useful only for very large volumes of >1000 l. In addition, this
is complicated and expensive.
[0044] According to the invention, a simpler removal of cell debris
from cell lysates of 1 l to 1000 l is enabled. This involves the
use of the filtration methods described in WO 93/11218.
[0045] Also, an endotoxin depletion or removal is already performed
in the separation of cell debris in an especially simple way
according to the invention. This is done by using the method
proposed in P 44 32 654.8. The lysate containing cell debris is
passed over filter layers of glass, silica gel, diatomaceous earth,
aluminium oxides, titanium oxides, zirconium oxides,
hydroxyapatite, and other inorganic minerals, such as perlite, or
filter layers of interlaced non-wovens made of fiber glass and
silica gel as well as cellulose, paper, pressed paper, interlaced
or bonded non-wovens made of polymers, especially polypropylene,
polyamides or polyester. Or it is passed over alumina or packed
diatomaceous earth or interlaced or bonded non-wovens made of fiber
glass and silica gel as well as cellulose, paper, pressed paper,
non-wovens made of paper. The fraction emerging from the filter
layer is collected and subsequently further treated according to
the invention.
[0046] Surprisingly, it has been shown that endotoxins are depleted
by such filtration. It is particularly preferred that the materials
forming the filter layer bear hydroxy groups, or are coated or
modified with organosilanes bearing or forming hydroxy groups, such
as 3
[0047] in particular, diol silica gel, diol diatomaceous earth
and/or diol perlite.
[0048] In particular, packed diatomaceous earth has proven useful
in the sample preparation for endotoxin depletion or removal.
[0049] The nucleic acid obtained by the process according to the
invention is also suited for use in gene therapy according to the
"antisense" or "sense" strategy. "Antisense" strategy makes use of
the tendency of, for example, MRNA to form hybrids with
complementary nucleic acids. The hybrids are inactive. Thus, the
"antisense" nucleic acid inactivates the MRNA. The "antisense" RNA
obtained according to the invention may be administered to the
subject to be treated continuously from outside or may be generated
inside the subject himself by correspondingly transformed cells.
The "sense" strategy involves a supplementation or assistance of,
e.g., mRNA which serves important functions. The RNA required is
administered to the subject to be treated. Due to its high purity,
the nucleic acid obtained by the process according to the invention
is also suited for use in so-called genetic vaccination
methods.
[0050] In a preferred embodiment of the process according to the
invention, the salts, which are necessary for eluting the nucleic
acids under conditions of high ionic strength, are removed by
treating with a mineral support material the fractions containing
nucleic acids and having the high salt concentrations. These
support materials essentially consist of non-modified inorganic
support materials, e.g., glass or powdered glass. The nucleic acid
will adsorb to such surfaces at high salt concentrations.
Thereafter, the adsorbed nucleic acid can be desorbed with
solutions of low ionic strength or demineralized water.
[0051] It has been found that the use of buffers containing
isopropanol instead of those containing ethanol is advantageous. As
proposed in P 44 03 693.0, particularly good transfection rates of
the nucleic acids prepared according to the invention can be
achieved by the use of buffers containing isopropanol.
[0052] According to the invention, a kit is also claimed containing
components necessary for performing the process according to the
invention. These include, in particular, reagents, also in
concentrated form for final mixing by the user, chromatographic
materials for the separation of the nucleic acids, aqueous
solutions (buffers, optionally also in concentrated form for final
adjusting by the user), and further auxiliaries, such as substances
for the removal of endotoxins, such as diatomaceous earth, or
chromatographic materials for desalting nucleic acids which have
been eluted with sodium chloride.
[0053] The anion exchange material which may be used in the process
according to the invention enables DNA preparations up to a
kilogramm scale, especially in the range of from 10 mg to 100 g of
DNA. An examination of the DNA which has been isolated by the
process according to the invention by means of HPLC analysis and
electron microscopy shows that such preparations are free of
proteins (endotoxins), genomic DNA and RNA. The invention will be
illustrated in more detail by the following examples.
2 Buffer P1: 100 .mu.g/ml RNase A, 50 mM Tris/HCl, (resuspension
buffer) 10 mM EDTA, pH 8.0 Buffer P2: 200 mM NaOH, 1% SDS (lysis
buffer) Buffer P3 3.0M KAc, pH 5.5 (neutralisation buffer) Buffer
QBT: 750 mM NaCl, 50 mM MOPS, 15% alcohol*, (equilibration buffer)
pH 7.0, 0.15% Triton X 100 Buffer QC: 1.0M NaCl, 50 mM MOPS, 15%
alcohol, (wash buffer) pH 7.0 Buffer QN: 1.6M NaCl, 50 mM MOPS, 15%
alcohol, (elution buffer) pH 8.5 TE: 10 mM Tris/HCl, 1 mM EDTA, pH
8.0 STE: 100 mM NaCl, 10 mM Tris/HCl, 1 mM EDTA, pH 8.0 Endotoxin
Removal 750 mM NaCl, 10% Triton X 100, Buffer: 50 mM MOPS, pH 7.0
*As the alcohols, isopropanol or ethanol are preferably used.
EXAMPLE 1
[0054] Isolation of 50 mg of pSVCFTR from 10 l of E. coli Culture
for the Aerosilation of CF Patients
[0055] The bacterial pellet resulting from 10 l of E. coli XL1 Blue
fermenter culture is resuspended in 500 ml each of buffers P1 and
P2. The mixture is incubated with 500 ml of buffer P3 on ice for 30
min and subsequently centrifuged at 20,000.times. g for 15 min. The
supernatant is filtered over a folded filter for clearing. The
filtered lysate is mixed with {fraction (1/10)} of its volume of
Endotoxin Removal Buffer (750 mM NaCl; 10% Triton X 114; 40 mM
MOPS, pH 7.0) and incubated on ice for 30 min. The lysate is now
pumped onto a chromatographic column of 26 mm.times.100 mm filled
with a QIAGEN anion exchanger (70-100 .mu.m particle size, 2.5
.mu.mol DEAE/g of chromatographic material) by means of a
peristaltic pump and adsorbed. The chromatographic column is
subsequently washed with 2000 ml of buffer QC at a flow rate of 15
ml/min. The plasmid DNA bound to the column is eluted with 280 ml
of buffer QN at a flow rate of 3 ml/min. The eluate obtained is
mixed with 200 ml of isopropanol and centrifuged at 20,000.times. g
for 30 min. The resulting pellet is resuspended in 40 ml of TE
buffer. The DNA thus purified is analyzed for purity by means of
electron microscopy (EM), HPLC analysis, photometric measurement,
agarose gel electrophoresis, and endotoxin test. This purification
method results in a high purity DNA with no detectable
contaminations of RNA, genomic DNA and endotoxins. The DNA thus
isolated is mixed with a liposome solution and administered to CF
patients by aerosilation in amounts of 10 .mu.g.
EXAMPLE 2
[0056] Isolation of 10 mg of pCMVlacZ using QIAGEN tip 10,000 for
the Injection of Plasmid DNA into Striated Muscle for the Treatment
of Muscular Dystrophy
[0057] Five liters of a DH5alph/pCMVlacZ overnight culture are
centrifuged, and the resulting pellet is resuspended in 125 ml of
P1, mixed with 125 ml of buffer P2, and incubated at room
temperature (RT) for 5 min. Then, 125 ml of buffer P3 is added,
mixing is performed, followed by incubation at 4.degree. C. for 30
min. The lysate is passed over a loose packing of diatomaceous
earth in a filtration column as described in P 44 32 654.8 and
subsequently spiked with {fraction (1/10)} of its volume of
Endotoxin Removal Buffer as in example 1 and incubated on ice for
30 min. The mixture is now spiked with 270 ml of isopropanol and
centrifuged at 20,000.times. g for 30 min. The resulting pellet is
dried ar RT for 10 min and resuspended in 5 ml of water. The
resuspended DNA is spiked with 25 ml of QC buffer. This mixture is
charged onto a DEAE silica gel column (26 mm.times.50 mm, 70-100
.mu.m, 2.0 .mu.mol/g) equilibrated with 75 ml of QBT buffer. The
column is then washed with 600 ml of QC buffer, and the DNA is then
eluted with 75 ml of QF buffer. The eluate is mixed with 52.5 ml of
isopropanol and centrifuged at 20,000.times. g for 30 min. The DNA
pellet is dried at RT for 10 min and resuspended in 1 ml of PBS.
The DNA solution can be used only for direct muscle injection.
EXAMPLE 3
[0058] Isolation of 100 mg of pXYHBV from a 20 l E. coli Culture
for use as "Genetic Hepatitis Vaccine"
[0059] The bacterial pellet resulting from a 20 l fermentation run
is resuspended in 1000 ml of buffer Pi, spiked with 1000 of buffer
P2 and incubated at RT for 5 min. After the addition of 1000 ml of
buffer P3, the mixture is incubated at 4.degree. C. for 30 min and
subsequently centrifuged at 20,000.times. g. The supernatant is
passed over a fiber glass filter, and the clear lysate is mixed
with 2250 ml of isopropanol and centrifuged at 20,000.times. g for
30 min. The resulting pellet is resuspended in 10 ml of water and
spiked with 90 ml of QC buffer. This mixture is pumped onto a
chromatographic column according to example 1 by means of a
peristaltic pump at a flow rate of 2 ml/min. The column is washed
at a flow rate of 15 ml/min, and the DNA is subsequently eluted
with 350 ml of QF buffer at a flow rate of 3 ml/min.
EXAMPLE 4
[0060] Removal of Endotoxin from DNA Preparations
[0061] The DNA prepared is adjusted to a final concentration of
0.1-1% with Triton X 114. Then, the DNA/Triton solution is
incubated on a "roller" at 4-7.degree. C. for 30 min. The solution
is heated to room temperature and centrifuged at 20,000.times. g
for 30 min or filtered. The supernatant is spiked with 0.7 volumes
of isopropanol and precipitated. The resulting pellet is dried and
resuspended in TE. The DNA thus treated is free of endotoxin.
EXAMPLE 5
[0062] Plasmid Preparation
[0063] A 150 ml HB 101 E. coli culture with pUC 18 plasmid DNA in
LB medium is centrifuged at 3000.times. g for 5 min to pelletize
the cells. The cell pellet is resuspended in 20 ml of 50 ml
Tris/HCl, 10 mM EDTA, pH 8.0, 100 .mu.g/ml RNase A. Twenty
milliliters of 0.2 M NaOH, 1% SDS are added to the cell suspension
for cell lysis, cautiously mixed and kept standing at room
temperature for 5 minutes. Then, 20 ml of 3 M potassium acetate, 2
M acetic acid is added for neutralisation, mixed, and incubated on
ice for 15 minutes, and the cell lysate is sucked through the
filter device according to the invention at a pressure difference
of 20 mbar to 800 mbar. Alternatively, the sample may be pressed
through the filter layers with a piston or by increased pressure.
After the filtration, the filtration device is removed, and the
filter cake with the cell fragments, denatured proteins and
precipitated SDS is discarded. The filtrated lysate is mixed with
{fraction (1/10)} of its volume of Endotoxin Removal Buffer (750 mM
NaCl; 10% Triton X 114; 40 mM MOPS, pH 7.0) and incubated on ice
for 30 min. The filtrate is completely sucked or pressed through
the anion exchange column to achieve adsorption of the DNA. The
extraction column is subsequently washed twice with 100 ml of 1 M
NaCl, 15% ethanol, 50 mM MOPS, pH 7.0, to remove RNA and proteins.
The DNA is eluted with 100 ml of 1.6 M NaCl, 15% ethanol, 50 mM
MOPS, pH 7.0. The eluted DNA is precipitated with alcohol for
desalting and concentrating, and the alcoholic pellet is pelletized
by centrifugation.
[0064] Alternatively, the alcoholic precipitate of the nucleic acid
may also be obtained by filtration. This has advantages when large
amounts of DNA must be prepared and the volumes to be handled are
larger than, for instance, 1 l .
EXAMPLE 6
[0065] A DNA template is transcribed into RNA via an in vitro
reaction. The reaction solution is adjusted to 750 mM NaCl and
purified over a QIAGEN.RTM. anion exchange column. The purified RNA
is subsequently used for in vitro or in vivo gene therapy.
EXAMPLE 7
[0066] Purification of 40 mg of pBR322 using DEAE Q Sepharose.RTM.
(Firm of Pharmacia)
[0067] The biomass from a 40 l fermenter culture of pBR322 was
lysed by alkaline lysis with 10 l each of buffers P1, P2, and P3.
Subsequently, the lysate was passed over a filter device consisting
of a loose packing and then incubated at 4.degree. C. for 30 min.
The DNA is now precipitated by the addition of 0.7 volumes of
isopropanol and resuspended in 20 ml of 10 mM Tris/HCl, pH 8.5, 1
mM EDTA, 50 mM NaCl. The resuspended DNA solution is charged onto a
DEAE Q Sepharose column with a bed volume of 200 ml. The DNA is
eluted with a gradient of 1 mM NaCl/ml, the buffers having the
following concentrations:
[0068] Buffer A: 10 mM Tris/HCl, 1 mM EDTA, 0.75 M NaCl, pH
8.0;
[0069] Buffer B: 10 mM Tris/HCl, 1 mM EDTA, 0.85 M NaCl.
[0070] The flow rate is 0.5 ml/min. The DNA is subsequently
precipitated with ethanol and resuspended in PBS buffer in a
concentration of 1 .mu.m/.mu.l.
* * * * *