U.S. patent application number 10/731726 was filed with the patent office on 2004-06-24 for method of preparing recombinant adeno-associated virus compositions.
This patent application is currently assigned to University of Florida Research Foundation. Invention is credited to Byrne, Barry J., Muzyczka, Nicholas, Zolotukhin, Sergei.
Application Number | 20040121444 10/731726 |
Document ID | / |
Family ID | 22201621 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121444 |
Kind Code |
A1 |
Zolotukhin, Sergei ; et
al. |
June 24, 2004 |
Method of preparing recombinant adeno-associated virus
compositions
Abstract
Disclosed are methods for the isolation and purification of
high-titer recombinant adeno-associated virus (rAAV) compositions.
Also disclosed are methods for reducing or eliminating the
concentration of helper adenovirus in rAAV samples. Methods are
disclosed that provide highly-purified rAAV stocks having titers up
to about 10.sup.13 particles/ml at particle-to-infectivity ratios
of less than 100 in processes that are accomplished about 24 hours
or less.
Inventors: |
Zolotukhin, Sergei;
(Gainesville, FL) ; Byrne, Barry J.; (Gainesville,
FL) ; Muzyczka, Nicholas; (Gainesville, FL) |
Correspondence
Address: |
EDWARDS & ANGELL, LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
University of Florida Research
Foundation
|
Family ID: |
22201621 |
Appl. No.: |
10/731726 |
Filed: |
December 9, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10731726 |
Dec 9, 2003 |
|
|
|
09621475 |
Jul 21, 2000 |
|
|
|
6660514 |
|
|
|
|
09621475 |
Jul 21, 2000 |
|
|
|
09321897 |
May 27, 1999 |
|
|
|
6146874 |
|
|
|
|
60086898 |
May 27, 1998 |
|
|
|
Current U.S.
Class: |
435/239 |
Current CPC
Class: |
Y10S 435/948 20130101;
C12N 2750/14151 20130101; Y10S 530/826 20130101; C12N 15/86
20130101; C12N 2750/14143 20130101; C12N 7/00 20130101; Y10S
435/803 20130101 |
Class at
Publication: |
435/239 |
International
Class: |
C12N 007/02 |
Claims
What is claimed is:
1. A method of isolating a recombinant adeno-associated virus,
comprising applying a sample containing recombinant
adeno-associated virus to an iodixanol gradient, and collecting
said recombinant adeno-associated virus from said gradient.
2. The method of claim 1, wherein said iodixanol gradient is a
discontinuous gradient.
3. The method of claim 2, wherein said iodixanol gradient comprises
an about 15% iodixanol step, an about 25% iodixanol step, an about
40% iodixanol step, and an about 60% iodixanol step.
4. The method of claim 3, wherein said recombinant adeno-associated
virus is collected from said 40% iodixanol step.
5. The method of claim 3, wherein said 15% iodixanol step further
comprises about 1 M NaCl.
6. The method of claim 1, wherein said iodixanol gradient is
subjected to centrifugation after applying said sample.
7. The method of claim 1, further comprising contacting said
recombinant adeno-associated virus with a matrix comprising
heparin, under conditions effective to permit binding of said virus
to said matrix, removing non-bound species from said matrix, and
eluting said virus from said matrix.
8. The method of claim 7, wherein said matrix comprises heparin
agarose type I or heparin agarose type II-S.
9. The method of claim 7, wherein said matrix is comprised within
an HPLC column.
10. The method of claim 7, wherein said virus is eluted from said
matrix with a solution comprising about 1 M NaCl.
11. The method of claim 1, further comprising contacting said
recombinant adeno-associated virus with a hydrophobic matrix, under
conditions effective to permit interaction of hydrophobic species
with said hydrophobic matrix, and collecting the non-interacting
virus from said hydrophobic matrix.
12. The method of claim 11, wherein said hydrophobic matrix
comprises phenyl groups.
13. The method of claim 12, wherein said hydrophobic matrix is
phenyl-sepharose.
14. The method of claim 1, further comprising applying said
recombinant adeno-associated virus to a cesium chloride equilibrium
density gradient, and collecting said recombinant adeno-associated
virus from said gradient.
15. The method of claim 1, further comprising contacting said
recombinant adeno-associated virus with at least a first ion
exchange chromatography medium, under conditions effective to
permit interaction of said virus with said medium, removing
non-interacting species from said medium, and eluting said virus
from said medium.
16. The method of claim 1, wherein said sample further comprises a
virus.
17. The method of claim 16, wherein said sample further comprises
an adenovirus.
18. The method of claim 1, wherein said sample further comprises at
least a first polypeptide or protein.
19. The method of claim 1, wherein said sample further comprises a
cell extract or a growth medium.
20. A method of isolating a recombinant adeno-associated virus,
comprising the steps of: a) centrifuging a sample containing
recombinant adeno-associated virus through an iodixanol gradient;
b) collecting from said iodixanol gradient at least a first
fraction comprising said recombinant adeno-associated virus; c)
contacting said at least a first fraction comprising said
recombinant adeno-associated virus with a matrix comprising
heparin, under conditions effective to permit binding of said virus
to said matrix; d) removing non-bound species from said matrix; and
e) eluting said virus from said matrix.
21. A method of isolating a recombinant adeno-associated virus,
comprising the steps of: a) centrifuging a sample containing
recombinant adeno-associated virus through an iodixanol gradient;
b) collecting from said iodixanol gradient at least a first
fraction comprising said recombinant adeno-associated virus; c)
contacting said at least a first fraction comprising said
recombinant adeno-associated virus with a matrix comprising
heparin, under conditions effective to permit binding of said virus
to said matrix; d) removing non-bound species from said matrix; e)
eluting said virus from said matrix; f) contacting the eluted virus
with a hydrophobic matrix, under conditions effective to permit
interaction of hydrophobic species with said hydrophobic matrix;
and g) collecting the non-interacting virus from said hydrophobic
matrix.
22. A method for reducing or eliminating adenovirus from a
recombinant adeno-associated virus composition contaminated with
adenovirus, comprising applying a sample containing recombinant
adeno-associated virus and adenovirus to an iodixanol gradient, and
collecting from said gradient at least a first fraction comprising
said recombinant adeno-associated virus.
23. A method of producing a recombinant adeno-associated virus
having a particle-to-infectivity ratio of less than about 100 to 1,
comprising the steps of: a) centrifuging a sample containing
recombinant adeno-associated virus through an iodixanol gradient;
b) collecting from said iodixanol gradient at least a first
fraction comprising said recombinant adeno-associated virus; c)
contacting said at least a first fraction comprising said
recombinant adeno-associated virus with a matrix comprising
heparin, under conditions effective to permit binding of said virus
to said matrix; d) removing non-bound species from said matrix; and
e) eluting said virus from said matrix.
24. Recombinant adeno-associated virus, prepared by applying a
sample containing recombinant adeno-associated virus to an
iodixanol gradient, and collecting said recombinant
adeno-associated virus from said gradient.
25. A kit comprising, in a suitable container, iodixanol, a matrix
comprising heparin and instructions for isolating recombinant
adeno-associated virus.
26. The kit of claim 25, wherein said iodixanol is formulated as an
iodixanol gradient.
27. The kit of claim 25, wherein said matrix comprises heparin
agarose type I or heparin agarose type II-S.
28. The kit of claim 25, further comprising a hydrophobic
matrix.
29. The kit of claim 28, wherein said hydrophobic matrix comprises
phenyl groups.
30. The kit of claim 29, wherein said hydrophobic matrix is
phenyl-sepharose.
Description
[0001] The present application claims the priority of U.S.
Provisional Patent Application Serial No. 60/086,898 filed May 27,
1998, the entire disclosure of which is incorporated herein by
reference without disclaimer. The government may have certain
rights in the present invention pursuant to grant numbers PO1
HL59412 and PO1 NS36302 from the National Institutes of Health.
1.0 BACKGROUND OF THE INVENTION
[0002] 1.1 Field of the Invention
[0003] The present invention relates generally to the field of
virology, and in particular, to methods for preparing
highly-purified, high-titer recombinant adeno-associated virus
compositions. In certain embodiments, the invention concerns the
use of equilibrium density centrifugation techniques, affinity
chromatographic media, and in certain embodiments anion- and
cation-exchange resins, to remove rAAV particles from solution and
to prepare highly purified viral stocks for use in a variety of
investigative, diagnostic and therapeutic regimens. Methods are
also provided for purifying rAAVs from solution and for reducing
the concentration of adenovirus in rAAV stocks.
[0004] 1.2 Description of Related Art
[0005] 1.2.1 Adeno-Associated Virus
[0006] Adeno-associated virus-2 (AAV) is a human parvovirus which
can be propagated both as a lytic virus and as a provirus (Cukor et
al., 1984; Hoggan et al., 1972). The viral genome consists of
linear single-stranded DNA (Rose et al., 1969), 4679 bases long
(Srivastava et al., 1983), flanked by inverted terminal repeats of
145 bases (Lusby et al., 1982). For lytic growth AAV requires
co-infection with a helper virus. Either adenovirus (Ad; Atchinson
et al., 1965; Hoggan, 1965; Parks et al., 1967) or herpes simplex
virus (HSV; Buller et al., 1981) can supply helper function.
Without helper, there is no evidence of AAV-specific replication or
gene expression (Rose et al., 1972; Carter et al., 1983; Carter et
al., 1983). When no helper is available, AAV can persist as an
integrated provirus (Hoggan, 1965; Berns et al., 1975; Handa et
al., 1977; Cheung et al., 1980; Berns et al., 1982).
[0007] Integration apparently involves recombination between AAV
termini and host sequences and most of the AAV sequences remain
intact in the provirus. The ability of AAV to integrate into host
DNA is apparently an inherent strategy for insuring the survival of
AAV sequences in the absence of the helper virus. When cells
carrying an AAV provirus are subsequently superinfected with a
helper, the integrated AAV genome is rescued and a productive lytic
cycle occurs (Hoggan, 1965).
[0008] AAV sequences cloned into prokaryotic plasmids are
infectious (Samulski et al., 1982). For example, when the wild type
AAV/pBR322 plasmid, pSM620, is transfected into human cells in the
presence of adenovirus, the AAV sequences are rescued from the
plasmid and a normal AAV lytic cycle ensues (Samulski et al.,
1982). This renders it possible to modify the AAV sequences in the
recombinant plasmid and, then, to grow a viral stock of the mutant
by transfecting the plasmid into human cells (Samulski et al.,
1983; Hermonat et al., 1984). AAV contains at least three
phenotypically distinct regions (Hermonat et al., 1984). The rep
region codes for one or more proteins that are required for DNA
replication and for rescue from the recombinant plasmid, while the
cap and lip regions appear to code for AAV capsid proteins and
mutants within these regions are capable of DNA replication
(Hermonat et al, 1984). It has been shown that the AAV termini are
required for DNA replication (Samulski et al., 1983).
[0009] The construction of two E. coli hybrid plasmids, each of
which contains the entire DNA genome of AAV, and the transfection
of the recombinant DNAs into human cell lines in the presence of
helper adenovirus to successfully rescue and replicate the AAV
genome has been described (Laughlin et al., 1983; Tratschin et al.,
1984a; 1984b).
[0010] 1.2.2 Conventional Methods for Preparing Recombinant AAV
[0011] Recombinant adeno-associated virus (rAAV) has been
demonstrated to be a useful vector for efficient and long-term gene
transfer in a variety of tissues, including lung (Flotte, 1993),
muscle (Kessler, 1996; Xiao and Samulski, 1996; Clark et al., 1997;
Fisher et al., 1997), brain (Kaplitt, 1994; Klein, 1998) retina
(Flannery, 1997; Lewin et al., 1998), and liver (Snyder, 1997). It
has also been demonstrated to evade the immune response of the host
by failing to transduce dendritic cells (Jooss et al., 1998). Phase
I clinical trails are underway for cystic fibrosis rAAV-mediated
gene therapy (Flotte et al., 1996; Wagner et al, 1998). Yet in
spite of these promising developments one of the problems that
remains to be solved is that vector production remains very
laborious.
[0012] Currently rAAV is most often produced by co-transfection of
rAAV vector plasmid and wt AAV helper plasmid into Ad-infected 293
cells (Hermonat and Muzyczka, 1984). Recent improvements in AAV
helper design (Li et al., 1997) as well as construction of
non-infectious mini-Ad plasmid helper (Grimm et al., 1998; Xiao et
al., 1998; Salvetti, 1998) have eliminated the need for Ad
infection, and made it possible to increase the yield of rAAV up to
10.sup.5 particles per transfected cell in a crude lysate. Scalable
methods of rAAV production that do not rely on DNA transfection
have also been developed (Chiorini et al., 1995; Conway et al.,
1997; Inoue and Russell, 1998; Clark et al., 1995). These methods,
which generally involve the construction of producer cell lines and
helper virus infection, are suitable for high-volume
production.
[0013] However, little progress has been made on the downstream
purification of rAAV. The conventional protocol involves the
stepwise precipitation of rAAV using ammonium sulfate, followed by
two or preferably, three rounds of CsCl density gradient
centrifugation. Each round of CsCl centrifugation involves
fractionation of the gradient and probing fractions for rAAV by
dot-blot hybridization or by PCR.TM. analysis. No only does it
require two weeks to complete, but the current protocol often
results in poor recovery of the vector and poor virus quality. The
growing demand for different rAAV stocks often strains the limited
capacities of vector production facilities. There is, therefore, a
clear need for a protocol that will reduce the preparation time
substantially without sacrificing the quality and/or purity of the
final product.
2.0 SUMMARY OF THE INVENTION
[0014] In a first embodiment, the invention concerns a method of
purifying a recombinant adeno-associated virus. In general, the
method comprises centrifuging a sample containing or suspected of
containing recombinant adeno-associated virus through at least a
first iodixanol gradient, and collecting the purified virus or at
least a first fraction comprising the recombinant adeno-associated
virus, from the gradient. Preferably the gradient is a
discontinuous gradient, although the inventors contemplate the
formulation of continuous iodixanol gradients that also provide
purification of rAAV compositions. In certain aspects of the
invention, multiple iodixanol gradients, for example at least a
second, at least a third and/or at least a fourth iodixanol
gradient, are used to purify the recombinant adeno-associated
virus.
[0015] In an exemplary discontinuous iodixanol gradient, the
gradient comprises an about 15% iodixanol step, an about 25%
iodixanol step, an about 40% iodixanol step, and an about 60%
iodixanol step. Optionally, the gradient may contain steps having
lower concentrations of iodixanol, and likewise, the gradient may
contain steps that have higher concentrations of iodixanol.
Naturally, the concentrations of each step do not need to be exact,
but can vary slightly depending upon the particular formulation and
preparation of each step. The inventors have shown that most rAAV
particles will band in an iodixanol gradient at a level
corresponding to a percentage of iodixanol approximately equal to
52%, although depending upon the number of viral particles loaded
on the gradient and the volume and capacity of the gradient, the
range of concentrations at which purified rAAV particles may be
found may range on the order of from about 50% to about 53%, or
from about 50% to about 54%, 55%, 56%, 57%, 58%, 59% and even up to
and including about 60% iodixanol. Likewise, the range of
concentrations at which the rAAV particles may be isolated
following centrifugation may be on the order of from about 55% down
to and including about 49%, about 48%, about 47%, about 46%, about
45%, about 44%, about 43%, about 42%, about 41% or about 40% or so
iodixanol. Naturally, all concentrations in the range of from about
40% to about 60% are contemplated to be useful in recovering
purified rAAV particles from the centrifuged gradient. As such, all
intermediate concentrations including about 41%, about 42%, about
43%, about 44%, about 45%, about 46%, about 47%, about 48%, about
49%, about 50%, about 51%, about 52%, about 53%, about 54%, about
55%, about 56%, about 57%, about 58%, and about 59% or so are
contemplated to be useful in the practice of the present invention
for recovering purified rAAV particles from the centrifuged
gradient.
[0016] When step gradients are utilized, it is convenient to
include in the gradient steps that encompass or "bracket" the range
of optimal recovery of virus. For example, in a 25%/40%/60% step
gradient, the 40% band comprises the virus, and this fraction is
then removed for recovery of the virus composition. The design of
both continuous and discontinuous gradients is well-known to those
of skill in the art, and those having benefit of the present
specification may readily prepare iodixanol gradients of sufficient
capacity and range to isolate a band of purified rAAV particles
from the gradient following centrifugation.
[0017] In certain embodiments, to improve the yield and/or recovery
of virus particles from such a gradient, one may add to one or more
steps of the gradient one or more salts to reduce or prevent
aggregation of the virus and any cellular debris or proteins,
polypeptides, etc. which may be present in the crude sample. In an
exemplary embodiment, the inventors have shown that the addition of
salt to the 15% iodixanol step in a discontinuous gradient improves
the recovery of virus particles from an iodixanol gradient. As an
example, the addition of NaCl to a final concentration of about 1 M
in the 15% step was found by the inventors to be particularly
advantageous in recovery of purified rAAV particles from the 40%
step of such a gradient. While addition of one or more salts to one
or more of the other steps in the gradient may be performed as
required, in most instances, the inventors have shown that the
presence of salt in other steps were either unnecessary or
unwarranted. In situations where one or more salts are added to a
layer which comprises the rAAV particles, following centrifugation
it may be desirable to remove or reduce the concentration of salt
in such a fraction prior to use of, or further purification of, the
rAAV. Such removal may readily be achieved by dialysis,
microconcentration, ultrafiltration, and the like.
[0018] In alternative embodiments, the inventors contemplate that
the gradient may optionally comprise one or more additional
compositions to permit further, or enhanced purification of rAAV
particles. Such compositions may include derivatives of iodixanol,
iodixanol analogs, iodixanol-derived compounds, and/or compounds
having centrifugation properties similar to, equal to, or superior
to, iodixanol-alone compositions. Depending upon the particular
composition added to the gradient, the relative position of the
purified particles in the gradient may vary from that in which
iodixanol alone is used (i.e. approximately 52% iodixanol), but
such variance is readily overcome in the design of the gradient,
and does not preclude the isolation of the rAAV from the particular
density in the gradient where such virus particles are banded
following centrifugation. Likewise, when one or more compositions
are added to the iodixanol gradient, the centrifugation time,
centrifugal force, and/or banding position within the gradient of
the viral particles may be varied depending upon the particular
application. Any such variations, improvements, or alterations in
the composition of the iodixanol gradient are also contemplated to
fall within the scope of this invention, and such modifications to
the gradient will be apparent to those of skill in the art given
the benefit of the teachings of the instant specification.
[0019] In a second embodiment, the invention relates to a method
for purifying rAAV particles that comprises contacting a sample
containing the virus with at least a first matrix that comprises
heparin, under conditions effective and for a period of time
sufficient to permit binding of the virus to the matrix, removing
any unbound proteins or contaminants from the matrix, and then
subsequently collecting or eluting the virus from the matrix. In
exemplary embodiments, the matrix comprises heparin agarose type I
or heparin agarose type II-S, although the inventors contemplate
the use of any heparin composition or combinations thereof
demonstrated to be effective in binding the rAAV, and thus removing
it from a solution that is contacted with such a matrix.
Preferably, the matrix is an affinity chromotographic medium, that
may be comprised within a column, a syringe, a microfilter, or
microaffinity column, or alternatively may be comprised within an
HPLC affinity column. The matrix may be formed of any material
suitable for the preparation of a heparin affinity matrix, and may,
for example, be formulated as a resin, bead, agarose, acrylamide,
glass, fiberglass, plastic, polyester, methacrylate, cellulose,
sepharose, sephacryl, and/or the like. In fact, the inventors
contemplate that the matrix may be fashioned out of any suitable
material that forms a solid or semi solid support, and that permits
the adsorption, ionic bonding, covalent linking, crosslinking,
derivatization, or other attachment of a heparin moiety to the
support matrix. Indeed, the art of affinity chromatographic medium
preparation is sufficiently advanced so that a skilled artisan
could readily prepare a suitable heparin affinity medium for use in
purifying the rAAV particles using the methods disclosed herein.
For example, the inventors have shown that an HPLC affinity column
containing a crosslinked polyhydroxylated polymer derivatized with
one or more heparin functional groups was useful in the
purification of rAAV from a solution contacted with such a
column.
[0020] Elution of the bound virus to the affinity column may be
achieved in any manner convenient to the skilled practitioner, and
may include, for example, the use of one or more elution buffers
such as a salt buffer, to collect the virus from the column. In an
exemplary embodiment, the inventors utilized a 1 M NaCl solution to
elute the virus from the column. Prior to elution, the column
comprising the bound virus may be washed with one or more washing
or equilibrating buffers prior to elution of the virus from the
column.
[0021] The use of an affinity column to purify rAAV particles may
be used alone, or may be combined with the iodixanol gradient as
described above to further increase the purification of the rAAV
composition. One or more affinity columns may be utilized prior to
the density gradient centrifugation purification method, and/or one
or more affinity columns may be utilized after the purification
through iodixanol gradients. In an exemplary embodiment, a cellular
lysate containing rAAV particles is subjected to iodixanol
centrifugation, and the fraction of the gradient containing the
partially-purified rAAV is then contacted with at least one heparin
affinity column to increase the total purity of the rAAV
preparation.
[0022] Likewise, following either or both of the aforementioned
purification methods, the rAAV composition obtained may be
subjected to further purification, dialysis, concentration, and/or
the like. In an exemplary embodiment, the partially-purified rAAV
preparation may be further purified by contacting a fraction or
sample containing or comprising recombinant adeno-associated virus
with a hydrophobic matrix, under conditions effective to permit
interaction of hydrophobic species (proteins or other contaminants)
with the hydrophobic matrix, and collecting the non-interacting
virus from the hydrophobic matrix. Preferred are hydrophobic
matrices that comprise phenyl groups, for example phenyl sepharose,
phenyl sepharose 6 fast flow (low sub) or phenyl sepharose 6 (high
sub). In certain embodiments, rAAV that has been partially purified
by heparin affinity chromatography is further purified by
hydrophobic interaction chromatography.
[0023] In other embodiments, the partially-purified rAAV
preparation may be further purified by subjecting the viral sample
to one or more cesium chloride equilibrium density gradients, and
collecting from the gradient(s) the fraction(s) comprising the
purified virus. The virus may then optionally be further purified
by dialysis, microfiltration, microconcentration, and/or
precipitation. Additionally, the virus may be further purified by
contacting the virus with one or more ion exchange chromatography
media, and eluting the virus from the media using one or more
suitable elution buffers. Such an ion exchange chromatography
medium may comprise a cation or an anion exchange medium. An
exemplary cation exchange medium comprises at least one
negatively-charged sulfonic group.
[0024] Contaminants that may be present in the sample containing
the recombinant adeno-associated virus include, but are not limited
to, viruses, such as adenovirus or herpes simplex virus, proteins,
polypeptides, peptides, nucleic acids, cell extracts, growth
medium, or combinations thereof. The methods of the present
invention serve to reduce or eliminate one or more, or in certain
embodiments all of the contaminants in a given recombinant
adeno-associated virus sample. In preferred embodiments, the rAAV
is about 70%, about 80%, about 90%, about 95%, about 98%, about
99%, about 99.5% or more pure as judged by any of a variety of
assays and analytical techniques that are known to those of skill
in the art, including, but not limited to gel electrophoresis and
staining and/or spectroscopy.
[0025] In certain embodiments, the invention provides methods for
the preparation of highly-purified rAAV compositions comprising
greater than about 10.sup.10 rAAV particles/ml. In exemplary
embodiments, such methods have been demonstrated useful in the
preparation of viral compositions comprising greater than about
10.sup.11, 10.sup.12, and even greater than about 10.sup.13 or
10.sup.14 particles/ml. In other embodiments, the invention
provides methods for the preparation of rAAV compositions having a
particle-to-infectivity ratio of less than about 100, and in
certain aspects less than about 90, about 80, about 70, about 60,
about 50 about 40, about 30, about 20 about 10, about 5, or in
certain exemplary embodiments rAAV compositions having a
particle-to-infectivity ratio of about 1.
[0026] The process for preparing highly-purified and/or
highly-infectious viral preparations generally comprise the steps
of centrifuging a sample containing recombinant adeno-associated
virus through an iodixanol gradient, collecting from the iodixanol
gradient at least a first fraction comprising the recombinant
adeno-associated virus, contacting the at least a first fraction
comprising the recombinant adeno-associated virus with a matrix
comprising heparin, under conditions effective to permit binding of
the virus to the matrix, removing non-bound species from the
matrix, and eluting the virus from the matrix. Other methods for
isolating rAAV provided by the present invention comprise the steps
of centrifuging a sample containing or suspected of containing
recombinant adeno-associated virus through an iodixanol gradient,
collecting the purified virus from the gradient, contacting the
virus collected from the gradient with a matrix comprising heparin,
under conditions effective to permit binding of the virus to the
matrix, collecting the virus from the matrix, subjecting the virus
collected from the matrix to at least a first cesium chloride
equilibrium density gradient, and collecting from the gradient a
fraction comprising the highly-purified rAAV composition.
[0027] Additional methods of isolating a recombinant
adeno-associated virus are also provided in the present invention.
These methods generally comprises the steps of centrifuging a
sample containing recombinant adeno-associated virus through an
iodixanol gradient, collecting from the iodixanol gradient at least
a first fraction comprising the recombinant adeno-associated virus,
contacting the at least a first fraction comprising the recombinant
adeno-associated virus with a matrix comprising heparin, under
conditions effective to permit binding of the virus to the matrix,
removing at least a first non-bound species from the matrix,
eluting the virus from the matrix, contacting the eluted virus with
a hydrophobic matrix, under conditions effective to permit
interaction of hydrophobic species with the hydrophobic matrix, and
collecting the non-interacting virus from the hydrophobic
matrix.
[0028] Further methods generally comprise the steps of centrifuging
a sample suspected of containing recombinant adeno-associated virus
through an iodixanol gradient, collecting the purified virus from
the gradient, contacting the virus collected from the gradient with
a first matrix comprising heparin, under conditions effective to
permit binding of the virus to the matrix, collecting the virus
from the first matrix, contacting the virus collected from the
first matrix with a second matrix comprising an anion exchange
medium, and collecting from the second matrix a fraction comprising
the purified virus.
[0029] In another embodiment, the invention provides a method of
preparing recombinant adeno-associated virus. The method generally
involves subjecting a sample suspected of containing recombinant
adeno-associated virus to centrifugation through an iodixanol
gradient, and collecting the virus from a fraction of the gradient
corresponding to a concentration of iodixanol of about 40%. Such a
gradient may be formed as described above, and may be prepared
either as a continuous or a discontinuous gradient. In the case of
discontinuous gradients, the gradient will preferably include at
least an about 15% iodixanol step, an about 25% iodixanol step, an
about 40% iodixanol step, and an about 60% iodixanol step, with the
virus being isolatable from the 40% iodixanol step following
centrifugation. Following recovery of the banded rAAV particles,
the virus may be further purified using the heparin affinity
chromatographic methods disclosed herein, and/or be optionally
further purified via CsCl gradient centrifugation, anion exchange
chromatography, cation exchange chromatography, affinity
chromatography, or precipitation.
[0030] The invention also provides methods for reducing or
eliminating adenovirus from a recombinant adeno-associated virus
composition contaminated with adenovirus. The method generally
comprises centrifuging a sample containing or suspected of
containing both recombinant adeno-associated virus and adenovirus
through one or more iodixanol gradients as described herein, and
collecting the recombinant adeno-associated virus from the
gradient. The concentration of adenovirus may be further reduced in
such a sample by a number of methods, including, but not limited
to, further purification on a heparin affinity column and/or a
hydrophobic interaction column, by heating the sample, or
alternatively, by anion exchange chromatography as described
herein.
[0031] A method for reducing the concentration of adenovirus in a
recombinant adeno-associated virus composition is also provided
that generally involves centrifuging a sample containing
recombinant adeno-associated virus through an iodixanol gradient,
collecting from the iodixanol gradient at least a first fraction
comprising the recombinant adeno-associated virus, contacting the
at least a first fraction comprising the recombinant
adeno-associated virus with a matrix comprising heparin, under
conditions effective to permit binding of the virus to the matrix,
removing any non-bound species from the matrix, and eluting the
virus from the matrix.
[0032] A further aspect of the invention is the preparation of a
high-titer rAAV composition. The method generally comprises the
steps of: centrifuging a sample or rAAV through an iodixanol
gradient, collecting the purified recombinant adeno-associated
virus from the gradient; contacting the partially-purified
recombinant adeno-associated virus collected from the gradient with
a matrix comprising heparin, under conditions effective to permit
binding of the recombinant adeno-associated virus to the matrix,
and collecting the recombinant adeno-associated virus from the
matrix. The purified rAAV composition eluted from the matrix may
also be optionally further purified, such as in the case of the
preparation of high-titer viral stocks, by contacting the sample
with a matrix comprising an anion exchange medium, under conditions
effective to permit binding of the recombinant adeno-associated
virus to the matrix, and collecting the purified recombinant
adeno-associated virus from the matrix, preferably by elution.
[0033] The present invention thus also provides recombinant
adeno-associated virus compositions, prepared by any one or more of
the methods described herein. Generally, the invention provides at
least a first recombinant adeno-associated virus composition,
prepared by applying a sample containing recombinant
adeno-associated virus to an iodixanol gradient, and collecting
from the gradient at least a first fraction comprising the
recombinant adeno-associated virus.
[0034] Also provided by the present invention are kits comprising
combinations of the recombinant adeno-associated virus isolation
media described herein. Generally, the kits comprise, in a suitable
container, iodixanol and a matrix comprising heparin. In certain
preferred aspects, the iodixanol is formulated as an iodixanol
gradient. In other kits of the present invention, the matrix
comprises heparin agarose type I or heparin agarose type II-S.
Additional kits of the invention further comprise a hydrophobic
matrix, such as a matrix comprising phenyl groups, exemplified by
phenyl sepharose.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0036] FIG. 1. rAAV purification flow chart.
[0037] FIG. 2. Iodixanol step gradient for the purification of
rAAV. Shown is a plot of the refractive index (vertical axis) of
one ml-fractions (fraction number, horizontal axis) collected from
the bottom of a tube after a 1 hour spin.
[0038] FIG. 3A and FIG. 3B. HPLC purification of the iodixanol
fraction of rAAV-UF5, monitored at 231 nm. The absorbance at 231 nm
(A.sub.231) is shown on the left vertical axis, time (min) is shown
on the horizontal axis, and the ratio of diluent B (%B) is shown on
the right vertical axis. FIG. 3A. POROS.RTM. HE/NM chromatography.
FIG. 3B. UNO.TM. S1 cation exchange chromatography. The dotted line
indicates the shape of the gradient. Elution time is shown in min
above the respective peaks.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0039] Recently, it has been shown that the transduction of cells
by wt AAV was mediated through the heparan sulfate proteoglycan
receptor (Summerford and Samulski, 1998). In order to develop an
efficient and simple protocol for purification of rAAV, the
inventors developed heparin affinity column chromatography, which
significantly simplifies and expedites the production of rAAV. To
efficiently bind the virus to the affinity media the inventors have
also introduced a new pre-purification technique--centrifugation of
the crude viral lysate through a pre-formed gradient of the
non-ionic gradient media iodixanol. The present invention provides
for the first time protocols which permit the completion of rAAV
purification in one day and produces viral stocks sufficiently pure
for pre-clinical and/or clinical studies. The inventors have shown
that use of these new purification techniques permit an increase in
the yield of purified virus by at least 10-fold over conventional
methods, resulting in highly-purified, high-titer stocks
(10.sup.12-10.sup.13 particles/ml), equivalent to at least about
10.sup.4-10.sup.5 particles per cell, as well as improved viral
infectivity and more rapid purification.
5.0 EXAMPLES
[0040] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 EXAMPLE 1
Methods for Production of RAAV Compositions
[0041] 5.1.1 Materials and Methods
[0042] 5.1.1.1 Cells
[0043] Low passage number (P29-35) 293 cells were propagated in
DMEM/10% FBS. The C12 cell line (Clark et al., 1995) was maintained
in the presence of 0.5 mg/ml G418, while the Cre8 cell line (Hardy
et al., 1997) was propagated in DMEM supplemented with 200 .mu.g/ml
G418.
[0044] 5.1.1.2 Construction of Recombinant Plasmids
[0045] The construction of pTR-UF5 was described earlier (Klein,
1998). To produce the vector containing the enhanced blue
fluorescent mutant of green fluorescent protein (gfp; Heim and
Tsien, 1996), the inventors have introduced the Tyr-145-Phe
mutation into pTR-UFB background (Zolotukhin et al., 1996) using
Quick Change site-Directed Mutagenesis kit (Stratagene, La Jolla,
Calif.). The resulting plasmid was termed pTR-UF6. To construct the
rAd-UF7 vector, the inventors substituted the rAAV cassette from
pTR-UF5 for the CMV promoter fragment in pAdlox (Hardy et al.,
1997). The infectious rAd-UF7 was rescued essentially as described
by Hardy et al. (1997). QC-PCR.TM. standard template pdl-neo was
constructed as described earlier (Conway et al., 1997). The primers
used to detect rAAV were:
1 5'-TATGGGATCGGCCATTGAAC-3' (SEQ ID NO:1) and
5'-CCTGATGCTCTTCGTCCAGA-3'. (SEQ ID NO:2)
[0046] 5.1.1.3 Production of RAAV
[0047] To produce rAAV, a triple co-transfection procedure was used
to introduce a rAAV vector plasmid (pTR-UF5 or pTR-UF6) together
with pACG2 AAV helper (Li et al., 1997) and pXX6 Ad helper (Xiao et
al., 1998) at a 1:1:1 molar ratio. Alternatively, rAAV vector
plasmid was co-transfected with the helper plasmid pDG carrying the
AAV rep and cap genes, as well as Ad helper genes, required for
rAAV replication/packaging (Grimm et al., 1998). Plasmid DNA used
in the transfection was purified by conventional alkaline
lysis/CsCl gradient protocol.
[0048] The transfection was carried out as follows: 293 cells (P33)
were split 1:2 the day prior to the experiment, so that, when
transfected, the cell confluence was about 75-80%. Ten 15-cm plates
were transfected as one batch. To make CaPO.sub.4-precipitate 180
.mu.g of pACG2 were mixed with 180 .mu.g of pTR-UF5 and 540 .mu.g
of pXX6 in a total volume of 12.5 ml of 0.25 M CaCl.sub.2. The old
media was removed from the cells and the formation of the
CaPO.sub.4-precipitate was initiated by adding 12.5 ml of
2.times.HBS pH 7.05 (pre-warmed at 37.degree. C.) to the
DNA/CaCl.sub.2 solution. The DNA was incubated for 1 min, at which
time the formation of the precipitate was stopped by transferring
the mixture into pre-warmed 200 ml of DMEM-10% FBS. Twenty-two ml
of the media was immediately dispensed into each plate and cells
were incubated at 37.degree. C. for 48 h. The
CaPO.sub.4-precipitate was allowed to stay on the cells during the
whole incubation period without compromising cell visibility.
Forty-eight hours post-transfection cells were harvested by
centrifugation at 1,140.times.g for 10 min; the media was discarded
unless specified otherwise. Cells were then lysed in 15 ml of 0.15
M NaCl--50 mM Tris HCl pH 8.5 by 3 freeze/thaw cycles in dry
ice-ethanol and 37.degree. C. baths. Benzonase (Nycomed Pharma A/S,
pure grade) was added to the mixture (50 U/ml final concentration)
and the lysate was incubated for 30 min at 37.degree. C. The crude
lysate was clarified by centrifugation at 3,700.times.g for 20 min
and the virus-containing supernatant was further purified by
iodixanol density gradient centrifugation.
[0049] 5.1.1.4 Conventional Purification Protocol
[0050] rAAV was purified essentially as described earlier (Snyder
et al., 1996) with the following modifications. The virus pellet
after the second ammonium sulfate cut was resuspended in total of
39 ml of 1.37 g/ml CsCl/PBS and subjected to an 18 h spin in 60 Ti
rotor (Beckman Instruments, Somerset, N.J.) at 255,600.times.g at
15.degree. C. The gradient was fractionated from the bottom of the
tube and aliquots of the middle ten fractions were screened for
rAAV by PCR.TM.. Positive fractions were pooled, diluted to 13 ml
with the CsCl solution of the same density and centrifuged in an 80
Ti rotor (Beckman Instruments, Somerset, N.J.) at 391,600.times.g
for 3.5 h at 15.degree. C. After fractionation of the gradient, the
positive fractions were identified by PCR.TM. and pooled. The virus
then was concentrated/dialyzed using the ULTRAFREE-15 centrifugal
filter device BIOMAX-100K (Millipore, Bedford, Mass.).
[0051] 5.1.1.5 Preparation of Iodixanol Density Gradient
[0052] A typical discontinuous step gradient was formed by
underlayering and displacing the less dense cell lysate with
Iodixanol
5,5'[(2-hydroxy-1-3-propanediyl)-bis(acetylamino]bis[N,N'-bis[2,3dihydrox-
ypropyl-2,4,6-triiodo-1,3-benzenecarboxamide], prepared using the
60% (w/v) sterile solution of OptiPrep (Nycomed). Specifically, 15
ml of the clarified lysate were transferred into a Quick-Seal
Ultra-Clear 25.times.89 mm centrifuge tube (Beckman Instruments,
Somerset, N.J.) using a syringe equipped with a 1.27.times.89 mm
spinal needle. Care was taken to avoid bubbles, which would
interfere with subsequent filling and sealing of the tube. A
two-channel variable speed peristaltic pump, Model EP-1 (Bio-Rad
Laboratories, Hercules, Calif.), was equipped with PharMed 1.6 mm
ID tubing with two additional 15 cm pieces of silicon 1.6 mm ID
tubing attached at both sides of the pump head frame assembly. Each
tubing line was equipped at both sides with a 100 .mu.l
microcapillary borosilicate glass pipet (Fisher, Pittsburgh, Pa.).
Two pipets at one end of both channels were simultaneously placed
into 50 ml screw cap conical tubes (Sarstedt).
[0053] Eighteen ml of the solution (9 ml per one centrifuge tube)
containing 15% iodixanol-1 M NaCl-PBS-MK (1.times.PBS-1 mM
MgCl.sub.2, 2.5 mM KCl) were transferred into the tube and the pump
was started at 4 ml/min. Both channels were primed with the
iodixanol solution down to the tip of the glass pipet at the other
end of the line, at which time the pump was stopped and the two
pipets were inserted into two centrifuge tubes containing cell
lysate. The tips of the pipets were placed at the bottom of the
tubes and the pump was started to dispense the first density step.
Care was taken to introduce no air bubbles into the tubing, which
could disturb the density layers. With about a drop of the first
density step solution left in the tube the pump was stopped and 12
ml of the second density step (6 ml per one centrifuge tube)
containing 25% iodixanol-PBS-MK-Phenol Red (2.5 .mu.l of 0.5% stock
solution per ml of the iodixanol solution) were added to the same
50 ml tube. The dispensing of the second step was resumed as
described above, followed by the third step, consisting of 10 ml (5
ml per one centrifuge tube) of 40% iodixanol-PBS-MK, and, finally,
by 10 ml (5 ml per one centrifuge tube) of 60% iodixanol containing
Phenol Red (at the same concentration as the 25% step, 0.01
.mu.g/ml). The two microcapillary pipets then were carefully
withdrawn and the tubes were filled with PBS-MK buffer. Therefore,
each gradient consisted of (from the bottom up): 5 ml 60%, 5 ml
40%, 6 ml 25%, 9 ml of 15% iodixanol, the last density step
containing 1 M NaCl.
[0054] Tubes were sealed and centrifuged in a Type 70 Ti rotor
(Beckman Instruments, Somerset, N.J.) at 350,000.times.g for 1 h at
18.degree. C. The Phenol Red serves to distinguish the alternating
density steps. About 4 ml of the clear 40% step was aspirated after
puncturing the tube on the side with a syringe equipped with an 18
gauge needle with the bevel uppermost. A similar amount was removed
as 0.75 to 1 ml fractions upon harvest. The virus was further
purified as described below and shown in FIG. 1.
[0055] 5.1.1.6 Purification of RAAV Using CSCL Gradient
Centrifugation
[0056] The rAAV-containing iodixanol fraction was further purified
using a conventional CsCl gradient. To form the gradient 4.5 ml of
virus in iodixanol were mixed with 35 ml of CsCl (1.37 g/ml in
PBS), transferred into a Quick-Seal 25.times.89 mm centrifuge tubes
(Beckman Instruments, Somerset, N.J.) and centrifuged in a Type 60
rotor (Beckman Instruments, Somerset, N.J.) at 214,800.times.g
overnight at 18.degree. C. The gradient was processed as described
above.
[0057] 5.1.1.7 Purification of RAAV USING Heparin Affinity
Chromatography
[0058] The binding, washing and elution conditions were identical
for all Heparin-ligand affinity media used. Typically, a pre-packed
2.5 ml Heparin agarose Type I column (Sigma Chemical, St. Louis,
Mo.) was equilibrated with 20 ml of PBS-MK under gravity.
Alternatively, the columns were placed inside 15 ml screw cap
conical tubes (Sarstedt) and spun in a low speed centrifuge Type
J6-HC (Beckman Instruments, Somerset, N.J.) at 200 rpm for 5 min.
After each spin the flowthrough was discarded and fresh buffer was
added to repeat the washing three more times. The iodixanol
fraction containing virus was applied to the pre-equilibrated
column under gravity and the column was washed with 10 ml of the
PBS-MK buffer either under gravity or in the spin column mode. The
rAAV was eluted with the same buffer containing 1 M NaCl under
gravity. After applying the elution buffer, the first 2 ml of the
eluant were discarded, and the virus was collected in the
subsequent 3.5 ml of the elution buffer. Conventional Heparin
columns that were not prepacked were loaded and eluted in a similar
manner.
[0059] Alternatively, the Heparin agarose columns were placed into
screw-type valves of the Visiprep Solid Phase Extraction (SPE)
Vacuum Manifold (Supelco). The manifold valves were equipped with
disposable Teflon valve liner guides, designed to eliminate the
possibility of cross-contamination from one sample to the next in
the same manifold port. Each guide was placed into 15 ml screw cap
conical tube (Sarstedt) used as the collection vessel. This
arrangement ensures that all surfaces that come in contact with the
sample can be replaced following each chromatography.
Chromatography was performed with house vacuum attached to the
manifold's vacuum gauge, using less than 1 cm H.sub.2O (-1'Hg)
vacuum. Precise flow control through each column was provided by
rotating the independent, screw-type valves built into the cover.
Up to 12 samples could be purified simultaneously using the 12-Port
Model manifold.
[0060] For the ACTI-Disk 50 filter disk chromatography, the binding
of the virus in 40% iodixanol was performed in the upward fashion,
i.e., the flow of the solution was directed against gravity from
the bottom part of the filter assembly towards the top using a
peristaltic pump. Once applied, the filter assembly was turned up
side down and chromatography was resumed in a regular downward
fashion with gravity.
[0061] 5.1.1.8 Purification of RAAV Using HPLC Chromatography
[0062] System Gold (Beckman Instruments, Somerset, N.J.) hardware
installed inside a biosafety cabinet was used to further purify the
iodixanol fraction of virus. Only biocompatible
polyetheretherketone (PEEK) tubing and fittings were used to
process the samples. The chromatography was monitored at 231 nm.
The virus in 4 to 5 ml of iodixanol was directly loaded onto a
column using 5 ml injection loop. When the volume of the sample
exceeded 5 ml, multiple successive injections were performed, each
followed by washing with 5 ml (injection loop dwell volume) of
mobile phase. Two different columns were successfully used to
purify the virus.
[0063] 5.1.1.9 UNO.TM. S1 Cation-Exchange Chromatography
[0064] UNO.TM. S1 column (Bio-Rad Laboratories, Hercules, Calif.)
contained "Continuous Bed" support (bed volume 1.3 ml) derivatized
with strongly acidic negatively charged-SO.sub.3 sulfonic groups.
The column was pre-equilibrated with solvent A (PBS-MK buffer). The
virus sample was loaded at 0.5 ml/min and the column was washed
with solvent A until the iodixanol-induced absorption was reduced
to near background levels. A 0-1 M gradient of NaCl in PBS-MK was
applied over 36 min (15 column volumes) and the virus was eluted as
a double UV absorption peak, which was collected manually.
[0065] 5.1.1.10 POROS.RTM. HE Heparin Affinity Chromatography
[0066] POROS.RTM. HE/M heparin column (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.) contained particles coated with a
crosslinked polyhydroxylated polymer (bed volume 1.7 ml)
derivatized with heparin functional groups. The chromatography
conditions were essentially the same as described for the UNO.TM.
S1 column, except that a 0-0.5 M Na.sub.2SO.sub.4 in PBS-MK
gradient was applied (15 column volumes) at a flow rate of 1
ml/min. A single UV absorption peak of a virus was collected
manually.
[0067] 5.1.1.11 Phenyl Sepharose Hydrophobic Interaction
Chromatography
[0068] Phenyl Sepharose (Pharmacia Biotech) is a highly
cross-linked agarose (6%, spherical) that is substituted with
approximately 20 .mu.mol (low sub) or 40 .mu.mol (high sub) of
phenyl per ml of gel. The column is equilibrated with a high ionic
strength buffer (salt concentration just below that employed for
salting out proteins, for example 1.7 M (NH.sub.4).sub.2SO.sub.4)
at a flow rate of about 400 cm/h. The rAAV does not interact with
the Phenyl Sepharose, and is eluted in the void volume of the
column, while certain contaminating proteins interact with the
column and are thus retained.
[0069] 5.1.1.12 Concentration of RAAV
[0070] The virus was concentrated and desalted by centrifugation
through a BIOMAX 100 K filter (Millipore, Bedford, Mass.) according
to the manufacturer's instructions. The high salt buffer was
changed by repeatedly diluting concentrated virus with Lactated
Ringer's solution and repeating the centrifugation.
[0071] 5.1.1.13 Quantitative Competitive PCR.TM. (QC-PCR.TM.) Assay
for Determining RAAV Physical Particles
[0072] The purified viral stock was first treated with DNase I to
digest any contaminating unpackaged DNA. Ten .mu.l of a purified
virus stock was incubated with 10 U of DNase I (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.) in a 100 .mu.l reaction mixture,
containing 50 mM Tris HCl, pH 7.5, 10 mM MgCl.sub.2 for 1 h at
37.degree. C. At the end of the reaction, 10 .mu.l of 10.times.
Proteinase K buffer (10 mM Tris HCl, pH 8.0, 10 mM EDTA, 1% SDS
final concentration) was added, followed by the addition of 1 .mu.l
of Proteinase K (18.6 mg/ml, Boehringer Mannheim Biochemicals,
Indianapolis, Ind.). The mixture was incubated at 37.degree. C. for
one h. Viral DNA was purified by phenol/chloroform extraction
(twice), followed by chloroform extraction and ethanol
precipitation using 10 .mu.g of glycogen as a carrier. The DNA
pellet was resuspended in 100 .mu.l of H.sub.2O and dilutions were
made to use in the QC-PCR.TM. assay.
[0073] The PCR.TM. reaction mixtures each contained 1 .mu.l of the
diluted viral DNA and two-fold serial dilutions of the internal
standard plasmid DNA pdl-neo. The most reliable range of the
dilution standard DNA was found to be between 1 and 100 pg. An
aliquot of each reaction was then analyzed by 2% agarose gel
electrophoresis, until two. PCR.TM. products were resolved. The
analog image of the ethidium bromide (EtBr)-stained gel was
digitized using an ImageStore 7500 system (UVP). The densities of
the target and competitor bands in each lane were measured using
ZERO-Dscan Image Analysis System, version 1.0 (Scanalytics) and the
respective ratios were plotted as a function of the standard DNA
concentration. A ratio of 1, at which the number of viral DNA
molecules equals the number of standard competitor DNA was used to
derive the respective DNA concentration of the virus stock, which
was the value of the line at the X intercept.
[0074] 5.1.1.14 Infectious Center Assay to Determine RAAV Virus
Titer
[0075] A modification of the previously published protocol
(McLaughlin et al., 1988) was used to measure the ability of the
virus to infect C12 cells (Clark et al., 1995), unpackage, and
replicate. Briefly, C12 cells were plated in a 96-well dish at
about 75% confluence and infected with Ad5 at the multiplicity of
infection (M.O.I.) of 20. One .mu.l of serially diluted rAAV to be
titered was added to each well, whereupon cells were incubated for
42 h. Cells infected with rAAV-UF5 were visually scored using the
fluorescence microscope. To calculate the titer by hybridization,
cells were harvested and processed essentially as described earlier
(McLaughlin et al., 1988).
[0076] 5.1.1.15 Protein Concentration
[0077] The protein concentration in rAAV samples was determined
using the NanoOrange.TM. Protein Quantitation Kit (Molecular
Probes). The fluorescence in the sample was measured using the
Laboratory Fluorometer Model TD-700 (Turner Designs). To estimate
the purity of various virus fractions, virus was electrophoresed on
12% SDS acrylamide gels for 5 hours at 200 volts under standard
buffer conditions and visualized by silver staining.
[0078] 5.1.2 Results
[0079] The history of the rAAV as a gene delivery vector is not
without controversy. While some investigators in the field report
efficient rAAV-mediated transduction, others have found strong
dependence of the transduction upon Ad helper virus contaminants
(Ferrari et al., 1996), wt AAV contaminants (McLaughlin et al,
1988; Samulski et al., 1989) or mitotic or growth state of the
cells being transduced (Russel et al., 1994). A pseudotransduction
artifact has been also reported when using crude rAAV viral
preparations (Alexander et al., 1997).
[0080] Some of the variability in rAAV transduction in vivo is
undoubtedly due to the intrinsic properties of the target cells.
Some targets for example, do not have the high affinity heparin
proteoglycan receptor (Summerford and Samulski, 1998) and others
may be incapable of efficiently synthesizing the transcriptionally
active form of the rAAV genome (Ferrari et al., 1996; Fisher et
al., 1996). However, much of the variation is also due to the
methods used for purifying rAAV and the contaminants that are
present in the final preparation. In general, there has been a
correlation between the success of AAV vectors and the ability to
generate high-titer virus free of contaminants. Under optimal
conditions, as few as 10-40 infectious particles of rAAV have been
found to be sufficient to transduce one cell in vivo (Klein et al.,
1998; Peel et al., 1997; Lewin et al, 1998).
[0081] Recent advances in design of wt AAV and mini Ad helper
plasmids have made it possible to produce high-titer rAAV free of
Ad contamination. Although the current transient transfection
protocol for producing rAAV yields up to about 10.sup.4-10.sup.5
rAAV particles per cell in crude lysates, relatively little
attention has been paid to downstream purification. Most
laboratories continue to use sequential CsCl centrifugation. Not
only does it take several weeks to complete, it often results in
loss of up to 90% of virus. Furthermore, the final stock is often
contaminated with cell or serum proteins, which may compromise
subsequent interpretation of the data by triggering an in vivo
immune response. While the quality of such vector preparations may
be useful in some laboratory studies, and perhaps even some
additional pre-clinical applications, they are unsuitable for
clinical studies using rAAV that require highly-purified vector
stocks containing few if any contaminating substances.
[0082] 5.1.2.1 Production of RAAV
[0083] To produce rAAV, the inventors used the transient
Ca-phosphate-mediated co-transfection protocol, delivering three
plasmids (rAAV vector pTR-UF5 (Zolotukhin et al., 1996), wt AAV
helper pACG2 (Li et al., 1997) and Ad helper pXX6 (Xiao et al.,
1998)). Alternatively the helper plasmid pDG was used to provide
all genes required to propagate rAAV (Grimm et al., 1998). To
streamline the protocol the CaPO.sub.4/DNA precipitate was left in
the media for the whole incubation period of 48 h. This did not
compromise cell viability, but did increase the transfection
efficiency at least two-fold. The transfection efficiency routinely
reached 60% as judged by GFP fluorescence. After harvesting the
cells, virus was extracted by freezing and thawing the cells and
clarified by low speed centrifugation. The use of sonication,
microfluidizing, and detergent extraction (for example,
deoxycholate) did not appear to significantly increase the viral
yield.
[0084] 5.1.2.2 Iodixanol Density Step Gradient
[0085] Tamayose and co-authors have recently described a
Cellulofine sulfate chromatography protocol as a method of
purification and concentration of the rAAV from the crude lysate
(Tamayose et al., 1996). However, using this method the inventors
repeatedly failed to quantitatively bind rAAV in the crude lysate.
It appeared that rAAV and cell proteins could form aggregates in
lysate. These complexes fail to display uniform biochemical
properties, which makes it difficult to develop a purification
strategy. It also leads to poor recovery of the virus at all
purification stages. Finally, this nonspecific interaction results
in contamination with Ad proteins even after several rounds of CsCl
gradient centrifugation.
[0086] The bulk purification of the crude is, therefore, a very
important stage in rAAV purification. In the conventional protocol
it is usually done by stepwise NH.sub.4SO.sub.4 precipitation
(Snyder et al., 1996). Although this simple procedure could be used
to concentrate the virus, the NH.sub.4SO.sub.4 precipitation makes
a poor purification step. The residual ammonium sulfate salt in the
protein pellet also interferes with subsequent ion-exchange
chromatography procedure. The dialysis at this purification stage
leads to the aggregation and precipitation of proteins, resulted in
poor recovery of rAAV. The combination of NH.sub.4SO.sub.4
precipitation and hydrophobic interaction Phenyl-sepharose
chromatography was also employed, although this approach also
failed to produce a purified virus without sizeable loss of the
infectivity. To solve the problem, the inventors introduced a new
step into rAAV production protocol--iodixanol density gradient,
which efficiently pre-purifies the virus from the crude cell
extract.
[0087] Iodixanol is an iodinated density gradient media originally
produced as an X-ray contrast compound for injection into humans
and, as such, it has been subjected to rigorous screening and
clinical testing. It is non-toxic to cells; indeed; cells can be
grown in the presence of 30% iodixanol for 3 days with no
subsequent effect on the viability of cells. Unlike CsCl and
sucrose gradients commonly used for fractionating macromolecules,
iodixanol solutions can be made iso-osmotic at all densities. This
property makes iodixanol an ideal media for analysis and downstream
purification steps. Because of its non-ionic and inert nature,
electrophoretic analysis and virus infectivity assays can be
carried out on gradient fractions directly in the presence of
iodixanol. Since the viscosity of iodixanol solutions is also lower
than those of sucrose of the same density, it is also possible to
use the iodixanol fractions directly in subsequent chromatography
purification steps without dialysis or dilution.
[0088] As mentioned earlier, rAAV aggregates with proteins in cell
lysate, which changes its buoyant density and makes it distribute
along the whole length of the gradient. This confounded initial
attempts to purify rAAV using discontinuous iodixanol gradients.
The inventors, however, devised a preformed multiple density step
gradient that included 1 M NaCl in the first 15% step. The
inventors reasoned that high concentrations of salt would
destabilize ionic interactions between macromolecules, and reduce
aggregation of rAAV particles with cell lysate material. High salt
concentrations were excluded, however, from the rest of the
iodixanol gradient in order to permit the virus to band under
iso-osmotic conditions, which was important for subsequent
purification steps.
[0089] The banding density of the purified rAAV-UF5 was
approximately 1.415 g/ml, which corresponded to an about 52%
concentration of iodixanol. The inventors therefore incorporated a
40% iodixanol step (1.21 g/ml) as a cut-off target step to
accommodate rAAV/protein complexes trailing at slightly lower
densities, followed by a 60% step that acts as a cushion for any
rAAV containing a full length genome. To locate the 40% density
step after the centrifugation, the inventors stained the upper 25%
and lower 60% density steps with Phenol Red dye.
[0090] A plot of the refractive index at the end of a 1 hour run is
shown in FIG. 2. rAAV was distributed through the 40% density step
and could be recovered by inserting a syringe needle at about 2 mm
below the 60%-40% density junction. The bulk of the rAAV bands
within the 40% density step (fractions 5-8, FIG. 2). The heavy band
at the 40%-to-25% density interface consisted mostly of cellular
proteins and contained less than 5% of input rAAV, as judged by
FCA. A small amount of the rAAV also bands at the 40%-60% density
junction (fraction 5, FIG. 2). Approximately 75-80% of the rAAV in
the crude lysate is recovered in the iodixanol fraction (Table
I).
[0091] The nucleic acid/protein ratio in the rAAV-UF5 is different
from wt AAV because of the size of the DNA packaged: 3400 bases in
rAAV-UF5 vs. 4680 in wt AAV, or approximately 73% of the wt AAV
size. Using the same protocol with no modifications, the inventors
purified about 15 different rAAV vectors with the size of the
packaged genome ranging from 3 to 5 kb. Regardless of the size,
there was no substantial difference in the banding pattern of rAAV.
Therefore, no modification of the protocol, accounting for the size
of rAAV genome, is required.
[0092] To determine the resolving capacity of the iodixanol
gradient, the inventors loaded into separate tubes virus-containing
lysates obtained from 1.56.times.10.sup.8 cells,
3.12.times.10.sup.8 cells, or 4.68.times.10.sup.8 cells,
corresponding to 5, 10 or 15 large 15-cm culture plates,
respectively. rAAV was aspirated as described, and aliquots of each
sample that were equivalent to 1.73.times.10.sup.6 cells were
subjected to SDS-gel electrophoresis. The three viral capsid
proteins VP1, VP2, and VP3 constituted the major protein species at
all concentrations, even in the tube with the most concentrated
lysate. In further studies, however, the inventors routinely loaded
the lysate from 10 plates per gradient. In the scale-up protocol
the viral lysate from 3.1.times.10.sup.9 cell (one hundred 15-cm
plates) could be pre-purified in one Ti 70 rotor during single
one-hour run. Such run could potentially produce 10.sup.14 virus
particles, or about 10.sup.12 infectious particles.
[0093] It is also possible to concentrate and purify rAAV from the
media supernatant using the iodixanol gradient (FIG. 1). To do
this, the inventors precipitated the bulk of proteins and virus
from the media using conventional precipitation with 50% ammonium
sulfate. The pellet was further resuspended in PBS-MK buffer and
subjected to regular iodixanol gradient purification. This
procedure, however, is optional, since at the time of harvesting
cells 48 hours post-transfection the majority of the virus (about
90%) (Grimm et al., 1998; Xiao et al., 1998) is associated with
cell pellet.
[0094] Iodixanol proved to be an excellent bulk purification method
that accomplished at least three things. Crude lysate was purified
by at least 100 fold and when Ad helper was present, Ad
contamination was reduced by a factor of 100. The virus was
concentrated in a non-ionic and relatively non-viscous medium that
could be loaded on virtually any kind of chromatographic matrix.
Finally, iodixanol prevented rAAV aggregation and the associated
loss of virus that accompanies most other bulk purification and
column chromatography methods. Typically, 70-80% of the starting
infectious units are recovered following iodixanol gradient
fractionation (Table I), and unlike other purification methods,
this step was more reproducible.
[0095] 5.1.2.3 Methods for Separating Adenovirus from RAAV
[0096] The production of rAAV by transient co-transfection with a
mini Ad plasmid is an efficient but laborious protocol. Although it
eliminates the problem of removing Ad virus from the rAAV crude
lysate, it requires up to 1 mg of plasmid DNA (combined), for
transfection of 10 plates. Furthermore, it is not readily amenable
to the industrial large-scale production using suspension cell
culture. An ideal production system would consist of rAAV proviral
cell line, induced to rescue and replicate by infection with a
helper virus carrying the rep/cap functions, such as an HSV
amplicon (Conway et al., 1997), or rAd. For downstream purification
the HSV helper could be separated from rAAV by simple filtration
due to the considerable size difference (Conway et al., 1997) or by
exposure to high salt. In case of Ad, rAAV is usually separated by
a combination of CsCl gradient centrifugation and heat treatment,
both approaches suffering from drawbacks. The inventors were
interested in whether the newly introduced iodixanol gradient could
be combined with ion exchange chromatography columns (FIG. 1) to
separate rAAV and Ad without heat inactivation of the latter.
[0097] To address this issue, the inventors prepared pTR-UF6. This
construct is identical to pTR-UF5 except that the gfp cDNA contains
a Tyr-145-Phe mutation in the pTR-UFB background described
previously (Zolotukhin et al., 1996) and fluoresces blue. At the
time of co-transfection of 293 cells with pTR-UF6 and pDG, they
were also infected with rAd-UF7 at an M.O.I. of 10. rAd-UF7 is a
recombinant E1-E3 deleted Ad vector that contains the gfp/neo
cassette from pTR-UF5 and fluoresces green. The use of these two
constructs together permitted the monitoring of infections with
rAAV (pTR-UF6) and rAd (rAD-UF7) in the same GFP fluorescence assay
by scoring for blue or green cells. Cells infected with rAAV
fluoresce blue, while cells infected with rAd (or both viruses)
fluoresce green.
[0098] Cells transfected with pTR-UF6 and infected with rAD-UF7
were processed exactly as described for the purification of rAAV
using iodixanol gradient. The gradient was fractionated after
puncturing the bottom of the tube and 25 .mu.l aliquots from each
fraction were subjected to the SDS acrylamide gel electrophoresis
and Western analysis with polyclonal anti-Ad antibodies. More than
99% of the Ad, as judged by the fluorescence assay, banded in the
gradient with densities lower than 1.4 g/ml. rAAV, on the other
hand, banded in fractions 5-8 (FIG. 2; densities of 1.4 to 1.415
g/ml) and were clearly separated from the Ad. The crude lysate
contained 4.5.times.10.sup.10 pfu of rAd-UF7 (as determined by the
fluorescence cell assay). After the iodixanol gradient the titer of
the rAd-UF7 dropped to 4.2.times.10.sup.8 pfu. Although iodixanol
gradient efficiently separated rAAV/rAd mixture and reduced the
titer of rAd by two logs, further purification steps were studied
to further separate rAd.
[0099] To reduce Ad contamination further, column chromatography
was used as a second step in purification following the iodixanol
gradient. To compare the effectiveness of the various column
chromatography steps, rAAV-UF5 was prepared from 1.times.10.sup.9
cells as described above, using pDG helper plasmid. The crude
lysate was purified using the iodixanol step gradient and
virus-containing fractions were pooled. The pooled fractions were
then split into equal portions and virus was purified using four
different methods illustrated in FIG. 1: (1) CsCl density gradient
centrifugation, (2) heparin affinity chromatography, (3) HPLC
heparin affinity chromatography, and (4) HPLC cation exchange
chromatography. The purification steps were monitored by measuring
rAAV titers, both physical and infectious, as well as protein
concentration in virus samples generated by each purification step
(Table 1). For purposes of comparison, a second batch of virus was
purified by the commonly used method of ammonium sulfate
precipitation followed by two consecutive CsCl gradients (Table
1).
[0100] 5.1.2.4 Heparin Affinity Chromatography
[0101] Heparinized supports have been successfully used for the
purification of many heparin-binding macromolecules, including
viruses such as CMV (Neyts et al., 1992). Heparin is the
glucosaminoglycan moiety covalently bound to the protein core of
proteoglycans (PG). It is closely related to heparan sulfate (HS),
which constitutes the glycosaminoglycan (GAG) chain of the HS
proteoglycan (HSPG). The latter has been shown to be a cell surface
receptor mediating AAV infection (Summerford and Samulski, 1998).
Covalent binding of heparin molecules to the matrix through its
reducing end mimics the orientation of the naturally occurring GAGs
(Nadcarni et al., 1994). To take advantage of the structural
similarities between heparin and HS, heparin affinity
chromatography was utilized to further purify rAAV.
[0102] Heparin is a heterogeneous carbohydrate molecule composed of
long unbranched polysaccharides modified by sulfations and
acetylations. The degree of sulfation strongly correlates with the
virus-binding capacity of HS (Herold et al., 1995). It, therefore,
was anticipated that heparinized matrices from different vendors
would display different affinity towards rAAV. Thus, to develop the
method the inventors tested several heparin ligand-containing
media, including ACTI-Disk 50 (Arbor Technologies, Inc.), Affi-Gel
Heparin Gel (Bio-Rad Laboratories, Hercules, Calif.),
Heparin-Agarose Type I, Heparin-Agarose Type II-S and, finally,
Heparin Agarose Type III-S, the last three manufactured by Sigma
Chemical, St. Louis, Mo. Although ACTI-Disk 50 was found to bind
rAAV quantitatively, it was not used in the actual production
protocol, since the manufacturer discontinued this product.
Affi-Gel Heparin gel and Heparin Agarose Type III-S columns failed
to bind at least 50% of the virus and, therefore, were excluded
from further consideration. Heparin-Agarose Type I and
Heparin-Agarose Type II-S pre-packed 2.5 ml columns were efficient
in retaining and subsequently releasing rAAV. The Type II-S column,
however, was found to be less selective, binding many cell proteins
along with the virus. The Heparin-Agarose Type I was the best among
those tested in terms of binding specificity and virus recovery,
and was used in further studies as described below.
[0103] rAAV-UF5 purity at different stages of purification was
analyzed by silver stained SDS acrylamide gel electrophoresis. The
iodixanol-purified fraction prepared from cells transfected with
pTR-UF5/pDG was directly applied to a Heparin-agarose Type I column
and eluted with 1 M NaCl as described above. The 1 M NaCl fraction
contained 35% of the input rAAV (Table 1), which was more than 95%
pure, as judged by the silver stained SDS gel analysis. The
Heparin-agarose affinity fraction of rAAV was consistently more
pure than virus purified by the conventional protocol using
ammonium sulfate, followed by two rounds of CsCl gradient
centrifugation.
2TABLE 1 SUMMARY OF RAAV-UF5 TITERS AND PROTEIN CONCENTRATION AT
DIFFERENT STEPS OF THE PURIFICATION PROTOCOL.sup.a Particles by
Particles by Infectious Infectious Particle- Infect. Particle
Infectious dot blot, QC PCR .TM., particles by particles by
to-infect. Units per recovery, particles Purification step
10.sup.11 10.sup.11 ICA, 10.sup.9 FCA, 10.sup.9 ratio.sup.b
Cell.sup.c %.sup.d yield, %.sup.c 1 3 .times. Frz./thaw lys. 57 103
69 62.7 90.8 209 100 100 2 Iodixanol 44 82 32.3 51 86 170 76 81 3
Iodixanol/CsCl 5.7 2.5 4 3.6 158 12 8.4 6 4 Iodixanol/ 20 63 32 35
56 117 35 56 Heparin agarose 5 Iodixanol/HPLC 15 16 12 20 73 67 26
32 POROS .RTM. HE/M 6 Iodixanol/HPLC 19 13 20 20 95 67 33 32 UNO
.TM. S1 7 2 .times. CsCl 7 6 4.8 2.9 241 1 .sup.aThe yield of rAAV
and protein concentrations in each row are normalized to 3 .times.
10.sup.8 cells (ten 15 cm plates). .sup.bThe
particle-to-infectivity ratio was calculated using numbers obtained
by dot blot assay and FCA. .sup.cCalculated using FCA
.sup.dCalculated using dot blot assay
[0104] 5.1.2.5 Purification of RAAV Using HPLC Chromatography
[0105] Two different HPLC columns, UNO.TM. S1 and POROS.RTM. HE/M
heparin, were tested to further purify the iodixanol fraction of
rAAV (FIG. 3A and FIG. 3B). Both columns were successful in
removing most of the protein contaminants that remained in the
iodixanol fraction. The UNO.TM. S1 purification yielded rAAV-UF5
that was more than 99% pure as judged by SDS acrylamide
electrophoresis. Curiously two rAAV peaks were obtained during
UNO.TM. S1 fractionation (FIG. 3B). Both peaks were found to
contain rAAV that was indistinguishable both by SDS-gel
electrophoresis analysis and by GFP fluorescence assay.
[0106] Both HPLC columns used in the study produced rAAV,
comparable both in terms of purity and yield. POROS.RTM. HE/M
column produced a slightly more infectious virus, which is not
surprising, since the purification process involves binding to
heparin, structurally similar to native AAV receptor. From the
practical point of view, HPLC Heparin column is easier to use, it
allows for a higher back pressure and, therefore, higher flow
rates. It also cleared off iodixanol in the flowthrough much faster
(30 min vs. 45 min, FIG. 3A and FIG. 3B). Finally, it performed
consistently, producing essentially identical chromatograms for as
many as 10 different virus runs (the maximum tried). This kind of
performance is very important for GMP validation of a production
protocol.
[0107] Having established that both the UNO.TM. S1 and POROS.RTM.
HE/M columns could be used successfully to purify rAAV, the
inventors determined whether they also would separate adenovirus
from AAV in preparations grown in the presence of Ad virus. To this
end, the rAAV-UF6/rAd-UF7 mixture (described above) was purified by
iodixanol gradient centrifugation and then subjected to HPLC
POROS.RTM. HE/M affinity chromatography under the conditions
described above. The majority of the contaminating rAD-UF7 was
found in the flowthrough. The peak of rAAV-UF6 contained
8.times.10.sup.5 pfu of rAd, as compared to 3.times.10.sup.10
infectious units (IU) of rAAV-UF6 particles. Thus, the rAd titer in
the mixed stock was decreased from 4.5.times.10.sup.10 in the crude
lysate, to 4.2.times.10.sup.8 in the iodixanol fraction, to the
8.times.10.sup.5 after the HPLC affinity step. The same degree of
separation was achieved with conventional chromatography using
Heparin-agarose Type I. In contrast, UNO.TM. S1 cation exchange
chromatography failed to separate rAd and rAAV. Additional data
indicates that the mixture could be further separated using UNO.TM.
Q1 anion exchange HPLC column.
[0108] 5.1.2.6 Iodixanol Plus CSCL Density Gradient
[0109] The use of an iodixanol step gradient followed by a CsCl
gradient was compared with the conventional use of two consecutive
CsCl gradients (Table 1). The iodixanol plus CsCl protocol produced
rAAV with purity that was comparable to iodixanol followed by
column chromatography. Both methods produced rAAV that was
significantly purer than virus that had undergone only two
consecutive CsCl gradients. However the rAAV produced by
conventional CsCl purification generally had higher
particle-to-infectivity ratios (200-1000) than the methods
described herein (Table 1). Furthermore, rAAV that had undergone
even one CsCl centrifugation (Table 1, row 3) had a higher
particle-to-infectivity ratio than virus that had not been exposed
to CsCl (Table 1, rows 4-6). These observations suggest that
treatment with CsCl leads to reduced viral infectivity.
[0110] Taken together, the data show that a combination of
iodixanol plus heparin affinity chromatography (either heparin
agarose or heparin HPLC) has unique advantages as a method for
purifying rAAV. To compare this method directly with the current
method for rAAV purification, a crude rAAV virus stock was prepared
and the two methods of purification were compared side by side with
the same starting material, i.e., ammonium sulfate fractionation
followed by two CsCl gradients vs. iodixanol fractionation followed
by heparin agarose chromatography (Table 2). A significant increase
in recovery of vector was seen with the iodixanol/heparin protocol,
resulting from an approximately 5 fold higher recovery of vector
particles and over a 100 fold increase in infectivity. Expressed as
the ratio of infectious particles to total particles, the virus
prepared by CsCl centrifugation had a significantly higher ratio
than virus prepared by the iodixanol protocol, approximately 1700
vs 67 (Table 1). Furthermore, as expected, the virus prepared by
the conventional CsCl method was significantly less pure than that
prepared by iodixanol/heparin.
3TABLE 2 COMPARISON OF IODIXANOL/HEPARIN AGAROSE AND
NH.sub.4SO.sub.4/CsCL PURIFICATION Particles Infectious
Particle-to- by QC Units by Infectivity Purificati n PCR .TM.
10.sup.11 FCA 10.sup.9 Ratio NH.sub.4SO.sub.4/2 .times. 0.2 0.012
1667 CsCl Iodixanol/Heparin 1.0 1.5 67 Agarose
[0111] Following iodixanol gradient fractionation, rAAV was
sufficiently free of cellular protein such that it displayed
reproducible chromatographic behavior during subsequent
purification. Two types of columns have been identified that are
capable of purifying rAAV approximately 10-100 fold, heparin
sulfate and sulfate cation exchange resins. Both types of material
could be used successfully in the HPLC format and displayed
recoveries of 40-70% (Table 1). By contrast, CsCl purification of
the iodixanol fraction resulted in the recovery of as little as 7%
of the starting infectious units. Therefore, methods have been
identified that increase the yield of infectious rAAV by at least
ten-fold in this step.
[0112] Importantly, neither iodixanol fractionation nor column
chromatography on heparin or cation exchange resins had a
significant effect on the particle-to-infectivity ratio of rAAV. In
contrast, the use of CsCl gradients generally had the detrimental
effect of increasing the particle-to-infectivity ratio. If CsCl
were the only method used for purification, the increase could be
dramatic. The particle-to-infectivity ratios of rAAV that had been
purified by iodixanol and heparin affinity ranged from as low as 26
to 73 (Table 1). The particle-to-infectivity ratio of rAAV that had
been purified by iodixanol and CsCl was approximately 158 (Table
1). Finally, virus that had been purified only by ammonium sulfate
fractionation and sequential CsCl centrifugation had
particle-to-infectivity ratios of 241 to 1600 (Tables 1 and 2).
[0113] Thus, the inventors have identified methods for producing
pure, high titer rAAV that are significantly better in yield and
quality of material produced than the conventional methods
currently in use. One of these methods, an iodixanol step gradient
followed by a conventional heparin agarose column has consistently
resulted in overall recoveries of greater than 50% of the starting
material, and produces virus that is better than 99% pure, with
particle-to-infectivity ratios less than 100:1. Furthermore, the
method allows the purification of rAAV in one day.
[0114] 5.1.2.7 Iodixanol PLUS Heparin Affinity and Phenyl Sepharose
Chromatography
[0115] The use of hydrophobic interaction chromatography (HIC) in
the further purification of rAAV was investigated using Phenyl
Sepharose gel (Pharmacia Biotech). rAAV that was initially purified
on an iodixanol gradient and Heparin-Sepharose chromatography, as
described above, was loaded onto a Phenyl Sepharose column. The
rAAV does not interact with the Phenyl Sepharose, and is present in
the supernatant (bulk purification) or elutes in the void volume
(column purification). Several proteins present in the rAAV sample
from the iodixanol/heparin purification, in particular several
proteins between 45 and 60 kDa and large proteins or aggregates of
greater than about 116 kDa, interacted with the Phenyl Sepharose,
and were retained in the gel.
[0116] 5.1.2.8 Characterization of the Purified RAAV
[0117] 5.1.2.8.1 RAAV Titering
[0118] An important index of virus quality is the ratio of the
physical particles to the infectious particles in a given
preparation. To characterize the purification steps and the quality
of the virus obtained using different methods, the inventors used
two independent assays to titer both physical and infectious rAAV
particles. For physical particle titers, the inventors used a
conventional dot-blot assay and a QC PCR.TM. assay. For the
infectivity titer, the inventors used fluorescence cell assay
(FCA), which scored for the expression of GFP, and infectious
center assay (ICA). In order to avoid adventitious contamination of
rAAV stocks with wt AAV, the use of wt AAV was eliminated from all
protocols, including the ICA. For the ICA and FCA, the inventors
used the C12 cell line (Clark et al., 1995), which contains
integrated wt AAV rep and cap genes. Ad5, which was used to
co-infect C12 along with rAAV, was titered using the same C12 cell
line in a serial dilution cytopathic effect (CPE) assay. The amount
of Ad producing well-developed CPE in 48 h on C 12 cells was used
to provide helper function in both the ICA and FCA assays.
[0119] Both physical particle titers and infectious particle
titers, each obtained by two independent titering methods, were
generally in agreement, differing in most cases by a factor of 2 or
less (Table 1). The particle-to-infectivity ratios ranged from 56
to 240. rAAV purified by iodixanol/Heparin affinity chromatography
had the lowest (Table 1, Rows 4 and 5). rAAV purified exclusively
by using CsCl centrifugation had the lowest infectivity, which is
probably due to the deleterious effect of hyper-osmotic conditions
of a gradient (Table 1, compare crude lysate in Row 1 and
CsCl-purified virus, Rows 3 and 7). In extreme cases some
CsCl-grade rAAV preparations had the respective ratios of 1000 or
higher, while HPLC/heparin affinity purified stocks had ratios as
low as 26.
[0120] 5.1.2.8.2 RAAV Recovery
[0121] To compare the effectiveness of the column chromatography
steps in a single study, rAAV-UF5 has been prepared from fifty 15
cm plates as described, using the pDG helper plasmid. The crude
lysate was pre-purified using 5 tubes of iodixanol gradient and
virus-containing fractions were pooled. The pooled fractions were
then split and virus was purified using 5 different methods (FIG.
1). The inventors monitored the purification steps by measuring
rAAV titers, both physical and infectious, as well as protein
concentration in virus samples (Table 1). The total amount of the
virus in the crude lysate was assumed to represent a 100% of virus,
available for purification. The iodixanol gradient centrifugation
step reduces the amount of protein in the sample 1,577 fold.
Therefore, the degree of purification achieved at the first
purification step is 1,214 times, if one takes into account the
yield of viral particles.
[0122] 5.1.2.8.3 Comparison of Helper Plasmids
[0123] Recently three independent groups described the construction
of a new generation of helper plasmids, pXX6, (Xiao et al., 1998),
pACG2 (Li et al., 1997), pDG (Grimm et al., 1998) and
pAd.DELTA.(Salvetti, 1998), which modulate the synthesis of
Rep78/68 and supply Ad helper functions from non-infectious,
non-packagable mini-Ad plasmids. The inventors had the opportunity
to evaluate side-by-side two systems, namely pACG2/pXX6 vs. pDG. In
the studies both systems performed well, the pACG2/pXX6 yielding
about 10.sup.15 particles of rAAV per ml of the purified stock per
starting size run of ten 15 cm plates, with the "wild-type"
replication-competent AAV contamination at about 3 to 4 logs lower
than recombinant virus titer. pDG, on the other hand, produced
somewhat lower titers, 3-4.times.10.sup.12 particles/ml, with no
detectable "wt" AAV contamination, as judged by the ICA, done on
293 cells with Ad5 helper.
[0124] In conclusion, the developed protocol is very efficient,
routinely yielding 30-40% of the total virus in the original crude
lysate. The recovery of the virus in conventional CsCl protocol in
the studies never exceeded 10%. The infectivity of
iodixanol/heparin-purified virus is exceptional with the
particle-to infectivity ratios consistently lower than 1:100. On
the other hand, the respective ratio for the CsCl-purified virus
stays within 1:200-1000 range. The inventors, therefore developed
the method which increases the overall yield of the infectious rAAV
by at least ten-fold.
[0125] In short, the inventors have developed protocols for the
purification of rAAV that are versatile and efficient. rAAV,
purified by any of these approaches, is highly infectious and
practically free of contaminants. It is affordable for an average
research lab (iodixanol/Heparin-agarose protocol), or it could be
adopted for a GMP production facility (iodixanol/HPLC
chromatography protocol). The use of such techniques make broader
gene therapy applications of rAAV feasible.
[0126] 6.0 References
[0127] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by
reference.
[0128] Alexander, Russel, Miller, "Transfer of contaminants in
Adeno-associated virus vector stocks can mimic transduction and
lead to artifactual results," Hum. Gene Ther. 8:1911-1920,
1997.
[0129] Atchinson et al., Science 194:754-756, 1965.
[0130] Berns et al., In: Virus Persistence, Mehay et al. (Ed.),
Cambridge Univ. Press, pp. 249-265, 1982.
[0131] Berns, Pinkerton, Thomas, Hoggan, "Detection of
adeno-associated virus (AAV)-specific nucleotide sequences in DNA
isolated from latently infected Detroit 6 cells," Virol.
68:556-560, 1975.
[0132] Buller, Janik, Sebring, Rose, "Herpes simplex virus types 1
and 2 completely help adenovirus-associated virus replication, J.
Virol. 40(1):241-247, 1981.
[0133] Carter et al., In: The Parvoviruses, K. I. Berns (Ed.),
Plenum, N.Y., pp. 67-128, 1983.
[0134] Carter et al., In: The Parvoviruses, K. I. Berns (Ed.),
Plenum, N.Y., pp. 153-207, 1983.
[0135] Cheung, Hoggan, Hauswirth, Berns, "Integration of the
adeno-associated virus genome into cellular DNA in latently
infected human Detroit 6 cells," J. Virol. 33:739-748, 1980.
[0136] Chiorini et al., "High-efficiency transfer of the T cell
co-stimulatory molecule B7-2 to lymphoid cells using high-titer
recombinant adeno-associated virus vectors," Hum. Gene Ther.
6:1531-1541, 1995.
[0137] Clark, Sferra and Johnson, "Recombinant adeno-associated
viral vectors mediate long-term transgene expression in muscle,"
Hum. Gene Ther. 8:659-669, 1997.
[0138] Clark, Voulgaropoulou, Fraley, Johnson, "Cell lines for the
production of recombinant Adeno-associated virus," Hum. Gene Ther.
6:1329-1341. 1995.
[0139] Conway, Zolotukhin, Muzyczka, Hayward, Byrne, "Recombinant
Adeno-associated virus Type 2 replication and packaging is entirely
supported by a Hopes Simplex virus Type 1 amplicon expressing rep
and cap," J. Virol. 71:8780-8789, 1997.
[0140] Cukor et al., In: The Paroviruses, K. I. Berns (Ed.),
Plenum, N.Y., pp. 33-66, 1984.
[0141] Ferrari, Samulski, Shenk, Samulski, "Second-strand synthesis
is a rate-limiting step for efficient transduction by recombinant
adeno-associated virus vectors," J. Virol. 1996:3227-3234,
1996.
[0142] Fisher et al., "Transduction with recombinant
adeno-associated virus for gene therapy is limited by
leading-strand synthesis," J. Virol. 70:520-532, 1996.
[0143] Fisher et al., "Recombinant adeno-associated virus for
muscle directed gene therapy," Nat. Med. 3:306-312, 1997.
[0144] Flannery, "Efficient photoreceptor-targeted gene expression
in vivo by recombinant adeno-associated virus," Proc. Natl. Acad.
Sci. USA 94:6916-6921, 1997.
[0145] Flotte, "Stable in vivo expression of the cystic fibrosis
transmembrane conductance regulator with an adeno-associated virus
vector," Proc. Natl. Acad. Sci. USA 90:10613-10617, 1993.
[0146] Flotte et al., "A phase I study of an adeno-associated
virus-CFTR gene vector in adult CF patients with mild lung
disease," Hum. Gene Ther. 7:1145-1159, 1996.
[0147] Grimm, Kern, Rittner, Kleinschmidt, "Novel tools for
production and purification or recombinant AAV vectors," Hum. Gene
Ther. 9:2745-2760, 1998.
[0148] Handa, Shiroki, Shimojo, "Establishment and characterization
of KB cell lines latently infected with adeno-associated virus type
1," Virol. 82:84-92, 1977.
[0149] Hardy, Kitamura, Harris-Stansil, Dai, Phipps, "Construction
of adenovirus vectors through Cre-lox recombination," J. Virol.
71:1842-1849, 1997.
[0150] Heim and Tsien, "Engineering green fluorescent protein for
improved brightness, longer wavelength and fluorescence resonance
energy transfer," Curr. Biol. 6:178-182, 1996.
[0151] Hermonat and Muzyczka, "Use of adeno-associated virus as a
mammalian DNA cloning vector: transduction of neomycin resistance
into mammalian tissue culture cells," Proc. Natl. Acad. Sci. USA
81:6466-6470, 1984.
[0152] Hermonat, Labow, Wright, Berns, Muzyczka, "Genetics of
adeno-associated virus: isolation and preliminary characterization
of adeno-associated virus type 2 mutants," J. Virol. 51:329-339,
1984.
[0153] Herold, Gerber, Polonsky, Belval, Shaklee, Holme,
"Identification of structural features of heparin required for
inhibition of Herpes Simplex virus Type 1 binding," Virol.
206:1108-1116, 1995.
[0154] Hoggan et al., In: Proceeding of the Fourth Lepetit
Colloquium, Cacoyac, Mexico, North Holland, Amsterdam, pp. 243-249,
1972.
[0155] Hoggan, Fed. Proc. 24:248, 1965.
[0156] Inoue and Russell, "Packaging cells based on inducible gene
amplification for the production of adeno-associated virus
vectors," J. Virol. 72:7024-7031, 1998.
[0157] Jooss, Yang, Fisher, Wilson, "Transduction of dendritic
cells by DNA viral vectors directs the immune response to transgene
products in muscle fibers," J. Virol. 727:4212-4223, 1998.
[0158] Kaplitt, "Long-term gene expression and phenotypic
correction using adeno-associated virus vectors in the mammalian
brain," Nat. Genet. 8:148-154, 1994.
[0159] Kessler, "Gene delivery to skeletal muscle results in
sustained expression and systemic delivery of a therapeutic
protein," Proc. Natl. Acad. Sci. USA 93:14082-14087, 1996.
[0160] Klein, "Neuron-specific transduction in the rat
septohippocampal or nigrostriatal pathway by recombinant
adeno-associated virus vectors," Exper. Neurol. 150:183-194,
1998.
[0161] Laughlin, Tratschin, Coon, Carter, "Cloning of infectious
adeno-associated virus genomes in bacterial plasmids," Gene
23:65-73, 1983.
[0162] Lewin et al., "Ribozyme rescue of photoreceptor cells in a
transgenic rat model of autosomal dominant retinitis pigmentosa,"
Nat. Med. 4:967-971, 1998.
[0163] Li, Samulski, Xiao, "Role for highly regulated rep gene
expression in adeno-associated virus vector production, J. Virol.
71:5236-5243, 1997.
[0164] Lusby and Berns, "Mapping of the 5' termini of two
adeno-associated virus 2 RNAs in the left half of the genome," J.
Virol. 41:518-526, 1982.
[0165] McLaughlin, Collis, Hermonat, Muzyczka, "Adeno-associated
virus general transduction vectors: analysis of proviral
structures," J. Virol. 62:1963-1973, 1988.
[0166] Nadcarni, Pervin, Linhardt, "Directional immobilization of
heparin to beaded supports," Anal. Biochem. 222:59-67, 1994.
[0167] Neyts, Snoeck, Schols, Balzarini, Esko, Van Schepdael,
DeClercq, "Sulfated polymers inhibit the interaction of human
cytomegalovirus with cell surface heparan sulfate," Virology
189:48-58, 1992.
[0168] Parks, Melnick, Rongey, Mayor, "Physical assay and growth
cycle studies of a defective adeno-satellite virus," J. Virol.
1:171-180, 1967.
[0169] Peel, "Efficient transduction of green fluorescent protein
in spinal cord neurons using adeno-associated virus vectors
containing cell type-specific promoters," Gene Ther. 4:16-24,
1997.
[0170] Rose and Koczot, "Adenovirus-associated virus
multiplication. VII. Helper requirement for viral deoxyribonucleic
acid and ribonucleic acid synthesis," J. Virol. 10:1-8, 1972.
[0171] Rose, Berns, Hoggan, Koczot, "Evidence for a single-stranded
adenovirus-associated virus genome: formation of a DNA density
hybrid on release of viral DNA," Proc. Natl. Acad. Sci. USA
64:863-869, 1969.
[0172] Russel, Miller, Alexander, Adeno-associated virus vectors
preferentially transduce cells in S phase," Proc. Natl. Acad. Sci.
USA 91:8915-8919, 1994.
[0173] Salvetti, "Factors influencing recombinant adeno-associated
virus production," Hum. Gene Ther. 9:695-706, 1998.
[0174] Samulski, Berns, Tan, Muzyczka, "Cloning of adeno-associated
virus into pBR322: rescue of intact virus from the recombinant
plasmid in human cells," Proc. Natl. Acad Sci. USA 79:2077-2080,
1982.
[0175] Samulski, Chang, Shenk, "Helper-free stocks of recombinant
adeno-associated viruses: normal integration does not require viral
gene expression," J. Virol. 63:3822-3828, 1989.
[0176] Samulski, Srivastava, Berns, Muzyczka, "Rescue of
adeno-associated virus from recombinant plasmids: gene correction
within the terminal repeats of AAV," Cell 33:135-143, 1983.
[0177] Snyder, "Persistent and therapeutic concentrations of human
factor IX in mice after hepatic gene transfer of recombinant AAV
vectors," Nat. Genet. 16:270-276, 1997.
[0178] Snyder, Xiao, Samulski, "Production of recombinant
adeno-associated viral vectors," In: Current Protocols in Human
Genetics (eds. Dracopoli et al.), John Wiley, New York, 1996.
[0179] Srivastava, Lusby, Berns, "Nucleotide sequence and
organization of the adeno-associated virus 2 genome," J. Virol.
45:555-564, 1983.
[0180] Summerford and Samulski, "Membrane-associated heparan
sulfate proteoglycan is a receptor for adeno-associated virus type
2 virions," J. Virol. 72:1438-1445, 1998.
[0181] Tamayose, Hirai, Shimada, "TA new strategy for large-scale
preparation of high-titer recombinant adeno-associated virus by
using packaging cell lines and sulfonated cellulose column
chromatography," Hum. Gene Ther. 7:507-513, 1996.
[0182] Tratschin, Miller, Carter, "Genetic analysis of
adeno-associated virus: properties of deletion mutants constructed
in vitro and evidence for an adeno-associated virus replication
function," J. Virol. 51:611-619, 1984a.
[0183] Tratschin, West, Sandbank, Carter, "A human parvovirus,
adeno-associated virus, as a eucaryotic vector: transient
expression and encapsidation of the procaryotic gene for
chloramphenicol acetyltransferase," Mol. Cell. Biol. 4:2072-2081,
1984b.
[0184] Wagner et al., "Efficient and persistent gene transfer of
AAV-CFTR in maxillary sinus," Lancet 351:1702-1703, 1998.
[0185] Xiao, Li and Samulski, "Efficient long-term gene transfer
into muscle tissue of immunocompetent mice by adeno-associated
virus vector," J. Virol. 70:8090-8108, 1996.
[0186] Xiao, Li, Samulski, "Production of high-titer recombinant
adeno-associated virus vectors in the absence of helper
Adenovirus," J. Virol. 72:2224-2232, 1998.
[0187] Zolotukhin, Potter, Hauswirth, Guy, Muzyczka, "A humanized
green fluorescent protein cDNA adapted for high level expression in
mammalian cells," J. Virol. 70:4646-4654, 1996.
[0188] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
Sequence CWU 1
1
2 1 20 DNA Artificial Sequence Description of Artificial
SequenceSYNTHETIC 1 tatgggatcg gccattgaac 20 2 20 DNA Artificial
Sequence Description of Artificial SequenceSYNTHETIC 2 cctgatgctc
ttcgtccaga 20
* * * * *