U.S. patent application number 14/505645 was filed with the patent office on 2015-08-27 for excipients for use in adeno-associated virus pharmaceutical formulations, and pharmaceutical formulations made therewith.
This patent application is currently assigned to Genzyme Corporation. The applicant listed for this patent is Genzyme Corporation. Invention is credited to Yero J. ESPINOZA, Hema S. SISTA.
Application Number | 20150238610 14/505645 |
Document ID | / |
Family ID | 26808304 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150238610 |
Kind Code |
A1 |
SISTA; Hema S. ; et
al. |
August 27, 2015 |
EXCIPIENTS FOR USE IN ADENO-ASSOCIATED VIRUS PHARMACEUTICAL
FORMULATIONS, AND PHARMACEUTICAL FORMULATIONS MADE THEREWITH
Abstract
Stable pharmaceutical compositions comprising recombinant
adeno-associated virus (AAV) virions are described. The
compositions provide protection against loss of recombinant AAV
vector genomes and transduceability under conditions such as
exposure to cycles of freezing and thawing and storage in glass or
polypropylene vials. The compositions comprise recombinant AAV
virions in combination with one or more dihydric or polyhydric
alcohols, and, optionally, a detergent, such as a sorbitan ester.
Also described are methods of using the compositions.
Inventors: |
SISTA; Hema S.; (Cupertino,
CA) ; ESPINOZA; Yero J.; (Alameda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Framingham |
MA |
US |
|
|
Assignee: |
Genzyme Corporation
Framingham
MA
|
Family ID: |
26808304 |
Appl. No.: |
14/505645 |
Filed: |
October 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13506638 |
May 4, 2012 |
8852607 |
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14505645 |
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|
12583679 |
Aug 24, 2009 |
8192975 |
|
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13506638 |
|
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|
10862036 |
Jun 4, 2004 |
7598070 |
|
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12583679 |
|
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|
10340389 |
Jan 10, 2003 |
6764845 |
|
|
10862036 |
|
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09453317 |
Dec 2, 1999 |
6759050 |
|
|
10340389 |
|
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60110689 |
Dec 3, 1998 |
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Current U.S.
Class: |
514/44R ;
435/235.1 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 15/86 20130101; A61K 48/0091 20130101; A61K 47/10 20130101;
A61K 47/26 20130101; C12N 2750/14143 20130101; A61K 38/4846
20130101; A61K 48/0008 20130101; A61K 9/0019 20130101; C12N
2750/14151 20130101 |
International
Class: |
A61K 47/26 20060101
A61K047/26; A61K 47/10 20060101 A61K047/10; A61K 48/00 20060101
A61K048/00 |
Claims
1. A pharmaceutical composition comprising recombinant
adeno-associated virus (AAV) virions and at least one dihydric or
polyhydric alcohol.
2. The pharmaceutical composition of claim 1, wherein the dihydric
or polyhydric alcohol is one or more alcohols selected from the
group consisting of polyethylene glycol, propylene glycol and
sorbitol.
3. The pharmaceutical composition of claim 2, wherein the one or
more alcohols is sorbitol and the sorbitol is present at a
concentration of about 0.1 wt. % to about 10 wt. %.
4. The pharmaceutical composition of claim 3, wherein sorbitol is
present at a concentration of about 1 wt. % to about 5 wt. %.
5. The pharmaceutical composition of claim 2, wherein the one or
more alcohols is polyethylene glycol and the polyethylene glycol is
present at a concentration of about 2 wt. % to about 40 wt. %.
6. The pharmaceutical composition of claim 5, wherein polyethylene
glycol is present at a concentration of about 10 wt. % to about 25
wt. %.
7. The pharmaceutical composition of claim 2, wherein the one or
more alcohols is propylene glycol and the propylene glycol is
present at a concentration of about 2 wt. % to about 60 wt. %.
8. The pharmaceutical composition of claim 7, wherein propylene
glycol is present at a concentration of about 5 wt. % to about 30
wt. %.
9. The pharmaceutical composition of claim 1, further comprising a
detergent.
10. The pharmaceutical composition of claim 9, wherein the
detergent is a nonionic detergent.
11. The pharmaceutical composition of claim 10, wherein the
detergent is a sorbitan ester.
12. The pharmaceutical composition of claim 11, wherein the
detergent is selected from the group consisting of
polyoxyethylenesorbitan monolaurate (TWEEN-20),
polyoxyethylenesorbitan monopalmitate (TWEEN-40),
polyoxyethylenesorbitan monostearate (TWEEN-60),
polyoxyethylenesorbitan tristearate (TWEEN-65),
polyoxyethylenesorbitan monooleate (TWEEN-80) and
polyoxyethylenesorbitan trioleate (TWEEN-85).
13. The pharmaceutical composition of claim 12, wherein the
detergent is polyoxyethylenesorbitan monolaurate (TWEEN-20) present
at a concentration of about 0.05 wt. % to about 5 wt. %.
14. The pharmaceutical composition of claim 12, wherein the
detergent is polyoxyethylenesorbitan monooleate (TWEEN-80), present
at a concentration of about 0.05 wt. % to about 5 wt. %.
15. A pharmaceutical composition comprising recombinant AAV virions
present in an amount sufficient to provide a therapeutic effect
when given in one or more doses, sorbitol present at a
concentration of about 1 wt. % to about 5 wt. % and a detergent
present at a concentration of about 0.1 wt. % to about 1 wt. %,
wherein the detergent is polyoxyethylenesorbitan monolaurate
(TWEEN-20) or polyoxyethylenesorbitan monooleate (TWEEN-80).
16-25. (canceled)
26. A method for protecting a recombinant AAV virion from loss of
activity resulting from storage of the virion in a glass vessel
comprising admixing the virion with a virion-stabilizing
composition comprising a dihydric or polyhydric alcohol.
27. The method of claim 26, wherein the dihydric or polyhydric
alcohol is one or more alcohols selected from the group consisting
of polyethylene glycol, propylene glycol and sorbitol.
28. The method of claim 27, wherein the one or more alcohols is
sorbitol.
29. The method of claim 27, wherein the one or more alcohols is
polyethylene glycol.
30. The method of claim 27, wherein the one or more alcohols is
propylene glycol.
31. The method of claim 26, wherein the virion-stabilizing
composition further comprises a detergent.
32. The method of claim 31, wherein the detergent is a sorbitan
ester.
33. The method of claim 32, wherein the sorbitan ester is selected
from the group consisting of polyoxyethylenesorbitan monolaurate
(TWEEN-20), polyoxyethylenesorbitan monopalmitate (TWEEN-40),
polyoxyethylenesorbitan monostearate (TWEEN-60),
polyoxyethylenesorbitan tristearate (TWEEN-65),
polyoxyethylenesorbitan monooleate (TWEEN-80) and
polyoxyethylenesorbitan trioleate (TWEEN-85).
34. The method of claim 26, wherein the recombinant AAV virion is
provided as a lyophilized preparation.
35. A method for protecting a recombinant AAV virion from loss of
activity resulting from storage of the virion in a glass vessel
comprising admixing the virion with a virion-stabilizing
composition comprising sorbitol and a sorbitan ester selected from
the group consisting of polyoxyethylenesorbitan monolaurate
(TWEEN-20) and polyoxyethylenesorbitan monooleate (TWEEN-80).
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. Ser. No.
13/506,638, filed May 4, 2012, now U.S. Pat. No. 8,852,607, which
is a continuation of U.S. Ser. No. 12/583,679, filed Aug. 24, 2009,
now U.S. Pat. No. 8,192,975, which is a continuation of U.S. Ser.
No. 10/862,036, filed Jun. 4, 2004, now U.S. Pat. No. 7,598,070,
which is a continuation of U.S. Ser. No. 10/340,389, filed Jan. 10,
2003, now U.S. Pat. No. 6,764,845, which is a divisional
application of U.S. Ser. No. 09/453,317, filed Dec. 2, 1999, now
U.S. Pat. No. 6,759,050, from which applications priority is
claimed pursuant to 35 U.S.C. .sctn.120. U.S. Ser. No. 09/453,317
claims the benefit under 35 U.S.C. .sctn.119(e)(1) to provisional
patent application Ser. No. 60/110,689, filed Dec. 3, 1998. The
above applications are incorporated herein by reference in their
entireties.
TECHNICAL FIELD
[0002] The present invention relates generally to DNA delivery
methods. More particularly, the invention relates to stable
pharmaceutical formulations comprising recombinant adeno-associated
virus (rAAV) virions that provide protection against loss of
transduceability due to manipulation, storage, transport, and the
like, of the formulation.
BACKGROUND
[0003] The commercialization of any chemical compound for use as a
pharmaceutical agent requires careful consideration of the
formulation in which the chemical compound will be prepared,
packaged and stored. The formulation must, of course, be compatible
with human and/or veterinary administration. The formulation must
be such that the agent retains potency for an extended period of
time. Indeed, the formulation itself must be stable over a long
period of time. The formulation must be compatible with techniques
used for its purification, as well as for the purification of the
agent contained within the formulation. Ultimately, the formulation
must be compatible with the material in which the agent will be
stored. If the agent must be frozen for stability, it is preferable
that the formulation provide some protection against inactivation
or denaturation due to freeze-thaw. In addition, the formulation
should provide a suitable milieu for various dilutions of the
agent.
[0004] Typically, pharmaceutical agents are stored as lyophilized
formulations in a sterile container. A pharmaceutical agent
formulation may be lyophilized if it is stable in such a nonaqueous
state. This is of particular importance if the formulation must be
stored frozen, as lyophilization minimizes the deleterious sequelae
that may occur when an aqueous preparation is frozen and
subsequently thawed. A glass vial is typically used because of the
compatibility of glass with presently used sterilization
techniques.
[0005] Adeno-associated virus (AAV) is a virus that readily
transduces many human tissue and cell types. Accordingly, AAV has
been used for gene therapy and nucleic acid immunization. The use
of AAV in these contexts requires consideration of the above
pharmaceutical formulation requirements. For example, it would be
preferred that an AAV-containing sample not be lyophilized because
of the possibility that small amounts of virus could become
aerosolized and inadvertently transduce an unintended host.
However, because AAV is known to be stable under a variety of
conditions that would inactivate most viruses, particularly
enveloped viruses, it was not previously believed that the
preparation of AAV formulations would be problematic.
[0006] It was unexpected, therefore, to find that the activity of
recombinant AAV (AAV) virions dropped significantly depending on
the formulation used for storage and the conditions to which the
formulation was exposed. It has been found, for example, that the
transduction activity of a rAAV formulation may depend on the
nature of the container, the constituents of the formulation, the
temperature of the formulation, as well as changes in temperature,
and the concentration of the rAAV virions stored.
[0007] It would, therefore, be a significant advancement in the art
to provide formulations for storing rAAV virions which would
preserve the activity of the rAAV virions for extended periods of
times in containers made of various materials, including glass.
DISCLOSURE OF THE INVENTION
[0008] The present invention is based on the discovery that Various
excipient compositions have a stabilizing effect on recombinant AAV
virions, such that less rAAV vector genomes are lost and higher
transduceability levels are achieved as compared with AAV
compositions that lack the excipients described herein. Various
forms of the different embodiments described herein can be
combined.
[0009] In one embodiment, then, a pharmaceutical composition
comprising rAAV virions is provided. The composition provides
protection against loss of rAAV vector genomes and transduceability
under conditions such as exposure to cycles of freezing and thawing
and storage in glass or polypropylene vials. The composition
comprises a dihydric or polyhydric alcohol, such as one or more of
sorbitol, polyethylene glycol, propylene glycol, and, optionally, a
detergent, such as a sorbitan ester.
[0010] In an additional embodiment, the pharmaceutical composition
comprises rAAV virions in an amount sufficient to provide a
therapeutic effect when given in one or more doses and sorbitol
present at a concentration of about 1 wt. % to about 5 wt. % and a
detergent present at a concentration of about 0.1 wt. % to about 1
wt. %, wherein the detergent is polyoxyethylenesorbitan monolaurate
(TWEEN-20) or polyoxyethylenesorbitan monooleate (TWEEN-80).
[0011] In yet other embodiments, a method for protecting a
recombinant AAV virion from loss of activity resulting from
exposure of the vision to a cycle of freezing and thawing, is
provided, as is a method for protecting a recombinant AAV virion
from loss of activity resulting from storage of the virion in a
glass vessel. The methods comprise admixing the virion with a
virion-stabilizing composition comprising a dihydric or polyhydric
alcohol. In particular embodiments, the alcohol is one or more
alcohols selected from the group consisting of polyethylene glycol,
propylene glycol and sorbitol. The compositions used in the methods
optionally include a detergent, such as a sorbitan ester.
[0012] In particular embodiments, the compositions used in the
methods comprise sorbitol and a sorbitan ester selected from the
group consisting of polyoxyethylenesorbitan monolaurate (TWEEN-20)
and polyoxyethylenesorbitan monooleate (TWEEN-80).
[0013] These and other embodiments of the subject invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0014] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0015] FIG. 1 is a bar graph illustrating the effect of
temperature, vector dilution and storage in a glass vial on
recombinant AAV vector (rAAV-hFIX) transduceability, as described
in the examples. The numbers on the x axis represent the storage
temperature; ppvial represents samples stored in a polypropylene
vial and gl vial represents samples stored in glass.
[0016] FIG. 2 depicts the results of experiments conducted as
described in Example 3 in which 293-HEK cells were transduced with
1.times.10.sup.8 vector genomes using samples stored in
polypropylene or glass, in 1% or 5% sorbitol, at various dilutions
as specified. The bars represent ng/ml of rAAV-hFIX and the line
graph above the bars represents data normalized for dilution.
[0017] FIG. 3 depicts the results of experiments conducted as
described in Example 3 in which 293-HEK cells were transduced with
5.times.10.sup.8 vector genomes using samples stored in
polypropylene or glass, in 1% or 5% sorbitol, at various dilutions
as specified. The bars represent ng/ml of rAAV-hFIX and the line
graph above the bars represents data normalized for dilution.
[0018] FIG. 4 depicts the results of experiments conducted at
-80.degree. C. or ambient (room) temperature for vector genome
count and transduceability assay in which transduction was done
using 1.times.10.sup.8 vector genomes as described in Example 3.
Maroon bars represent results of experiments done at room
temperature and light blue bars represent experiments done at
-80.degree. C.
[0019] FIG. 5 depicts the results of experiments conducted at
-80.degree. C. or ambient (room) temperature for vector genome
count and transduceability assay in which transduction was done
using 5.times.10.sup.8 vector genomes as described in Example 3.
Maroon bars represent results of experiments done at room
temperature and light blue bars represent experiments done at
-80.degree. C.
[0020] FIG. 6 depicts the results obtained in the experiment
described in Example 4 using the parameters given in Table 4. Green
bars represent experiments conducted using 1.times.10.sup.8 vector
genomes; red bars represent experiments conducted using
5.times.10.sup.8 vector genomes; pink bars represent experiments
conducted using 1.times.10.sup.9 vector genomes; dark blue bars
represent experiments conducted using 5.times.10.sup.9 vector
genomes.
[0021] FIG. 7 depicts the results obtained in the experiment
described in Example 4 using the parameters given in Table 4 and
regraphed as vector genomes. Dark blue bars represent experiments
conducted using media as the diluent; pink bars represent
experiments conducted using 0.1% TWEEN-20 as the diluent; yellow
bars represent experiments conducted using 0.2% TWEEN-20 as the
diluent; red bars represent experiments conducted using 0.5%
TWEEN-20 as the diluent; green bars represent experiments conducted
using 0.1% TWEEN-80 as the diluent; brown bars represent
experiments conducted using 0.2% TWEEN-80 as the diluent; lavender
bars represent experiments conducted using 0.5% TWEEN-80 as the
diluent; teal blue bars represent experiments conducted using 2%
PEG-3350 as the diluent; turquoise blue bars represent experiments
conducted using 3% PEG-3350 as the diluent; purple-striped bars
represent experiments conducted using 2.25% glycine as the diluent;
light blue bars represent experiments conducted using 0.1%
TWEEN-20+2% PEG-3350+2.25% glycine as the diluent; blue bars
represent experiments conducted using 0.1% TWEEN-80+2%
PEG-3350+2.25% glycine as the diluent. In all cases, the excipient
included 1% sorbitol.
[0022] FIG. 8 depicts the results obtained in the experiment
described in Example 5 to determine the effect of formulation
composition on the stability of recombinant AAV vectors using the
parameters described in Table 5. Light blue bars represent
experiments conducted using media as the diluent; yellow bars
represent experiments conducted using 10% propylene glycol as the
diluent; pink bars represent experiments conducted using 25%
propylene glycol as the diluent; turquoise blue bars represent
experiments conducted using 50% propylene glycol as the diluent;
dark blue bars represent experiments conducted using 18% PEG-400 as
the diluent; light brown bars represent experiments conducted using
25% propylene glycol+0.2% TWEEN-20 as the diluent; blue bars
represent experiments conducted using 25% propylene glycol+0.2%
TWEEN-80 as the diluent.
[0023] FIG. 9 depicts the results obtained in the experiment
described in Example 6 to determine the effect of various
excipients and storage conditions on the stability of recombinant
AAV vectors, as described in Table 6. Light blue bars represent
experiments conducted using media as the diluent and storage in
glass at -80.degree. C.; red bars represent experiments conducted
using 0.5% TWEEN-80 as the diluent and storage in glass at
-80.degree. C.; green-striped bars represent experiments conducted
using 1% sorbitol as the diluent and storage in glass at
-80.degree. C.; yellow bars represent experiments conducted using
media as the diluent and storage in polypropylene at -80.degree.
C.; gray bars represent experiments conducted using 0.5% TWEEN-80
as the diluent and storage in polypropylene at -80.degree. C.;
purple-striped bars represent experiments conducted using 1%
sorbitol as the diluent and storage in polypropylene at -80.degree.
C.; dark blue bars represent experiments conducted using media as
the diluent and storage in glass at +4.degree. C.; blue bars
represent experiments conducted using 0.5% TWEEN-80 as the diluent
and storage in glass at +4.degree. C.; blue-striped bars represent
experiments conducted using 1% sorbitol as the diluent and storage
in glass at +4.degree. C.; mustard yellow bars represent
experiments conducted using media as the diluent and storage in
polypropylene at +4.degree. C.; light yellow bars represent
experiments conducted using 0.5% TWEEN-80 as the diluent and
storage in polypropylene at +4.degree. C.; pink-striped bars
represent experiments conducted using 1% sorbitol as the diluent
and storage in polypropylene at +4.degree. C.
[0024] FIG. 10 depicts the results obtained in the experiment
described in Example 7 to determine the effect of various
excipients on loss of rAAV vector activity in samples stored in
glass or polypropylene vials. Lavender bars represent experiments
conducted using media as the diluent and storage in glass at
-80.degree. C.; light blue-striped bars represent experiments
conducted using 1% sorbitol as the diluent and storage in glass at
-80.degree. C.; dark blue-striped bars represent experiments
conducted using 10% propylene glycol (PG) as diluent and storage in
glass at -80.degree. C.; gray bars represent experiments conducted
using 25% PG as diluent and storage in glass at -80.degree. C.;
blue bars represent experiments conducted using 10% PG+0.2%
TWEEN-80 as diluent and storage in glass at -80.degree. C.; yellow
bars represent experiments conducted using 25% PG+0.2% TWEEN-80 as
diluent and storage in glass at -80.degree. C.; brown-striped bars
represent experiments conducted using 10% PG+0.5% TWEEN-80 as
diluent and storage in glass at -80.degree. C.; purple-striped bars
represent experiments conducted using 25% PG+0.5% TWEEN-80 as
diluent and storage in glass at -80.degree. C.; dark purple bars
represent experiments conducted using media as the diluent and
storage in polypropylene at +4.degree. C.; pink bars represent
experiments conducted using 1% sorbitol as the diluent and storage
in polypropylene at +4.degree. C.; light yellow bars represent
experiments conducted using 10% PG as diluent and storage in
polypropylene at +4.degree. C.; turquoise blue bars represent
experiments conducted using 25% PG as diluent and storage in
polypropylene at +4.degree. C.; dark green bars represent
experiments conducted using 10% PG+0.2% TWEEN-80 as diluent and
storage in polypropylene at +4.degree. C.; light blue bars
represent experiments conducted using 25% PG+0.2% TWEEN-80 as
diluent and storage in polypropylene at +4.degree. C.; green bars
represent experiments conducted using 10% PG+0.5% TWEEN-80 as
diluent and storage in polypropylene at +40.degree. C.; dark blue
bars represent experiments conducted using 25% PG+0.5% TWEEN-80 as
diluent and storage in polypropylene at +4.degree. C. The number
"118" in the figure represents a specific experiment number.
[0025] FIG. 11 depicts the results obtained in the experiment
described in Example 8 to determine the effect of 5% sorbitol,
alone and in combination with various excipients on the stability
of recombinant AAV vectors after a freeze/thaw cycle of samples
stored in a glass vial or a polypropylene tube. Light blue bars
represent experiments conducted using media as the diluent (without
sorbitol) and storage in glass at -80.degree. C.; pink bars
represent experiments conducted using 5% sorbitol and storage in
glass at -80.degree. C.; yellow bars represent experiments
conducted using 5% sorbitol+0.1% TWEEN-80 and storage in glass at
-80.degree. C.; green bars represent experiments' conducted using
5% sorbitol+0.25% TWEEN-80 and storage in glass at -80.degree. C.;
light brown bars represent experiments conducted using 5%
sorbitol+0.5% TWEEN-80 and storage in glass at -80.degree. C.;
turquoise blue bars represent experiments conducted using media as
the diluent (without soibitol) and storage in polypropylene at
-80.degree. C.; gray bats represent experiments conducted using 5%
sorbitol and storage in polypropylene at -80.degree. C.; blue bars
represent experiments conducted using 5% sorbitol+0.1% TWEEN-80 and
storage in polypropylene at -80.degree. C.; lime green bars
represent experiments conducted using 5% sorbitol+0.25% TWEEN-80
and storage in polypropylene at -80.degree. C.; red bars represent
experiments conducted using 5% sorbitol+0.5% TWEEN-80 and storage
in polypropylene at -80.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of virology,
microbiology, molecular biology and recombinant DNA techniques
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (Current Edition); DNA Cloning: A Practical
Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide
Synthesis (N. Gait, ed., Current Edition); Nucleic Acid
Hybridization (B. Hames & S. Higgins, eds., Current Edition);
Transcription and Translation (B. Hames & S. Higgins, eds.,
Current Edition); CRC Handbook of Parvoviruses, vol. I.& II (P.
Tijssen, ed.); Fundamental Virology, 2nd Edition, vol. I & II
(B. N. Fields and D. M. Knipe, eds.); Freshney Culture of Animal
Cells, A Manual of Basic Technique (Wiley-Liss, Third Edition); and
Ausubel et al. (1991) Current Protocols in Molecular Biology (Wiley
Inter science, NY).
[0027] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0028] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise.
A. DEFINITIONS
[0029] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0030] By "vector" is meant any genetic element, such ifs a
plasmid, phage, transposon, cosmid, chromosome, virus, virion,
etc., which is capable of replication when associated with the
proper control elements and which can transfer gene sequences
between cells. Thus, the term includes cloning and expression
vehicles, as well as viral vectors.
[0031] By "AAV vector" is meant a vector derived from an
adeno-associated virus serotype, including without limitation,
AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. AAV vectors can have
one or more of the AAV wild-type genes deleted in whole or part,
preferably the rep and/or cap genes (described below), but retain
functional flanking ITR sequences (also described below).
Functional ITR sequences are necessary for the rescue, replication
and packaging of the AAV virion. Thus, an AAV vector is defined
herein to include at least those sequences required in cis for
replication and packaging (e.g., functional ITRs) of the virus. The
ITRs need not be the wild-type nucleotide sequences, and may be
altered, e.g., by the insertion, deletion or substitution of
nucleotides, so long as the sequences provide for functional
rescue, replication and packaging.
[0032] By "recombinant virus" is meant a virus that has been
genetically altered, e.g., by the addition or insertion of a
heterologous nucleic acid construct into the particle.
[0033] By "AAV virion" is meant a complete virus particle, such as
a wild-type (wt) AAV virus particle (comprising a linear,
single-stranded AAV nucleic acid genome associated with an AAV
capsid protein coat). In this regard, single-stranded AAV nucleic
acid molecules of either complementary sense, e.g., "sense" or
"antisense" strands, can be packaged into any one AAV virion and
both strands are equally infectious.
[0034] A "recombinant AAV virion," or "rAAV virion" is defined
herein as an infectious, replication-defective virus composed of an
AAV protein shell, encapsidating a DNA molecule of interest which
is flanked on both sides by AAV ITRs. An rAAV virion is produced in
a suitable host cell which has had an AAV vector, AAV helper
functions and accessory functions introduced therein. In this
manner, the host cell is rendered capable of encoding AAV
polypeptides that are requited for packaging the AAV vector
(containing a recombinant nucleotide sequence of interest) into
recombinant virion particles for subsequent gene delivery.
[0035] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are known in the art. See, e.g., Graham et
al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular
Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New
York, Davis et al. (1986) Basic Methods in Molecular Biology,
Elsevier, and Chu et al. (1981) Gene 1:197. Such techniques can be
used to introduce one or more exogenous DNA moieties, such as a
plasmid vector and other nucleic acid molecules, into suitable host
cells. The term refers to both stable and transient uptake of the
genetic material.
[0036] The term "transduction" denotes the delivery of a DNA
molecule to a recipient cell either in vivo or in vitro, via a
replication-defective viral vector, such as via a recombinant AAV
virion.
[0037] By "DNA" is meant a polymeric form of deoxyribonucleotides
(adenine, guanine, thymine, or cytosine) in double-stranded or
single-stranded form, either relaxed and supercoiled. This term
refers only to the primary and secondary structure of the molecule,
and does not limit it to any particular tertiary forms. Thus, this
term includes single- and double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular DNA molecules, sequences may be described herein
according to the normal convention of giving only the sequence in
the 5' to 3' direction along the nontranscribed strand of DNA
(i.e., the strand having the sequence homologous to the mRNA). The
term captures molecules that include the four bases adenine,
guanine, thymine, or cytosine, as well as molecules that include
base analogs which are known in the art.
[0038] A "gene" or "coding sequence" or a sequence which "encodes"
a particular protein, is a nucleic acid molecule which is
transcribed (in the case of DNA) and translated (in the case of
mRNA) into a polypeptide in vitro or in vivo when placed under the
control of appropriate regulatory sequences. The boundaries of the
gene are determined by a start codon at the 5' terminus
(corresponding to the amino terminal of the encoded protein) and a
translation stop codon at the 3' (corresponding to the carboxy
terminal of the encoded protein) terminus. A gene can include, but
is not limited to, cDNA from prokaryotic or eukaryotic mRNA,
genomic DNA sequences from prokaryotic or eukaryotic DNA, and even
synthetic DNA sequences. A transcription termination sequence will
usually be located 3' to the gene sequence.
[0039] The term "control elements" refers collectively to promoter
regions, polyadenylation signals, transcription termination
sequences, upstream regulatory domains, origins of replication,
internal ribosome entry sites ("IRES"), enhancers, and the like,
which collectively provide for the replication, transcription and
translation of a coding sequence in a recipient cell. Not all of
these control elements need always be present so long as the
selected coding sequence is capable of being replicated,
transcribed and translated in an appropriate host cell.
[0040] The term "promoter region" is used herein in its ordinary
sense to refer to a nucleotide region comprising a DNA regulatory
sequence, wherein the regulatory sequence is derived from a gene
that is capable of binding RNA polymerase and initiating
transcription of a downstream (3'-direction) coding sequence.
[0041] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control elements operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control elements need not be contiguous with
the coding sequence and can be on the same (cis) or different
(trans) nucleic acid molecule from the coding sequence, so long as
they function to direct the expression thereof. Thus, for example,
intervening untranslated yet transcribed sequences can be present
between a promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0042] For the purpose of describing the relative position of
nucleotide sequences in a particular nucleic acid molecule
throughout the instant application, such as when a particular
nucleotide sequence is described as being situated "upstream,"
"downstream," "3'" or "5'" relative to another sequence, it is to
be understood that it is the position of the sequences in the
"sense" or "coding" strand of a DNA molecule that is being referred
to as is conventional in the art.
[0043] By "polyhydric alcohol" is meant an alcohol containing three
or more hydroxyl groups. Generally, alcohols having three hydroxyl
groups (trihydric) are glycerols, while those with more than three
hydroxyl groups are sugar alcohols. A "dihydric alcohol" is one
having two hydroxyl groups. Examples of polyhydric and dihydric
alcohols are given below.
B. GENERAL METHODS
[0044] The present invention provides stable pharmaceutical
compositions comprising rAAV virions. The compositions remain
stable and active even when subjected to freeze/thaw cycling and
when stored in containers made of various materials, including
glass.
[0045] Recombinant AAV virions containing a heterologous nucleotide
sequence of interest can be used for gene delivery, such as in gene
therapy applications, for the production of transgenic animals, in
nucleic acid vaccination, ribozyme and antisense therapy, as well
as for the delivery of genes in vitro, to a variety of cell
types.
[0046] Generally, rAAV virions are introduced into the cells of a
subject using either in vivo or in vitro transduction techniques.
If transduced in vitro, the desired recipient cell will be removed
from the subject, transduced with rAAV virions and reintroduced
into the subject. Alternatively, syngeneic or xenogeneic cells can
be used where those cells will not generate an inappropriate immune
response in the subject.
[0047] Suitable methods for the delivery and introduction of
transduced cells into a subject have been described. For example,
cells can be transduced in vitro by combining recombinant rAAV
virions with the cells e.g., in appropriate media, and screening
for those cells harboring the DNA of interest using conventional
techniques such as Southern blots and/or PCR, or by using
selectable markers. Transduced cells can then be formulated into
pharmaceutical compositions, described more fully below, and the
composition introduced into the subject by various routes, such as
by intramuscular, intravenous, intra arterial, subcutaneous and
intraperitoneal injection, or by injection into smooth muscle,
using e.g., a catheter, or directly into an organ.
[0048] For in vivo delivery, the rAAV virions will be formulated
into a pharmaceutical composition and will generally be
administered parenterally, e.g., by intramuscular injection
directly into skeletal muscle, intra articularly, intravenously or
directly into an organ.
[0049] Appropriate doses will depend on the subject being treated
(e.g., human or nonhuman primate or other mammal), age and general
condition of the subject to be treated, the severity of the
condition being treated, the mode of administration of the rAAV
virions, among other factors. An appropriate effective amount can
be readily determined by one of skill in the art.
[0050] Thus, a "therapeutically effective amount" will fall in a
relatively broad range that can be determined through clinical
trials. For example, for in vivo injection, i.e., injection
directly to the subject, a therapeutically effective dose will be
on the order of from about 10.sup.5 to 10.sup.16 of the rAAV
virions, more preferably 10.sup.8 to 10.sup.14 rAAV virions. For in
vitro transduction, an effective amount of rAAV virions to be
delivered to cells will be on the order of 10.sup.5 to 10.sup.13,
preferably 10.sup.8 to 10.sup.13 of the rAAV virions. If the
composition comprises transduced cells to be delivered back to the
subject, the amount of transduced cells in the pharmaceutical
compositions will be from about 10.sup.4 to 10.sup.10 cells, more
preferably 10.sup.5 to 10.sup.8 cells. The dose, of course, depends
on the efficiency of transduction, promoter strength, the stability
of the message and the protein encoded thereby, etc. Effective
dosages can be readily established by one of ordinary skill in the
art through routine trials establishing dose response curves.
[0051] Dosage treatment may be a single dose schedule or a multiple
dose schedule to ultimately deliver the amount specified above.
Moreover, the subject may be administered as many doses as
appropriate. Thus, the subject may be given, e.g., 10.sup.5 to
10.sup.16 rAAV virions in a single dose, or two, four, five, six or
more doses that collectively result in delivery of, e.g., 10.sup.5
to 10.sup.16 rAAV virions. One of skill in the art can readily
determine an appropriate number of doses to administer.
[0052] Pharmaceutical compositions will thus comprise sufficient
genetic material to produce a therapeutically effective amount of
the protein of interest, i.e., an amount sufficient to reduce or
ameliorate symptoms of the disease state in question or an amount
sufficient to confer the desired benefit. Thus, rAAV virions will
be present in the subject compositions in an amount sufficient to
provide a therapeutic effect when given in one or more doses. The
rAAV virions can be provided as lyophilized preparations and
diluted in the virion-stabilizing compositions for immediate or
future use. Alternatively, the rAAV virions may be provided
immediately after production and stored for future use.
[0053] The pharmaceutical compositions will also contain a
pharmaceutically acceptable excipient. Such excipients include any
pharmaceutical, agent that does not itself induce the production of
antibodies harmful to the individual receiving the composition, and
which may be administered without undue toxicity. Pharmaceutically
acceptable excipients include, but are not limited to, liquids such
as water, saline, glycerol and ethanol. Pharmaceutically acceptable
salts can be included therein; for example, mineral acid salts such
as hydrochlorides, hydrobromides, phosphates, sulfates, and the
like; and the salts of organic acids such as acetates, propionates,
malonates, benzoates, and the like. Additionally, auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, may be present in such vehicles. A
thorough discussion of pharmaceutically acceptable excipients is
available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co.,
N.J. 1991).
[0054] Preferred excipients confer a protective effect on the rAAV
virion such that loss of rAAV virions, as well as transduceability
resulting from formulation procedures, packaging, storage,
transport, and the like, is minimized. These excipient compositions
are therefore considered "virion-stabilizing" in the sense that
they provide higher rAAV virion titers and higher transduceability
levels than their non-protected counterparts, as measured using
standard assays, such as the assays described in the experimental
section. These compositions therefore demonstrate "enhanced
transduceability levels" as compared to compositions lacking the
particular excipients described herein, and are therefore more
stable than their non-protected counterparts.
[0055] Excipients that are used to protect the rAAV virion from
activity degradative conditions include, but are not limited to,
detergents, proteins, e.g., ovalbumin and bovine serum albumin,
amino acids, e.g., glycine, polyhydric and dihydric alcohols, such
as but not limited to polyethylene glycols (PEG) of varying
molecular weights, such as PEG-200, PEG-400, PEG-600, PEG-1000,
PEG-1450, PEG-3350, PEG-6000, PEG-8000 and any molecular weights in
between these values, with molecular weights of 1500 to 6000
preferred; propylene glycols (PG), sugar alcohols, such as a
carbohydrate, preferably, sorbitol. The detergent, when present;
can be an anionic, a cationic, a zwitterionic or a nonionic
detergent. A preferred detergent is a nonionic detergent. More
preferably, the nonionic detergent is a sorbitan ester, e.g.,
polyoxyethylenesorbitan monolaurate (TWEEN-20)
polyoxyethylenesorbitan monopalmitate (TWEEN-40),
polyoxyethylenesorbitan monostearate (TWEEN-60),
polyoxyethylenesorbitan tristearate (TWEEN-65),
polyoxyethylenesorbitan monooleate (TWEEN-80),
polyoxyethylenesorbitan trioleate (TWEEN-85), preferably TWEEN-20
and/or TWEEN-80. These excipients are commercially available from a
number of vendors, such as Sigma, St. Louis, Mo.
[0056] The amount of the various excipients present will vary and
is readily determined by one of skill in the art. For example, a
protein excipient, such as BSA, if present, will generally be
present at a concentration of between 1.0 wt. % to about 20 wt. %,
preferably 10 wt. %. If an amino acid such as glycine is used in
the formulations, it will generally be present at a concentration
of about 1 wt. % to about 5 wt. %. A carbohydrate, such as
sorbitol, if present, will be present at a concentration of about
0.1 wt. % to about 10 wt. %, preferably between about 0.5 wt. % to
about 15 wt. %, more preferably about 1 wt. % to about 5 wt. %. If
PG is present, it will generally be present on the order of about 2
wt. % to about 40 wt. %, preferably about 10 wt. % top about 25 wt.
%. If propylene glycol is used in the subject formulations, it will
typically be present at a concentration of about 2 wt. % to about
60 wt. %, preferably about 5 wt. % to about 30 wt. %. If a
detergent such as a sorbitan ester (TWEEN) is present, it will
generally be present at a concentration of about 0.05 wt. % to
about 5 wt. %, preferably between about 0.1 wt. % and about 1 wt.
%.
[0057] In one-preferred embodiment, an aqueous virion-stabilizing
formulation comprises a carbohydrate, such as sorbitol, at a
concentration of between 0.1 wt. % to about 10 wt. %, preferably
between about 1 wt. % to about 5 wt. %, and a detergent, such as a
sorbitan ester (TWEEN) at a concentration of between about 0.05 wt.
% and about 5 wt. %, preferably between about 0.1 wt. % and about 1
wt. %. Virions are generally present in the composition in an
amount sufficient to provide a therapeutic effect when given in one
or more doses, as defined above.
C. EXPERIMENTAL
[0058] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0059] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
MATERIALS AND METHODS
Production of Recombinant AAV Virions
[0060] Recombinant AAV virions can be produced using the method
described in commonly owned U.S. Pat. No. 5,622,856 to Natsoulis,
the disclosure of which is incorporated herein by reference.
[0061] Briefly, the method includes the steps of: introducing an
AAV vector into a suitable host cell; introducing an AAV helper
construct into the host cell to express essential AAV helper
functions; expressing viral helper functions in the host cell; and
culturing the cell to produce rAAV virions. The AAV vector and AAV
helper constructs can be transfected into the host cell, either
sequentially or simultaneously, using techniques known to those of
skill in the art. The expression of viral helper functions can be
provided by infecting the host cell with a suitable helper virus
selected from the group of adenoviruses, herpesviruses and vaccinia
viruses. The viral helper functions transactivate AAV promoters
present in the AAV helper construct that direct the transcription
and translation of AAV rep and cap regions. Thus, rAAV virions
harboring a selected heterologous nucleotide sequence are formed
and can be purified from the preparation using known.
[0062] The supernatant obtained from the host cell is titered for
rAAV viral production either by dot blot to calculate the number of
viral genomes or by transducing cells with the rAAV thus produced
and harvested, and assaying for .beta.-galactosidase activity to
determine functional units as indicating rAAV LacZ
transduceability. Transducing vector titers can be determined by
infecting 293 cells, or any cell competent for transfection with
AAV, with a dilution series of the rAAV virions. After 24 hours,
the cells are fixed and stained with X-Gal. Sanes et al. (1986)
EMBO 5:3133-3142. The titer is calculated by quantifying the number
of blue cells.
[0063] Construction of pAAVLacZ--
[0064] An AAV vector carrying the lacZ gene (pAAV-lacZ) was
constructed as follows. The AAV coding region of pSub201 (Samulski
et al. (1987) J. Virol 61:3096-3101), between the XbaI sites, was
replaced with EcoRI linkers, resulting in plasmid pAS203. The EcoRI
to HindIII fragment of pCMV.beta. (CLONETECH) was rendered blunt
ended and cloned in the Klenow treated EcoRI site of pAS203 to
yield pAAV-lacZ.
Example 1
Effect of Freezing/Thaw Cycle on Recombinant AAV Activity
[0065] This experiment was done to determine the effect of a
freeze/thaw cycle on rAAV activity. As shown in the table, about
75% of the activity is lost if no agent is added to the rAAV before
it is frozen. The addition of bovine serum albumin (BSA) or
polyoxyethylenesorbitan monolaurate (TWEEN-20) alone improved the
recovery (about 50%). Sorbitol, however, completely protected the
sample from freeze/thaw inactivation. These experiments were
performed in polypropylene vials.
[0066] AAV-lacZ was chromatographed on an ion exchange column. A
small volume from each of the fractions from the column was assayed
for blue cell activity on the same day, prior to freezing the
sample. The peak fraction contained 47% of the initial load on the
column.
[0067] The remainder of each fraction was split into 4 portions and
the following excipients added to each portion:
[0068] Portion a--no excipient;
[0069] Portion b--BSA to a final concentration of 10%;
[0070] Portion c--sorbitol to a final concentration of 5%;
[0071] Portion d--TWEEN-20 to a final concentration of 0.5%;
[0072] These samples were frozen, thawed a few days later and
assayed for blue cell activity. The results are summarized in Table
1
TABLE-US-00001 TABLE 1 Sample Excipient % Yield Active fraction,
pre-freeze None 47% Active fraction, post freeze-thaw None 15%
Active fraction, post freeze-thaw BSA 27% Active fraction, post
freeze-thaw Sorbitol 52% Active fraction, post freeze-thaw TWEEN-20
29%
[0073] These results indicate that a single freeze/thaw cycle can
result in a significant reduction in rAAV activity. This reduction
in activity can be abrogated by addition of protective agents such
as proteins, polyhydric alcohols and detergents, all of which are
believed to act by different mechanisms. In this experiment,
sorbitol had the greatest protective effect; essentially no loss of
activity following freezing and thawing was observed in the
sorbitol-containing sample.
Example 2
Effect of Vector Dilution, Temperature and Storage in Glass Vials
on Recombinant AAV Activity
[0074] Initial stability experiments were conducted to determine
the effect of vector dilution, recovery from polypropylene (pp)
vials and glass (gl) vials and the effect of temperature
(-80.degree. C., 2-8.degree. C., 37.degree. C.) on storage
stability and the effect of the addition of sorbitol on stability
and recovery.
[0075] Samples of rAAV in a 1% sorbitol solution in phosphate
buffered saline (PBS) were assayed undiluted as well as diluted
1-fold to about 10-fold (1.25 ml-12.5 ml) within 1% sorbitol/DPBS
buffer. 0.5 ml aliquots of each member of the dilution series were
placed in different storage conditions.
[0076] Following freezing, the samples were thawed and analyzed for
vector genomes and for transduceability as described in Example 1.
For transduceability studies, the vector genome titer used in the
experiment was calculated using the starting concentration of
undiluted vector. Each sample wag diluted in complete Dulbecco's
Modified Eagle Medium (DMEM) such that 1.times.10.sup.8 or
5.times.10.sup.8 vector genomes could be added to 298-HEK cells in
a volume of 10 to 20 .mu.L.
[0077] The stability-indicating assay used was loss of
transduceability. Loss of transduceability was measured by human
factor IX protein (hFIX) production following transduction with
rAAV of 293 human embryonic kidney (HEK) cells. In this assay,
different amounts of rAAV-hFIX vector were added to HEK cells and
the ability to transduce was measured. The factor IX protein
produced and secreted as a consequence of infection was measured
using an ELISA technique. The results are reported in ng/mL of
human-factor IX protein (hFIX).
[0078] As can be seen by the results depicted in FIG. 1, storing
rAAV in glass regardless of the temperature that it was stored at
caused the rAAV activity to drop. In this experiment it appears
that activity and temperature are inversely related--i.e., the
lower the temperature the higher the activity.
Example 3
Effect of Storage in Glass on Recombinant AAV Activity
[0079] The results of the experiment described in Example 0.2
indicate that storage of rAAV in glass resulted in a loss of
activity. This experiment was designed to examine whether this was
due merely to the fact that rAAV was adsorbed to the glass
vial.
[0080] In order to rule out this possibility, the number of genomes
(the number of DNA molecules encapsulated in AAV as determined by
Southern Blot dot) added to the glass and polypropylene vials was
determined. The rAAV was allowed to sit in the glass and
polypropylene vials in various formulations and temperatures. Two
aliquots of rAAV virus were taken: the first used to recount the
number of genomes recovered and the second to determine activity
(functional units as opposed to genomes) as determined in Example
1. The results indicate that when rAAV is stored in glass the
activity drops significantly. This occurs at various dilutions of
rAAV.
[0081] rAAV samples were frozen undiluted as well as diluted
5-fold, 50-fold or 100-fold. The diluent used was such that the
final concentration of sorbitol was either 5% or 1%. Duplicate
samples were placed in glass vials and polypropylene tubes. Samples
were placed at -80.degree. C. or at ambient temperature. Following
freezing, the samples were thawed and analyzed for vector genomes
and for transduceability as described above. For transduceability
studies, the vector genome titer used in the experiment was
calculated using the starting concentration of undiluted vector.
Each sample was diluted in complete DMEM such that 1.times.10.sup.8
or 5.times.10.sup.8 vector genomes could be added to 293-HEK cells
in a volume of 10 to 20 .mu.L.
[0082] The stability-indicating assays used were loss of vector
genomes and loss of transduceability. Loss of vector genomes was
measured using a dot blot assay. This assay involves extraction of
vector DNA from the sample, denaturing the DNA, and loading it on a
nylon membrane. By hybridizing this DNA to a complimentary
radioactive DNA probe, the number of vector genomes can be
calculated by comparison to a standard. Loss of transduceability
was measured as described in Example 2.
[0083] Table 2 lists the data obtained for 293-HEK cells transduced
with 1.times.10.sup.8 vector genomes. The data is shown for the
samples incubated at -80.degree. C. Column 2 shows the
transduceability, column 3 is the measured vector genomes/ml and
column 4 is the data in column 3 multiplied by the dilution factor.
In the table, "ppS" indicates polypropylene stock container and
"ppC" indicates polypropylene container.
TABLE-US-00002 TABLE 2 3 4 2 Vector Normalized 1 ng/ml Genomes/
Vector Sample hFIX ml Genomes 5%, ppS, -80.degree. C., stock 11.6
1.5 .times. 10.sup.12 1.5 .times. 10.sup.12 5%, ppC, -80.degree.
C., 1:5 6.6 2.3 .times. 10.sup.11 1.2 .times. 10.sup.12 5%, glass,
-80.degree. C., 1:5 3.8 1.9 .times. 10.sup.11 9.5 .times. 10.sup.11
1%, ppC, -80.degree. C., 1:5 4.2 1.9 .times. 10.sup.11 9.5 .times.
10.sup.11 1%, glass, -80.degree. C., 1:5 2.1 1.7 .times. 10.sup.11
8.5 .times. 10.sup.11 5%, ppC., -80.degree. C., 1:50 5.9 2.5
.times. 10.sup.10 1.3 .times. 10.sup.12 5%, glass, -80.degree. C.,
1:50 4.5 1.5 .times. 10.sup.10 7.5 .times. 10.sup.11 1%, ppC,
-80.degree. C., 1:50 4.3 1.7 .times. 10.sup.10 8.5 .times.
10.sup.11 1%, glass, -80.degree. C., 1:50 4.6 1.9 .times. 10.sup.10
9.5 .times. 10.sup.11 5%, ppC, -80.degree. C., 1:100 5.5 1.5
.times. 10.sup.10 1.5 .times. 10.sup.12 5%, glass, -80.degree. C.,
1:100 4.0 9.3 .times. 10.sup.9 9.3 .times. 10.sup.11 1%, ppC,
-80.degree. C., 1:100 4.6 1.2 .times. 10.sup.10 1.2 .times.
10.sup.12 1%, glass, -80.degree. C., 1:100 2.4 1.1 .times.
10.sup.10 1.1 .times. 10.sup.12
[0084] FIG. 2 depicts the data for the vector genome and
transduceability results in which 1.times.10.sup.8 vector genomes
were used for the transduceability assay. Vector genomes obtained
experimentally have been normalized for the dilution to compare
with the transduceability results. In addition, the vector genome
results have been divided by 1.times.10.sup.11 to produce
manageable numbers for the graph.
[0085] Table 3 and FIG. 3 depict the results of experiments
conducted as described above except that the transduceability assay
for 293-HEK cells transduced with 5.times.10.sup.8 vector
TABLE-US-00003 TABLE 3 3 4 2 Vector Normalized 1 ng/ml Genomes/
Vector Sample hFIX ml Genomes 5%, ppS, -80.degree. C., stock 78.8
1.5 .times. 10.sup.12 1.5 .times. 10.sup.12 5%, ppC, -80.degree.
C., 1:5 48.3 2.3 .times. 10.sup.11 1.2 .times. 10.sup.12 5%, glass,
-80.degree. C., 1:5 37.1 1.9 .times. 10.sup.11 9.5 .times.
10.sup.11 1%, ppC, -80.degree. C., 1:5 29.9 1.9 .times. 10.sup.11
9.5 .times. 10.sup.11 1%, glass, -80.degree. C., 1:5 23.9 1.7
.times. 10.sup.11 8.5 .times. 10.sup.11 5%, ppC, -80.degree. C.,
1:50 11.3 2.5 .times. 10.sup.10 1.3 .times. 10.sup.12 5%, glass,
-80.degree. C., 1:50 18.5 1.5 .times. 10.sup.10 7.5 .times.
10.sup.11 1%, ppC, -80.degree. C., 1:50 8.6 1.7 .times. 10.sup.10
8.5 .times. 10.sup.11 1%, glass, -80.degree. C.; 1:50 4.2 1.9
.times. 10.sup.10 9.5 .times. 10.sup.11 5%, ppC, -80.degree. C.,
1:100 11.2 1.5 .times. 10.sup.10 1.5 .times. 10.sup.12 5%, glass,
-80.degree. C., 1:100 30.7 9.3 .times. 10.sup.9 9.3 .times.
10.sup.11 1%, ppC, -80.degree. C., 1:100 14.5 1.2 .times. 10.sup.10
1.2 .times. 10.sup.12 1%, glass, -80.degree. C., 1:100 7.1 1.1
.times. 10.sup.10 1.1 .times. 10.sup.12
[0086] FIG. 4 and FIG. 5 depict the results of experiments
conducted at -80.degree. C. or ambient temperature for vector
genome count and transduceability assay in which transduceability
was done using 1.times.10.sup.8 vector genomes (FIG. 4) and
5.times.10.sup.8 vector genomes (FIG. 5.) These results from the
transduceability experiments indicate that as the vector is diluted
it decreases in infective capability. It can also be seen that
samples prepared in 1% sorbitol have reduced transduceability
compared to those prepared in 5% sorbitol. The results also
indicate that storage in glass at -80.degree. C. results in reduced
transduceability compared to polypropylene.
[0087] Thus, it appears that there can be a physical loss of vector
in the container under the conditions examined in these
experiments. However, from the vector genome data it can be
observed that there is no observable change in the number of vector
genomes. The % correlation of variance (% CV) in the range of
vector genomes (see Tables 2 and 3) is 22.4, well within the
variability of the dot blot assay. However, the % CV for the
transduceability for both titers, i.e., 1.times.10.sup.8 vector
genomes and 5.times.10.sup.8 vector genomes, is greater than the
variability of the transduceability assay (about 30%). Accordingly;
it appears that, rather than a loss in vector genome number, the
vector per se is losing transduceability.
Example 4
The Effect of Added Excipients on the Stability of Recombinant
AAV-I
[0088] These experiments were designed to study the effect of
different formulations containing 1% sorbitol and various
concentrations of TWEEN-20, TWEEN-80, polyethylene glycol (PEG),
glycine and combinations thereof. Virus placed in growth media was
used as a baseline. The tubes used throughout were polypropylene.
The samples were maintained in the formulations for 1 hr at room
temperature before transducing culture cells.
[0089] The stability of the AAV vector was measured using the loss
of transduceability assay described in Example 2 to further explore
the effect of added excipients. Samples were diluted in media or in
the buffer excipient to give concentrations of 1.times.10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9 or 5.times.10.sup.9 in 15 .mu.L.
Samples were placed in polypropylene tubes for about 1 hour and
then used to transduce 293-HEK cells.
[0090] The formulations of excipients and results are given in
ng/ml of human factor IX in Table 4 and the results listed therein
are depicted in FIG. 6 and FIG. 7. In all cases, the excipient
included 1% sorbitol.
TABLE-US-00004 TABLE 4 Diluent Concentration (All experimental
excipients contained 1% Sorbitol) (ng/ml) Media 30.5 194.5 353.1
615.8 0.1% TWEEN-20 26.4 178.6 347.7 616.9 0.2% TWEEN-20 31.8 180.6
384.4 623.9 0.5% TWEEN-20 30.2 194.6 355.8 654.6 0.1% TWEEN-80 29.2
178.1 360.8 595.9 0.2% TWEEN-80 35.6 186.4 399.8 669.0 0.5%
TWEEN-80 29.8 183.6 344.3 588.6 2% PEG-3350 25.0 164.5 331.5 604.4
3% PEG-3350 28.6 162.0 354.9 556.0 2.25% glycine 14.9 115.6 240.8
516.3 0.1% TWEEN-20 + 2% PEG + 2.25% glycine 23.5 143.5 329.9 533.1
0.1% TWEEN-80 + 2% PEG + 2.25% glycine 24.0 140.6 329.5 532.3
Particles/well 1 .times. 10.sup.8 5 .times. 10.sup.8 1 .times.
10.sup.9 5 .times. 10.sup.9
[0091] These data indicate that the addition of TWEEN seems to have
stabilized rAAV, and PEG and glycine do little and may even reduce
the overall activity of rAAV.
Example 5
The Effect of Added Excipients on the Stability of Recombinant
AAV-II
[0092] The stability of the AAV vector was measured as described in
Example 4. The formulations of excipients and results are given in
Table 5 and the results listed therein are depicted in FIG. 8. In
all cases, the excipient included 1% sorbitol.
TABLE-US-00005 TABLE 5 Diluent Concentration All experimental
excipients contained 1% Sorbitol (ng/ml) Media 28.5 192.2 269.2
607.0 10% Propylene Glycol 26.0 144.7 313.3 607.0 25% Propylene
Glycol 20.9 170.8 292.4 568.1 50% Propylene Glycol 26.9 163.4 256.5
600.1 18% PEG-400 14.8 94.2 224.7 701.1 25% Propylene Glycol + 0.2%
TWEEN-20 49.7 235.7 352.3 834.9 25% Propylene Glycol + 0.2%
TWEEN-80 47.5 314.2 542.6 919.9 Particles/well 1 .times. 10.sup.8 5
.times. 10.sup.8 1 .times. 10.sup.9 5 .times. 10.sup.9
Example 6
The Effect of Added Excipients on the Stability of Recombinant AAV:
Comparison of Glass and Polypropylene Vials
[0093] This experiment was designed to study the effect of 1%
sorbitol and TWEEN-80 on the activity of rAAV stored in glass vials
compared to the effect on activity of rAAV stored in polypropylene
vials at two temperatures (4.degree. C. and -80.degree. C.).
[0094] The stability of the AAV vector was measured as described
using 5.times.10.sup.7, 1.times.10.sup.8 or 5.times.10.sup.8
particles/well. Each sample was done in a glass vial (GV) or a
polypropylene tube (PT), and stored at room temperature (+4.degree.
C.) or at -80.degree. C. overnight. The formulations of excipients
and results are given in Table 6 and the results listed therein are
depicted in FIG. 9.
TABLE-US-00006 TABLE 6 Diluent Concentration (ng/ml) Media, glass,
-80.degree. C. 23.6 42.8 243.3 0.5% TWEEN-80, glass, -80.degree. C.
23.5 46.7 254.7 1% sorbitol, glass, -80.degree. C. 6.2 8.9 59.2
Media, polypropylene, -80.degree. C. 26.4 48.1 251.1 0.5% TWEEN-80,
polypropylene, -80.degree. C. 30.2 56.0 228.2 1% sorbitol,
polypropylene, -80.degree. C. 12.2 23.3 185.9 Media, glass,
+4.degree. C. 19.7 34.9 247.1 0.5% TWEEN-80, glass, +4.degree. C.
31.1 58.7 282.2 1% sorbitol, glass, +4.degree. C. 7.0 14.5 111.9
Media, polypr4ylene, +4.degree. C. 18.1 30.8 212.7 0.5% TWEEN-80,
polypropylene, +4.degree. C. 23.3 46.6 248.6 1% sorbitol,
polypropylene, +4.degree. C. 15.2 31.9 200.9 Particles/well 5
.times. 10.sup.7 1 .times. 10.sup.8 5 .times. 10.sup.8
[0095] These data indicate that 1% sorbitol does not provide a
significant protective effect for rAAV activity when-stored in
glass vials. When sorbitol is used alone, it provides a protective
effect on rAAV stored in polypropylene vials. There is also an
apparent protective effect when the sample is stored at 4.degree.
C. rather than at -80.degree. C. The inclusion of TWEEN in the
formulation reverses the reduced activity caused by storage of an
rAAV sample in a glass vial.
Example 7
The Effect of Added Excipients on the Stability of Recombinant
AAV-III: Comparison of Storage in Glass and Polypropylene Vials
[0096] The stability of the AAV vector was measured as described in
Example 5. The formulations of excipients and results are given in
FIG. 10. The data indicate that neither propylene glycol (PG) nor
sorbitol alone protect against loss of activity of an rAAV sample
stored in a glass vial. When PG and TWEEN were combined, loss of
activity was minimized and, in fact, it appears that PG and TWEEN
together may have a synergistic effect on activity.
Example 8
The Effect of Added Excipients on the Stability of Recombinant AAV:
the Effect of 5% Sorbitol
[0097] These experiments were designed to study the effect of 5%
sorbitol, in combination with various concentrations of TWEEN on
the activity of rAAV.
[0098] The stability of the AAV vector after a freeze/thaw cycle
was measured as described in Example 6 using 1.times.10.sup.8,
5.times.10.sup.8 or 1.times.10.sup.9 particles/well. Each sample
was done in a glass vial or a polypropylene tube, and stored at
-80.degree. C. overnight. The formulations of excipients and
results are given in FIG. 11.
[0099] A formulation containing 5% sorbitol provided a partial
protective effect against loss of rAAV activity when the sample was
stored in a glass vial. However, the addition of TWEEN provided a
significantly greater protective effect.
[0100] Thus, formulations for enhancing the stability of
recombinant AAV preparations are described. Although preferred
embodiments of the subject invention have been described in some
detail, it is understood that obvious variations can be made
without departing from the spirit and the scope of the invention as
defined by the appended claims.
* * * * *