U.S. patent application number 15/494125 was filed with the patent office on 2017-10-26 for regulatable adeno-associated virus (aav) vector.
The applicant listed for this patent is Georg-August-Universitat Gottingen Stiftung Offentlichen Rechts, Universtitatsmedizin. Invention is credited to Sebastian KUGLER.
Application Number | 20170304464 15/494125 |
Document ID | / |
Family ID | 55952959 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170304464 |
Kind Code |
A1 |
KUGLER; Sebastian |
October 26, 2017 |
REGULATABLE ADENO-ASSOCIATED VIRUS (AAV) VECTOR
Abstract
The present invention relates to regulatable adeno-associated
virus (AAV) vectors as well as to their use in gene therapy. It
further relates to corresponding nucleic acid molecules, host
cells, non-human transgenic animals, pharmaceutical compositions
and kits.
Inventors: |
KUGLER; Sebastian;
(Gottingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Georg-August-Universitat Gottingen Stiftung Offentlichen Rechts,
Universtitatsmedizin |
Gottingen |
|
DE |
|
|
Family ID: |
55952959 |
Appl. No.: |
15/494125 |
Filed: |
April 21, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2830/85 20130101;
C12N 2830/002 20130101; C12N 2830/702 20130101; A61K 48/0008
20130101; A61K 48/005 20130101; C12N 2750/14143 20130101; C12N
2830/008 20130101; C12N 2830/42 20130101; C12N 2840/60 20130101;
C12N 2830/34 20130101; C12N 2830/40 20130101; C12N 2830/50
20130101; C12N 2840/007 20130101; A61K 48/0058 20130101; A61K 48/00
20130101; C12N 15/86 20130101; C07K 14/475 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86; A61K 48/00 20060101
A61K048/00; C07K 14/475 20060101 C07K014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2016 |
EP |
16166637.5 |
Claims
1. An adeno-associated virus (AAV) vector comprising (i) a first
expression cassette directing the expression of a regulator protein
under the control of a first promoter, wherein the regulator
protein is activated in the presence of an activator molecule, and
(ii) a second expression cassette directing the expression of a
molecule of interest, wherein the second expression cassette
comprises a promoter region, and the expression of the molecule of
interest is induced by binding of the activated regulator protein
to the promoter region, wherein the first expression cassette and
the second expression cassette are arranged in a tail-to-head
configuration.
2. The AAV vector of claim 1, wherein the first promoter has one or
more of the following features: (i) it is a constitutive promoter;
(ii) it is selected from the group consisting of cell-specific
promoters, tissue-specific promoters and organ-specific promoters;
(iii) it is selected from the group consisting of human synapsin 1
gene (hSYN1) promoter, tubulin alpha 1 (Ta1) promoter, glial
fibrillary acidic protein (GFAP) promoter, cytomegalovirus (CMV)
promoter, human beta-actin-CMV hybrid promoter and functional
fragments or variants of any of the foregoing.
3. The AAV vector of claim 1, wherein the regulator protein is a
gene switch fusion protein comprising a GAL4 DNA binding domain, a
truncated progesterone receptor ligand binding domain and a p65
transactivation domain from NF-kappaB.
4. The AAV vector of claim 3, wherein the activator molecule is
mifepristone (Mfp).
5. The AAV vector of claim 1, wherein the first expression cassette
comprises, in 5' to 3' direction, the first promoter, a coding
sequence for the regulator protein and a first polyadenylation
signal sequence.
6. The AAV vector of claim 5, wherein the first expression cassette
further comprises a synthetic intron arranged between the coding
sequence for the regulator protein and the first polyadenylation
signal sequence.
7. The AAV vector of claim 1, wherein the promoter region comprises
one or more binding sites for the activated regulator protein and a
second promoter.
8. The AAV vector of claim 7, wherein the promoter region further
comprises a synthetic intron.
9. The AAV vector of claim 7, wherein the one or more binding sites
for the activated regulator protein are GAL4 binding sites.
10. The AAV vector of claim 7, wherein the second promoter is a
minimal promoter which is induced by the binding of the activated
regulator protein to the one or more binding sites for the
activated regulator protein.
11. The AAV vector of claim 10, wherein the second promoter is a
minimal promoter comprising a TATA sequence and/or an mRNA
initiation sequence.
12. The AAV vector of claim 1, wherein the molecule of interest is
a therapeutically active peptide or protein or a therapeutically
active oligo- or polynucleotide.
13. The AAV vector of claim 12, wherein the molecule of interest is
a neurotrophic factor.
14. The AAV vector of claim 13, wherein the neurotrophic factor is
glial cell line-derived neurotrophic factor (GDNF).
15. The AAV vector of claim 1, wherein the second expression
cassette comprises, in 5' to 3' direction, the promoter region, a
coding sequence for the molecule of interest and a second
polyadenylation signal sequence.
16. The AAV vector of claim 1, wherein the first expression
cassette and the second expression cassette are separated by a
nucleotide sequence comprising an insulator element.
17. The AAV vector of claim 16, wherein the insulator element is a
transcription blocker comprising a transcription pause site and a
polyadenylation signal sequence.
18. The AAV vector of claim 1, wherein the AAV vector comprises the
nucleotide sequence represented by SEQ ID NO: 1 or a functional
variant thereof, wherein the functional variant has a nucleotide
sequence which is at least 80% or at least 85% or at least 90% or
at least 95% identical to SEQ ID NO: 1.
19. The AAV vector of claim 1, wherein, in the absence of the
activator molecule, the molecule of interest is not expressed in a
host or is expressed in a host at a level which is at most 10-fold
or at most 5-fold or at most 4-fold or at most 3-fold or at most
2-fold increased as compared to the normal expression level of the
molecule of interest in the host, wherein the host is a cell,
tissue or organ.
20. An adeno-associated virus (AAV) vector construct comprising (i)
a first expression cassette directing the expression of a regulator
protein under the control of a first promoter, wherein the
regulator protein is activated in the presence of an activator
molecule, and (ii) a second expression cassette comprising a
multiple cloning site allowing the insertion of a coding sequence
for a molecule of interest, wherein the second expression cassette
comprises a promoter region, and the expression of the molecule of
interest is induced by binding of the activated regulator protein
to the promoter region, wherein the first expression cassette and
the second expression cassette are arranged in a tail-to-head
configuration.
21. The AAV vector construct of claim 12, wherein the AAV vector
construct comprises the nucleotide sequence represented by SEQ ID
NO: 2 or a functional variant thereof, wherein the functional
variant has a nucleotide sequence which is at least 80% or at least
85% or at least 90% or at least 95% identical to SEQ ID NO: 2.
22. A method for treating, ameliorating or preventing a disease or
disorder in a subject, comprising (a) introducing into the subject
the AAV vector of claim 1; and (b) administering to the subject the
activator molecule to induce expression of the molecule of
interest, wherein the molecule of interest is a therapeutically
active peptide or protein or a therapeutically active oligo- or
polynucleotide.
23. The method of claim 22, wherein the molecule of interest is a
neurotrophic factor and the disease or disorder is selected from
the group consisting of Parkinson's disease, Huntington's disease,
spinal cord lesion and an amyloid-related disorder.
24. The method of claim 23, wherein the neurotrophic factor is
glial cell line-derived neurotrophic factor (GDNF).
25. The method of claim 23, wherein the amyloid-related disorder is
selected from the group consisting of Alzheimer's disease, cerebral
amyloid angiopathy, dementia, motor neuropathy, Down's syndrome,
Creutzfeld Jacob disease, transmissible spongiform
encephalopathies, hereditary cerebral hemorrhage with amyloidosis
Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy
body disease, mixed dementias, head trauma, familial Danish
Dementia, familial British Dementia, inclusion body myositis (IBM),
neuronal disorder related to aging, and chronic pain.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims the benefit of priority from
European Patent Application No. 16 166 637.5, filed on Apr. 22,
2016, which is hereby incorporated by reference in its
entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] A sequence listing containing SEQ ID NOs:1-2 is submitted
herewith and is specifically incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to regulatable
adeno-associated virus (AAV) vectors as well as to their use in
gene therapy. It further relates to corresponding nucleic acid
molecules, host cells, non-human transgenic animals, pharmaceutical
compositions and kits.
BACKGROUND OF THE INVENTION
[0004] Gene therapy in its current configuration is an irreversible
process. Typically, a potentially therapeutic transgene is inserted
into target cells/tissues by means of gene transfer with a viral
vector. From this time on, the transgene is expressed without
external control over its expression level and without the option
to shut off transgene expression in case of unforeseen side effects
or sufficient therapeutic success.
[0005] Attempts to overcome this limitation have generated several
regulatable gene transfer systems, one of which is the gene switch
(GS), whose principles are described in FIG. 1. A constitutive
promoter (which may be cell-type specific or ubiquitously active)
expresses the GS fusion protein, which consists of a GAL4 DNA
binding domain, a truncated progesterone receptor ligand binding
domain as drug binding domain, and the p65 transactivation domain
from NF-kappaB. Upon binding of the GS fusion protein to the small
molecule drug mifepristone (Mfp, a synthetic steroid), it dimerizes
and binds to a polynucleotide sequence consisting of several GAL4
DNA binding sites. Consequently, the p65 moiety of the GS fusion
protein recruits the basic cellular transcription machinery,
allowing expression of the therapeutic factor from a minimal
promoter. Withdrawal of Mfp disables binding of the GS fusion
protein to its DNA target sequence, and thus ceases transgene
expression.
[0006] The GS system was described for plasmids, transgenic cells,
adenoviral and herpes simplex gene transfer vectors and for
transgenic animals during the 1990's (Wang Y. et al., Proc. Natl.
Acad. Sci. USA. 1994, 91:8180-8184; Wang Y. et al., Gene Ther.
1997, 4:432-441; Burcin M. M. et al., Proc. Natl. Acad. Sci. USA.
1999, 96:355-360; Oligino T. et al., Gene Ther. 1998, 5:491-496).
As adeno-associated viral vectors (AAVs) are especially well-suited
gene therapy tools due to their proven safety record in human
clinical trials (Bartus R. T. et al., Neurology. 2013,
80:1698-1701; Chtarto A. et al., Br J Clin Pharmacol. 2013,
76:217-232), the inventor has adopted the GS system to AAV vectors.
Using the brain as target tissue and Parkinson's disease as a
target disease, the inventor demonstrated successful therapeutic
treatment of motor symptoms in a rat model of Parkinson's disease
(Tereshchenko J. et al., Neurobiol Dis. 2014, 65:35-42; Maddalena
A. et al., Mol Ther Nucleic Acids. 2013, 2:e106). In this approach,
the inventor was able to show that short-term induced expression of
the neurotrophic factor GDNF (glial cell line-derived neurotrophic
factor, Kordower J. H. et al., Mov Disord. 2013, 28:96-109)
resulted in long-term recovery from motor impairments in this
model.
[0007] A functional GS system usually requires gene transfer of two
expression cassettes into target cells: one cassette expressing the
GS fusion protein and a second cassette expressing the therapeutic
factor from the regulated minimal promoter. Accordingly, the
above-mentioned studies were conducted with a two-vector system, in
which the GS expression cassette was contained in one virus, while
the regulated GDNF expression cassette was contained in a second
virus. This configuration was necessary to prevent leaky expression
of GDNF in the non-Mfp-induced state (Maddalena A. et al., Mol Ther
Nucleic Acids. 2013, 2:e106). While such a two-vector system offers
flexibility in terms of adjusting the ratio of GS expression
cassette versus the GDNF expression cassette, it appears unlikely
that such a double vector formulation could be approved by
authorities for human gene therapy.
[0008] A one-vector system described in Maddalena A. et al. (Mol
Ther Nucleic Acids. 2013, 2:e106) exhibits leaky expression of GDNF
in the non-Mfp-induced state. Other regulated vector systems, e.g.,
as described in Liu Y. et al. (Molecular Therapy. 2008,
16(3):474-480) and Naidoo J. et al. (Neurology Research Int. 2012,
267, No. 5202), are based on the use of active transcriptional
silencers, such as the Tet repressor, and/or are based on viruses,
which will hamper or prevent the clinical use of these systems.
[0009] Thus, there is a need in the art for a regulated AAV
vector-based expression system with at least the following
features: [0010] all elements of the system (e.g., GS system) for
regulated expression of a therapeutic molecule (e.g., a
neurotrophic factor, such as GDNF) should be contained in one
vector genome; [0011] after injection of this vector into the
patient (e.g., into a specific tissue/organ, such as the brain), it
should not express the therapeutic molecule in the absence of the
activator molecule (e.g., Mfp); [0012] after application of the
activator molecule, levels of the therapeutic molecule should
increase substantially to levels of therapeutic value; and [0013]
after withdrawal of the activator molecule levels of the
therapeutic molecule should decline to background levels again.
SUMMARY OF THE INVENTION
[0014] In one aspect, the present invention relates to an
adeno-associated virus (AAV) vector comprising
(i) a first expression cassette directing the expression of a
regulator protein under the control of a first promoter, wherein
the regulator protein is activated in the presence of an activator
molecule, and (ii) a second expression cassette directing the
expression of a molecule of interest, wherein the second expression
cassette comprises a promoter region, and the expression of the
molecule of interest is induced by binding of the activated
regulator protein to the promoter region, wherein the first
expression cassette and the second expression cassette are arranged
in a tail-to-head configuration.
[0015] In one embodiment, the first promoter has one or more of the
following features:
(i) it is a constitutive promoter; (ii) it is selected from the
group consisting of cell-specific promoters, tissue-specific
promoters and organ-specific promoters; (iii) it is selected from
the group consisting of human synapsin 1 gene (hSYN1) promoter,
tubulin alpha 1 (Ta1) promoter, glial fibrillary acidic protein
(GFAP) promoter, cytomegalovirus (CMV) promoter, human
beta-actin-CMV hybrid promoter and functional fragments or variants
of any of the foregoing.
[0016] In one embodiment, the regulator protein is a gene switch
fusion protein comprising a GAL4 DNA binding domain, a truncated
progesterone receptor ligand binding domain and a p65
transactivation domain from NF-kappaB.
[0017] In one embodiment, the activator molecule is mifepristone
(Mfp).
[0018] In one embodiment, the first expression cassette comprises,
in 5' to 3' direction, the first promoter, a coding sequence for
the regulator protein and a first polyadenylation signal sequence,
wherein, optionally, the first expression cassette further
comprises a synthetic intron arranged between the coding sequence
for the regulator protein and the first polyadenylation signal
sequence.
[0019] In one embodiment, the promoter region comprises one or more
binding sites for the activated regulator protein, a second
promoter and, optionally, a synthetic intron.
[0020] In one embodiment, the one or more binding sites for the
activated regulator protein are GAL4 binding sites.
[0021] In one embodiment, the second promoter is a minimal promoter
which is induced by the binding of the activated regulator protein
to the one or more binding sites for the activated regulator
protein.
[0022] In one embodiment, the second promoter is a minimal promoter
comprising a TATA sequence and/or an mRNA initiation sequence.
[0023] In one embodiment, the molecule of interest is a
therapeutically active peptide or protein or a therapeutically
active oligo- or polynucleotide.
[0024] In one embodiment, the molecule of interest is a
neurotrophic factor.
[0025] In one embodiment, the second expression cassette comprises,
in 5' to 3' direction, the promoter region, a coding sequence for
the molecule of interest and a second polyadenylation signal
sequence.
[0026] In one embodiment, the first expression cassette and the
second expression cassette are separated by a nucleotide sequence
comprising an insulator element.
[0027] In one embodiment, the insulator element is a transcription
blocker comprising a transcription pause site and a polyadenylation
signal sequence.
[0028] In one embodiment, the AAV vector comprises the nucleotide
sequence represented by SEQ ID NO: 1 or a functional variant
thereof, wherein the functional variant has a nucleotide sequence
which is at least 80% or at least 85% or at least 90% or at least
95% identical to SEQ ID NO: 1.
[0029] In one embodiment, in the absence of the activator molecule,
the molecule of interest is not expressed in a host or is expressed
in a host at a level which is at most 10-fold or at most 5-fold or
at most 4-fold or at most 3-fold or at most 2-fold increased as
compared to the normal expression level of the molecule of interest
in the host, wherein the host is a cell, tissue or organ.
[0030] In another aspect, the present invention relates to an
adeno-associated virus (AAV) vector construct comprising
(i) a first expression cassette directing the expression of a
regulator protein under the control of a first promoter, wherein
the regulator protein is activated in the presence of an activator
molecule, and (ii) a second expression cassette comprising a
multiple cloning site allowing the insertion of a coding sequence
for a molecule of interest, wherein the second expression cassette
comprises a promoter region, and the expression of the molecule of
interest is induced by binding of the activated regulator protein
to the promoter region, wherein the first expression cassette and
the second expression cassette are arranged in a tail-to-head
configuration.
[0031] In one embodiment, the AAV vector construct comprises the
nucleotide sequence represented by SEQ ID NO: 2 or a functional
variant thereof, wherein the functional variant has a nucleotide
sequence which is at least 80% or at least 85% or at least 90% or
at least 95% identical to SEQ ID NO: 2.
[0032] In another aspect, the present invention relates to the AAV
vector as defined above, wherein the molecule of interest is a
therapeutically active peptide or protein or a therapeutically
active oligo- or polynucleotide, for use as a medicament.
[0033] In another aspect, the present invention relates to the AAV
vector as defined above, wherein the molecule of interest is a
neurotrophic factor, for use in treating, ameliorating or
preventing a disease or disorder selected from the group consisting
of Parkinson's disease, Huntington's disease, spinal cord lesion
and an amyloid-related disorder, wherein, preferably, the
amyloid-related disorder is selected from the group consisting of
Alzheimer's disease (e.g., sporadic Alzheimer's disease or familial
Alzheimer's disease), cerebral amyloid angiopathy, dementia, motor
neuropathy, Down's syndrome, Creutzfeld Jacob disease,
transmissible spongiform encephalopathies, hereditary cerebral
hemorrhage with amyloidosis Dutch type, HIV-related dementia,
fronto-temporal dementia, Lewy body disease, mixed dementias, head
trauma, familial Danish Dementia, familial British Dementia,
inclusion body myositis (IBM), neuronal disorder related to aging,
and chronic pain.
[0034] In another aspect, the present invention relates to the use
of the AAV vector as defined above for the preparation of a
medicament for treating, ameliorating or preventing a disease or
disorder in a subject, wherein the molecule of interest is a
therapeutically active peptide or protein or therapeutically active
oligo- or polynucleotide.
[0035] In another aspect, the present invention relates to a method
for treating, ameliorating or preventing a disease or disorder in a
subject, comprising
(a) introducing into the subject the AAV vector as defined above;
and (b) administering to the subject the activator molecule to
induce expression of the molecule of interest, wherein the molecule
of interest is a therapeutically active peptide or protein or
therapeutically active oligo- or polynucleotide.
[0036] In some embodiments of said use or said method, the molecule
of interest is a neurotrophic factor and the disease or disorder is
selected from the group consisting of Parkinson's disease,
Huntington's disease, spinal cord lesion and an amyloid-related
disorder, wherein, preferably, the amyloid-related disorder is
selected from the group consisting of Alzheimer's disease (e.g.,
sporadic Alzheimer's disease or familial Alzheimer's disease),
cerebral amyloid angiopathy, dementia, motor neuropathy, Down's
syndrome, Creutzfeld Jacob disease, transmissible spongiform
encephalopathies, hereditary cerebral hemorrhage with amyloidosis
Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy
body disease, mixed dementias, head trauma, familial Danish
Dementia, familial British Dementia, inclusion body myositis (IBM),
neuronal disorder related to aging, and chronic pain.
[0037] In another aspect, the present invention relates to a host
cell comprising the AAV vector as defined above.
[0038] In another aspect, the present invention relates to a
non-human transgenic animal comprising the AAV vector as defined
above.
[0039] In another aspect, the present invention relates to a
pharmaceutical composition comprising the AAV vector as defined
above or the host cell as defined above.
[0040] In yet another aspect, the present invention relates to a
kit comprising the AAV vector as defined above or the AAV vector
construct as defined above or the host cell as defined above or the
pharmaceutical composition as defined above.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 shows the components of the gene switch (GS)
system.
[0042] FIG. 2 shows different one-vector GS-GDNF genomes tested by
the inventor. SV40=simian virus 40 polyadenylation site;
Intron=polynucleotide containing splice donor and acceptor sites;
GS=cDNA of the gene switch fusion protein; hSYN1=fragment of human
synapsin 1 gene promoter; uasTATA=regulated promoter consisting of
six GAL4 binding sites and a minimal TATA promoter plus splice
donor and acceptor sites; GDNF=cDNA of GDNF; BGH=bovine growth
hormone polyadenylation site; TB=polynucleotide containing
synthetic transcription pause and polyadenylation site.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Although the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodologies, protocols and reagents described herein
as these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention, which will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
ordinary skill in the art. Preferably, the terms used herein are
defined as described in "A multilingual glossary of
biotechnological terms (IUPAC Recommendations)", H. G. W.
Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta,
CH-4010 Basel, Switzerland, (1995). The practice of the present
invention will employ, unless otherwise indicated, conventional
methods of chemistry, biochemistry, cell biology, immunology, and
recombinant DNA techniques which are explained in the literature in
the field (cf., e.g., Molecular Cloning: A Laboratory Manual,
3.sup.rd Edition, J. Sambrook et al. eds., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor 2000).
[0044] In the following, certain elements of the present invention
will be described. These elements may be listed with specific
embodiments, however, it should be understood that they may be
combined in any manner and in any number to create additional
embodiments. The variously described examples and preferred
embodiments should not be construed to limit the present invention
to only the explicitly described embodiments. This description
should be understood to support and encompass embodiments, which
combine the explicitly described embodiments with any number of the
disclosed and/or preferred elements. Furthermore, any permutations
and combinations of all described elements in this application
should be considered disclosed by the description of the present
application unless the context indicates otherwise.
[0045] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated member, integer or step or group
of members, integers or steps but not the exclusion of any other
member, integer or step or group of members, integers or steps
although in some embodiments such other member, integer or step or
group of members, integers or steps may be excluded, i.e. the
subject-matter consists in the inclusion of a stated member,
integer or step or group of members, integers or steps. The terms
"a" and "an" and "the" and similar reference used in the context of
describing the invention (especially in the context of the claims)
are to be construed to cover both the singular and the plural,
unless otherwise indicated herein or clearly contradicted by
context. Recitation of ranges of values herein is merely intended
to serve as a shorthand method of referring individually to each
separate value falling within the range. Unless otherwise indicated
herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as"), provided herein is intended merely to better
illustrate the invention and does not pose a limitation on the
scope of the invention otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element essential to the practice of the invention.
[0046] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0047] The present invention provides an adeno-associated virus
(AAV) vector comprising
(i) a first expression cassette directing the expression of a
regulator protein under the control of a first promoter, wherein
the regulator protein is activated in the presence of an activator
molecule, and (ii) a second expression cassette directing the
expression of a molecule of interest, wherein the second expression
cassette comprises a promoter region, and the expression of the
molecule of interest is induced by binding of the activated
regulator protein to the promoter region, wherein the first
expression cassette and the second expression cassette are arranged
in a tail-to-head configuration.
[0048] As used herein, the term "adeno-associated virus (AAV)
vector" means an AAV viral particle containing an AAV vector genome
(which, in turn, comprises the first and second expression
cassettes referred to herein). It is meant to include AAV vectors
of all serotypes, preferably AAV-1 through AAV-9, more preferably
AAV-1, AAV-2, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and
combinations thereof. AAV vectors resulting from the combination of
different serotypes may be referred to as hybrid AAV vectors. In
one embodiment, the AAV vector is selected from the group
consisting of AAV-1, AAV-2, AAV-4, AAV-5 and AAV-6, and
combinations thereof. In one embodiment, the AAV vector is an AAV-5
vector. In one embodiment, the AAV vector is an AAV-5 vector
comprising AAV-2 inverted terminal repeats (ITRs). Also included in
the present invention are AAV vectors comprising variants of the
naturally occurring viral proteins, e.g., one or more capsid
proteins. In one embodiment, the one or more variant capsid
proteins comprise the substitution of one or more amino acid
residues, thereby modifying, preferably improving, the vector
transduction properties. Suitable variants of AAV viral proteins
are known to the skilled person.
[0049] The term "expression cassette", as used herein, refers to a
nucleotide sequence which directs the cell's machinery (or any
other transcription active system, such as an in vitro
transcription/translation system) to express/make a particular
functional product. Generally, an expression cassette comprises a
promoter sequence, an open reading frame (also referred to as
coding region) coding, e.g., for a peptide or protein, and a 3'
untranslated region (3' UTR), which preferably contains a
polyadenylation signal sequence. An expression cassette according
to the present invention may further comprise a 5' untranslated
region (5' UTR), which is located 3' of the promoter (region) and
5' of the coding region.
[0050] The term "tail-to-head configuration", as used herein, means
that, in the AAV vector, (i) the 3' end of the first expression
cassette is adjacent to the 5' end of the second expression
cassette or (ii) the 3' end of the second expression cassette is
adjacent to the 5' end of the first expression cassette. In other
words, the first expression cassette and the second expression
cassette are oriented in the same transcriptional direction. In a
preferred embodiment, the 3' end of the first expression cassette
is adjacent to the 5' end of the second expression cassette.
[0051] In accordance with the present invention, "adjacent" can be
"directly adjacent" or "indirectly adjacent". The term "indirectly
adjacent", as used herein, refers to the situation where the first
expression cassette and the second expression cassette are
separated by a nucleotide sequence. In one embodiment, the
nucleotide sequence consists of less than 1500 nucleotides, less
than 1000 nucleotides, less than 500 nucleotides, less than 250
nucleotides, less than 200 nucleotides, or less than 150
nucleotides. In one embodiment, the nucleotide sequence comprises,
essentially consists of or consists of an insulator element.
[0052] The term "insulator element", as used herein, refers to a
nucleotide sequence, preferably a synthetic nucleotide sequence,
reducing or preventing expression of the molecule of interest in
the absence of the activator molecule. Such nucleotide sequences
are known to a person skilled in the art. In one embodiment, the
insulator element is a transcription blocker (TB) comprising,
essentially consisting of or consisting of a transcription pause
site and a polyadenylation signal sequence (also referred to herein
as a polyadenylation site).
[0053] In one embodiment, the first promoter is a constitutive
promoter. The term "constitutive promoter", as used herein, refers
to an unregulated promoter that allows for continual transcription
of its associated gene.
[0054] In one embodiment, the first promoter is selected from the
group consisting of human synapsin 1 gene (hSYN1) promoter, tubulin
alpha 1 (Ta1) promoter, glial fibrillary acidic protein (GFAP)
promoter, cytomegalovirus (CMV) promoter, human beta-actin-CMV
hybrid promoter and functional fragments or variants of any of the
foregoing.
[0055] In one embodiment, the first promoter allows for ubiquitous
expression of the regulator protein and is preferably selected from
the group consisting of cytomegalovirus (CMV) promoter, human
beta-actin-CMV hybrid promoter and functional fragments or variants
of any of the foregoing.
[0056] In one embodiment, the first promoter is selected from the
group consisting of cell-specific promoters, tissue-specific
promoters and organ-specific promoters. In one embodiment, the
promoter is a neuron-specific promoter or an astrocyte-specific
promoter. In one embodiment, the neuron-specific promoter is
selected from the group consisting of human synapsin 1 gene (hSYN1)
promoter, tubulin alpha 1 (Ta1) promoter and functional fragments
or variants of any of the foregoing. In one embodiment, the
neuron-specific promoter is human synapsin 1 gene (hSYN1) promoter
or a functional fragment or variant thereof. In one embodiment, the
astrocyte-specific promoter is glial fibrillary acidic protein
(GFAP) promoter or a functional fragment or variant thereof.
[0057] The term "regulator protein", as used herein, refers to a
protein (e.g., a fusion protein), which, upon interaction with an
activator molecule (in particular, upon binding of the activator
molecule to the regulator protein), undergoes a conformational
change to an activated state and binds to the promoter region of
the second expression cassette, thereby inducing the expression of
the molecule of interest. According to the present invention, the
regulator protein does not bind to the promoter region of the
second expression cassette in its inactive state. In one
embodiment, the regulator protein is a fusion protein comprising a
(truncated) progesterone receptor ligand binding domain. In one
embodiment, the fusion protein is a gene switch fusion protein.
Suitable gene switch fusion proteins that can be used in accordance
with the present invention are described, for example, in WO
2002/24899 A2, WO 2009/045370 A2, Wang Y. et al., Proc. Natl. Acad.
Sci. USA. 1994, 91:8180-8184, and Wang Y. et al., Gene Ther. 1997,
4:432-441. In one embodiment, the regulator protein is a gene
switch fusion protein comprising, essentially consisting of or
consisting of a GAL4 DNA binding domain (e.g., amino acids 1 to 93
of S. cerevisiae GAL4), a truncated progesterone receptor ligand
binding domain (e.g., amino acids 640 to 914 of human progesterone
receptor) and a p65 transactivation domain from NF-kappaB (e.g.,
amino acids 283 to 551 of the human p65 subunit of NF-kappaB).
[0058] The term "activator molecule", as used herein, refers to a
molecule (e.g., a small molecule compound) that activates the
regulator protein in vivo and is, preferably, pharmaceutically
acceptable. Preferably, the activator molecule is a ligand of the
regulator protein, wherein activation of the regulator protein may,
for example, comprise the dimerization of the regulator protein.
Suitable activator molecules that can be used in accordance with
the present invention are described, for example, in WO 2002/24899
A2, WO 2009/045370 A2, Wang Y. et al., Proc. Natl. Acad. Sci. USA.
1994, 91:8180-8184, and Wang Y. et al., Gene Ther. 1997, 4:432-441.
In one embodiment, the activator molecule is an anti-progestin. In
one embodiment, the activator molecule is mifepristone (Mfp;
RU486).
[0059] A commercially available gene switch system that can be used
in accordance with the present invention is the GeneSwitch.TM.
System (ThermoFisher Scientific).
[0060] The AAV vector of the present invention may comprise one or
more introns. The term "intron" as used herein refers to a sequence
encoded in a DNA sequence that is transcribed into an RNA molecule
by RNA polymerase but is removed by splicing to form the mature
messenger RNA. A "synthetic intron" refers to a sequence that is
not initially replicated from a naturally occurring intron sequence
and generally will not have a naturally occurring sequence, but
will be removed from an RNA transcript during normal
post-transcriptional processing. Preferably, the synthetic intron
includes consensus sequences for the 5' splice site, 3' splice
site, and, optionally, the branch point. Such synthetic intron,
when introduced into the AAV vector of the present invention, may
direct the splicing of RNA transcripts in a highly efficient and
accurate manner, thereby minimizing cryptic splicing and maximizing
production of the desired gene product, e.g., the regulator protein
and/or the molecule of interest. In one embodiment, the synthetic
intron is an IVS8 synthetic intron.
[0061] In one embodiment, the AAV vector of the present invention
does not comprise a(n) (active) transcriptional silencer, such as a
Tet repressor.
[0062] In one embodiment, the first expression cassette comprises
or essentially consists of, in 5' to 3' direction, the first
promoter, a coding sequence for the regulator protein and a first
polyadenylation signal sequence. Optionally, the first expression
cassette further comprises a synthetic intron arranged between the
coding sequence for the regulator protein and the first
polyadenylation signal sequence and/or a synthetic intron located
in the 5' UTR, if present.
[0063] In one embodiment, the promoter region in the second
expression cassette comprises, essentially consists of or consists
of one or more binding sites for the activated regulator protein, a
second promoter and, optionally, a synthetic intron.
[0064] In one embodiment, the one or more binding sites for the
activated regulator protein are GAL4 binding sites, also referred
to herein as GAL4 upstream activating sequences (UAS). In one
embodiment, the promoter region comprises six GAL4 binding
sites.
[0065] In one embodiment, the second promoter is a minimal promoter
which is induced by the binding of the activated regulator protein
to the one or more binding sites for the activated regulator
protein. The term "minimal promoter", as used herein, refers to
minimal portion of a promoter required to properly initiate
transcription. In one embodiment, the second promoter is a minimal
promoter comprising, essentially consisting of or consisting of a
TATA sequence and/or an mRNA initiation sequence. In one
embodiment, the second promoter is a minimal promoter comprising
the adenovirus E1b TATA sequence.
[0066] In one embodiment, the second expression cassette comprises,
essentially consists of or consists of, in 5' to 3' direction, the
promoter region, a coding sequence for the molecule of interest and
a second polyadenylation signal sequence. The second expression
cassette may also comprise one or more synthetic introns, e.g., in
the promoter region and/or arranged between the coding sequence for
the molecule of interest and the second polyadenylation signal
sequence.
[0067] According to the present invention, the first
polyadenylation signal sequence and the second polyadenylation
signal sequence may be the same or different. In one embodiment,
the first polyadenylation signal sequence and the second
polyadenylation signal sequence are independently selected from the
group consisting of a simian virus 40 (SV40) polyadenylation signal
sequence, a bovine growth hormone (bGH) polyadenylation signal
sequence and a human growth hormone (hGH) polyadenylation signal
sequence. In one embodiment, the first polyadenylation signal
sequence is a simian virus 40 (SV40) polyadenylation signal
sequence, and the second polyadenylation signal sequence is a
bovine growth hormone (bGH) polyadenylation signal sequence.
[0068] In the context of the present invention, the term "DNA"
relates to a molecule, which comprises deoxyribonucleotide residues
and preferably is entirely or substantially composed of
deoxyribonucleotide residues. "Deoxyribonucleotide" relates to a
nucleotide, which lacks a hydroxyl group at the 2'-position of a
.beta.-D-ribofuranosyl group. The term "DNA" comprises isolated DNA
such as partially or completely purified DNA, essentially pure DNA,
synthetic DNA, and recombinantly generated DNA and includes
modified DNA, which differs from naturally occurring DNA by
addition, deletion, substitution and/or alteration of one or more
nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of a DNA or
internally, for example at one or more nucleotides of the DNA.
Nucleotides in DNA molecules can also comprise non-standard
nucleotides, such as non-naturally occurring nucleotides or
chemically synthesized nucleotides. These altered DNAs can be
referred to as analogs or analogs of naturally occurring DNA.
[0069] The term "molecule of interest", as used herein, refers to a
macromolecule, such as but not limited to RNA, a peptide and a
polypeptide or protein.
[0070] In one embodiment, the molecule of interest is a
therapeutically active peptide or protein or a therapeutically
active oligo- or polynucleotide. The term "peptide", as used
herein, generally relates to substances which include at least 2,
at least 3, at least 4, at least 6, at least 8, at least 10, at
least 12 or at least 14 and preferably up to 8, 10, 12, 14, 16, 18,
20, 25, 30, 50, or 100 consecutive amino acids which are connected
together by peptide bonds. The terms "polypeptide" and "protein",
as used herein, relate to large peptides, preferably peptides
having more than 100 amino acids, but the terms "peptide",
"polypeptide" and "protein" may be used interchangeably herein. The
term "oligonucleotide", as used herein, refers to short DNA or RNA
molecules, preferably with 30 or less nucleotide residues. The term
"polynucleotide", as used herein, refers to long DNA or RNA
molecules, preferably with more than 30 nucleotide residues. In one
embodiment, the oligo- or polynucleotide is an RNA oligo- or
polynucleotide.
[0071] In the context of the present invention, the term "RNA"
relates to a molecule, which comprises ribonucleotide residues and
preferably is entirely or substantially composed of ribonucleotide
residues. "Ribonucleotide" relates to a nucleotide with a hydroxyl
group at the 2'-position of a .beta.-D-ribofuranosyl group. The RNA
may, for example, be a ribozyme, an antisense RNA or an miRNA.
[0072] The term "therapeutically active", as used herein, refers to
a molecule of interest which has a therapeutic/pharmacologic effect
when administered appropriately to a subject suffering from a
disease or disorder. Such therapeutic/pharmacologic effect is one
that is expected to be related to a beneficial effect on the course
or a symptom of the disease or disorder.
[0073] In one embodiment, the molecule of interest is a
neurotrophic factor. The term "neurotrophic factor", as used
herein, refers to proteins that are involved in or responsible for
the growth and survival of developing neurons and the maintenance
of mature neurons. In one embodiment, the neurotrophic factor is
selected from the group consisting of glial cell line-derived
neurotrophic factor (GDNF), brain-derived neurotrophic factor
(BDNF), neurotrophin 3 (NT-3), nerve growth factor (NGF), neurturin
(NRTN), artemin (ARTN) and persephin (PSPN). GDNF, NRTN, ARTN and
PSPN are members of the GDNF family of ligands (GFL), which may be
preferred neurotrophic factors in accordance with the present
invention. In one embodiment, the neurotrophic factor is GDNF.
[0074] The molecule of interest may, in accordance with the present
invention, also be a peptide or protein or a oligo- or
polynucleotide, which is not therapeutically active. Such molecule
of interest may, for example, be a fluorescent protein, such as
(E)GFP, RFP, YFP and derivatives thereof, or a luminescent protein,
such as luciferase and derivatives thereof.
[0075] In one embodiment, the AAV vector comprises the nucleotide
sequence represented by SEQ ID NO: 1 (or a nucleotide sequence
complementary to SEQ ID NO: 1) or a functional variant thereof,
wherein the functional variant has a nucleotide sequence which is
at least 80% or at least 85% or at least 90% or at least 95%
identical to SEQ ID NO: 1 (or to the nucleotide sequence
complementary to SEQ ID NO: 1).
[0076] In one embodiment, the AAV vector further comprises inverted
terminal repeats (ITRs), e.g., AAV-2 ITRs, flanking the first and
second expression cassettes (or the nucleotide sequence defined
above, if applicable).
[0077] The present invention also provides an AAV vector as defined
herein, wherein the molecule of interest is a therapeutically
active peptide or protein or a therapeutically active oligo- or
polynucleotide, for use as a medicament. The term "medicament", as
used herein, refers to a substance/composition used in therapy,
i.e., in treating, ameliorating or preventing a disease or
disorder. According to the invention, the terms "disease" or
"disorder" refer to any pathological state. In one embodiment, the
disease or disorder is a neurological or neurodegenerative
disease.
[0078] The present invention also provides an AAV vector as defined
herein, wherein the molecule of interest is a neurotrophic factor,
for use in treating, ameliorating or preventing a disease or
disorder selected from the group consisting of Parkinson's disease,
Huntington's disease, spinal cord lesion and an amyloid-related
disorder, wherein, preferably, the amyloid-related disorder is
selected from the group consisting of Alzheimer's disease (e.g.,
sporadic Alzheimer's disease or familial Alzheimer's disease),
cerebral amyloid angiopathy, dementia, motor neuropathy, Down's
syndrome, Creutzfeld Jacob disease, transmissible spongiform
encephalopathies, hereditary cerebral hemorrhage with amyloidosis
Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy
body disease, mixed dementias, head trauma, familial Danish
Dementia, familial British Dementia, inclusion body myositis (IBM),
neuronal disorder related to aging, and chronic pain.
[0079] In one embodiment, the neurotrophic factor is selected from
the group consisting of GDNF, BDNF, NT-3, NGF, NRTN, ARTN and PSPN,
and the AAV vector is for use in treating, ameliorating or
preventing a disease or disorder selected from the group consisting
of Parkinson's disease, Huntington's disease and an amyloid-related
disorder, wherein, preferably, the amyloid-related disorder is
selected from the group consisting of Alzheimer's disease (e.g.,
sporadic Alzheimer's disease or familial Alzheimer's disease),
cerebral amyloid angiopathy, dementia, motor neuropathy, Down's
syndrome, Creutzfeld Jacob disease, transmissible spongiform
encephalopathies, hereditary cerebral hemorrhage with amyloidosis
Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy
body disease, mixed dementias, head trauma, familial Danish
Dementia, familial British Dementia, inclusion body myositis (IBM),
neuronal disorder related to aging, and chronic pain. In one
embodiment, the disease or disorder is Parkinson's disease.
[0080] In one embodiment, the neurotrophic factor is BDNF, and the
AAV vector is for use in treating, ameliorating or preventing a
spinal cord lesion.
[0081] The present invention further provides the use of the AAV
vector as defined herein for the preparation of a medicament for
treating, ameliorating or preventing a disease or disorder in a
subject, wherein the molecule of interest is a therapeutically
active peptide or protein or therapeutically active oligo- or
polynucleotide.
[0082] The present invention further provides a method for
treating, ameliorating or preventing a disease or disorder in a
subject, comprising
(a) introducing into the subject the AAV vector as defined herein;
and (b) administering to the subject the activator molecule to
induce expression of the molecule of interest, wherein the molecule
of interest is a therapeutically active peptide or protein or
therapeutically active oligo- or polynucleotide.
[0083] According to the present invention, step (a) may be
performed in vivo (by introducing the AAV vector directly into
cells of the subject) or at least partially ex vivo (by
transferring the AAV vector into isolated cells of the subject or
non-autologous cells and introducing the modified cells into the
subject or into a different subject). Said introducing may be
performed by any suitable method, either systemically (e.g.,
orally, intravenously, sublingually, transdermally) or locally
(e.g., intraperitoneally, intrathecally, intraventricularly or by
direct injection into the target tissue or organ). In one
embodiment, the AAV vector is introduced into the subject by
injection, e.g., by direct injection into the target tissue or
organ (e.g., the brain).
[0084] The activator molecule may be administered by any suitable
method, either systemically (e.g., orally, intravenously,
sublingually, transdermally) or locally (e.g., intraperitoneally,
intrathecally, intraventricularly or by direct injection into the
tissue or organ where the AAV vector was introduced).
Administration of the activator molecule can occur once or several
times, continuously or intermittently.
[0085] The optimal time interval between step (a) and step (b) can
be determined for each type of cell/tissue/organ and disease or
disorder using only routine techniques.
[0086] The term "subject", as used herein, relates to any organism
such as a vertebrate, particularly any mammal, including both a
human and another mammal, e.g., an animal such as a rodent, a
rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a
non-human primate (e.g., a monkey). The rodent may be a mouse, rat,
hamster, guinea pig, or chinchilla. Preferably, the subject is a
human. In one embodiment, a subject is a subject with or suspected
of having a disease or disorder, in particular a disease or
disorder as disclosed herein, also designated "patient" herein.
[0087] In some embodiments of said use or said method, the molecule
of interest is a neurotrophic factor and the disease or disorder is
selected from the group consisting of Parkinson's disease,
Huntington's disease, spinal cord lesion and an amyloid-related
disorder, wherein, preferably, the amyloid-related disorder is
selected from the group consisting of Alzheimer's disease (e.g.,
sporadic Alzheimer's disease or familial Alzheimer's disease),
cerebral amyloid angiopathy, dementia, motor neuropathy, Down's
syndrome, Creutzfeld Jacob disease, transmissible spongiform
encephalopathies, hereditary cerebral hemorrhage with amyloidosis
Dutch type, HIV-related dementia, fronto-temporal dementia, Lewy
body disease, mixed dementias, head trauma, familial Danish
Dementia, familial British Dementia, inclusion body myositis (IBM),
neuronal disorder related to aging, and chronic pain.
[0088] The present invention also provides nucleic acid molecules,
in particular DNA molecules, comprised in the AAV vector of the
invention. In one embodiment, said nucleic acid molecule is an AAV
vector genome comprising the first expression cassette and the
second expression cassette as defined herein, wherein the first
expression cassette and the second expression cassette are arranged
in a tail-to-head configuration. Such nucleic acid molecules are,
e.g., provided in the form of an adeno-associated virus (AAV)
vector construct, which, preferably, allows the production of the
AAV vector of the invention by methods known in the art (e.g., as
described in Tereshchenko J. et al., Neurobiol Dis. 2014, 65:35-42;
Maddalena A. et al., Mol Ther Nucleic Acids. 2013, 2:e106; and
Drinkut A. et al., Mol Ther. 2012, 20:534-543). Such AAV vector
construct may, for example, be in the form of a plasmid (referred
to as AAV vector plasmid) or in the form of a linear (expression)
construct. The term "AAV vector plasmid", as used herein, is meant
to refer to a double stranded circular nucleic acid molecule that
contains at least a functional portion of an AAV nucleic acid
molecule.
[0089] The present invention further provides an adeno-associated
virus (AAV) vector construct comprising
(i) a first expression cassette directing the expression of a
regulator protein under the control of a first promoter, wherein
the regulator protein is activated in the presence of an activator
molecule, and (ii) a second expression cassette comprising a
multiple cloning site allowing the insertion of a coding sequence
for a molecule of interest, wherein the second expression cassette
comprises a promoter region, and the expression of the molecule of
interest is induced by binding of the activated regulator protein
to the promoter region, wherein the first expression cassette and
the second expression cassette are arranged in a tail-to-head
configuration.
[0090] Such AAV may be referred to as an empty AAV vector
construct, i.e., without a coding sequence for a molecule of
interest. Preferably, all elements of this empty AAV vector
construct are as defined herein in connection with the AAV
vector.
[0091] In one embodiment, the AAV vector construct comprises the
nucleotide sequence represented by SEQ ID NO: 2 (or a nucleotide
sequence complementary to SEQ ID NO: 2) or a functional variant
thereof, wherein the functional variant has a nucleotide sequence
which is at least 80% or at least 85% or at least 90% or at least
95% identical to SEQ ID NO: 2 (or to the nucleotide sequence
complementary to SEQ ID NO: 2).
[0092] In one embodiment, the AAV vector construct further
comprises inverted terminal repeats (ITRs), e.g., AAV-2 ITRs,
flanking the first and second expression cassettes (or the
nucleotide sequence defined above, if applicable).
[0093] In one embodiment, the AAV vector construct comprises a
coding sequence for a molecule of interest, which is inserted into
said multiple cloning site.
[0094] In one embodiment, the AAV vector construct does not
comprise a(n) (active) transcriptional silencer, such as a Tet
repressor.
[0095] Preferably, the AAV vectors of the present invention ensure
that, in the absence of the activator molecule, the molecule of
interest is not expressed in a host or is expressed in a host at a
level which is at most 10-fold or at most 5-fold or at most 4-fold
or at most 3-fold or at most 2-fold increased as compared to the
normal expression level of the molecule of interest in the host,
wherein the host is a cell, tissue or organ.
[0096] The terms "part" or "fragment" or "portion" are used
interchangeably herein and refer to a continuous element. For
example, a part of a structure, such as an amino acid sequence or
nucleotide sequence, refers to a continuous element of said
structure. For example, a part or fragment of a nucleotide sequence
preferably comprises a sequence of at least 6, in particular at
least 8, at least 12, at least 15, at least 20, at least 30, at
least 50, at least 100, at least 150, at least 160, at least 170,
at least 180, at least 190 or at least 200 consecutive nucleotides
of the nucleotide sequence.
[0097] For the purposes of the present invention, "variants" of an
amino acid sequence or nucleotide sequence comprise amino
acid/nucleotide insertion variants, amino acid/nucleotide addition
variants, amino acid/nucleotide deletion variants and/or amino
acid/nucleotide substitution variants. Amino acid/nucleotide
insertion variants comprise insertions of single or two or more
amino acids/nucleotides in a particular amino acid sequence or
nucleotide sequence. In the case of amino acid/nucleotide sequence
variants having an insertion, one or more amino acid/nucleotide
residues are inserted into a particular site in an amino acid
sequence or nucleotide sequence, although random insertion with
appropriate screening of the resulting product is also possible.
Amino acid/nucleotide addition variants comprise N- and/or
C-terminal fusions of one or more amino acids/nucleotides, such as
1, 2, 3, 5, 10, 20, 30, 50, or more amino acids/nucleotides. Amino
acid/nucleotide deletion variants are characterized by the removal
of one or more amino acids/nucleotides from the sequence, such as
by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino
acids/nucleotide. The deletions may be in any position of the amino
acid sequence or nucleotide sequence, for example at the N- and/or
C-terminus. Amino acid/nucleotide deletion variants that comprise
the deletion at the N-terminal and/or C-terminal end of the amino
acid sequence or nucleotide sequence are also called N-terminal
and/or C-terminal truncation variants. Amino acid/nucleotide
substitution variants are characterized by at least one residue in
the sequence being removed and another residue being inserted in
its place. In one embodiment, the amino acid/nucleotide
substitution variant comprises the substitution of up to 10, 9, 8,
7, 6, 5, 4, 3 or 2 amino acids/nucleotides.
[0098] The term "variant", as used herein in connection with a
promoter, may also refer to mutants, species variants and
homologues of said promoter, including those, which occur
naturally.
[0099] Alternatively or additionally, a "variant" as used herein,
can be characterized by a certain degree of sequence identity to
the parent amino acid sequence or nucleotide sequence from which it
is derived. More precisely, an amino acid sequence variant in the
context of the present invention may exhibit at least 80% sequence
identity to its parent amino acid sequence. A nucleotide sequence
variant in the context of the present invention may exhibit at
least 80% sequence identity to its parent nucleotide sequence. The
term "at least 80% identical to", as used herein, refers to a
sequence identity of at least 80%, at least 81%, at least 82%, at
least 83%, at least 84%, at least 85%, at least 86%, at least 87%,
at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, or at least 99% to the respective
parent/reference amino acid sequence or to the respective
parent/reference nucleotide sequence. Preferably, the amino acid
sequence in question and the parent/reference amino acid sequence
exhibit the indicated sequence identity over the entire length of
the parent/reference amino acid sequence. Preferably, the
nucleotide sequence in question and the parent/reference nucleotide
sequence exhibit the indicated sequence identity over the entire
length of the parent/reference nucleotide sequence.
[0100] The similarity of nucleotide and amino acid sequences, i.e.,
the percentage of sequence identity, can be determined via sequence
alignments. Such alignments can be carried out with several
art-known algorithms, preferably with the mathematical algorithm of
Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877), with hmmalign (HMMER package,
http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson J.
D. et al. Nucleic Acids Res. 1994, 22:4673-80) available e.g. on
http://www.ebi.ac.uk/Tools/clustalw/ or on
http://www.ebi.ac.uk/Tools/clustalw2/index.html or on
http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw-
.html. Preferred parameters used are the default parameters as they
are set on http://www.ebi.ac.uk/Tools/clustalw/ or
http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of
sequence identity (sequence matching) may be calculated using e.g.
BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is
incorporated into the BLASTN and BLASTP programs of Altschul et al.
J. Mol. Biol. 1990, 215:403-410. BLAST polynucleotide searches are
performed with the BLASTN program, score=100, word length=12, to
obtain polynucleotide sequences that are homologous to those
nucleic acids which encode F, N, or M2-1. BLAST protein searches
are performed with the BLASTP program, score=50, word length=3, to
obtain amino acid sequences homologous to the F polypeptide, N
polypeptide, or M2-1 polypeptide. To obtain gapped alignments for
comparative purposes, Gapped BLAST is utilized as described in
Altschul et al. Nucleic Acids Res. 1997, 25:3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs are used. Sequence matching analysis may
be supplemented by established homology mapping techniques like
Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62)
or Markov random fields.
[0101] The terms "functional fragment" and "functional variant", as
used herein in connection with a promoter, refers to a fragment or
variant of a promoter which is functional in the sense that it has
the same or essentially the same activity (in particular properly
initiating transcription and/or cell-/tissue-/organ-specificity, if
applicable) as said promoter. The term "functional variant", as
used herein in connection with the nucleotide sequence comprised in
an AAV vector of the present invention (e.g., the nucleotide
sequence of SEQ ID NO: 1 or SEQ ID NO: 2), refers to a variant of
said nucleotide sequence which is functional in the sense that it
allows the regulatable expression of a molecule of interest to the
same or essentially the same degree as the AAV vector comprising
said nucleotide sequence (e.g., the nucleotide sequence of SEQ ID
NO: 1 or SEQ ID NO: 2), wherein, preferably, in the absence of the
activator molecule, the molecule of interest is not expressed in a
host or is expressed in a host at a level which is at most 10-fold
or at most 5-fold or at most 4-fold or at most 3-fold or at most
2-fold increased as compared to the normal expression level of the
molecule of interest in the host, wherein the host is a cell,
tissue or organ.
[0102] The present invention also provides a host cell comprising
the AAV vector of the present invention or a nucleic acid molecule
of the present invention, e.g., an AAV vector construct as defined
herein. Such host cell may either be a prokaryotic cell (e.g., a
bacterial cell) or a eukaryotic cell (e.g., a fungal, plant or
animal cell). Preferably, the host cell is an isolated host cell.
In one embodiment, the host cell is a producer cell (or producer
cell line) allowing the production of the AAV vector of the present
invention, e.g., based on an AAV vector construct as defined herein
and with co-transfection of suitable helper constructs, e.g.,
helper plasmids (see, for example, US 2004/0235174 A1).
[0103] Suitable producer cells are known to a person skilled in the
art and include, for example, HEK293 cells.
[0104] The present invention further provides a non-human
transgenic animal comprising the AAV vector of the present
invention. The term "non-human transgenic animal", as used herein,
relates, in particular, to non-human mammals, e.g., a rodent, a
rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a
non-human primate (e.g., a monkey). The rodent may be a mouse, rat,
hamster, guinea pig, or chinchilla.
[0105] The present invention further provides a pharmaceutical
composition comprising the AAV vector or the host cell of the
present invention. A pharmaceutical composition in accordance with
the present invention may further comprise one or more carriers
and/or excipients, all of which are preferably pharmaceutically
acceptable. The term "pharmaceutically acceptable", as used herein,
refers to the non-toxicity of a material, which, preferably, does
not interact with the action of the active component of the
pharmaceutical composition, i.e., the AAV vector or host cell of
the present invention. In particular, "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopoeia, European
Pharmacopoeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to an organic or inorganic component, of a natural or synthetic
nature, in which the active component is combined in order to
facilitate, enhance or enable application. According to the
invention, the term "carrier" also includes one or more compatible
solid or liquid fillers, diluents or encapsulating substances,
which are suitable for administration to a subject. Possible
carrier substances (e.g., diluents) are, for example, sterile
water, Ringer's solution, Lactated Ringer's solution, physiological
saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl
alcohol), phosphate-buffered saline (PBS), Hank's solution, fixed
oils, polyalkylene glycols, hydrogenated naphthalenes and
biocompatible lactide polymers, lactide/glycolide copolymers or
polyoxyethylene/polyoxy-propylene copolymers. In one embodiment,
the carrier is PBS. The resulting solutions or suspensions are
preferably isotonic to the blood of the recipient. Suitable
carriers and their formulations are described in greater detail in
Remington's Pharmaceutical Sciences, 17.sup.th ed., 1985, Mack
Publishing Co. The term "excipient", as used herein, is intended to
include all substances which may be present in a pharmaceutical
composition and which are not active ingredients, such as salts,
binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol,
mannitol), lubricants, thickeners, surface active agents,
preservatives, emulsifiers, buffer substances, stabilizing agents,
flavouring agents or colorants.
[0106] The present invention further provides a kit comprising the
AAV vector or a nucleic acid molecule, e.g., the AAV vector
construct, or the host cell or the pharmaceutical composition of
the present invention. As used herein, the term "kit" (also
referred to as "kit of parts") refers to an article of manufacture
comprising one or more containers and, optionally, a data carrier.
Said one or more containers may be filled with one or more of the
means or reagents disclosed herein, e.g. one container with an AAV
vector of the present invention and one container with the
corresponding activator molecule. Additional containers may be
included in the kit that contain, e.g., diluents, buffers and
further reagents. Said data carrier may be a non-electronical data
carrier, e.g., a graphical data carrier such as an information
leaflet, an information sheet, a bar code or an access code, or an
electronical data carrier such as a floppy disk, a compact disk
(CD), a digital versatile disk (DVD), a microchip or another
semiconductor-based electronical data carrier. The access code may
allow the access to a database, e.g., an internet database, a
centralized, or a decentralized database. Said data carrier may
comprise instructions for the use of the kit in accordance with the
present invention.
[0107] The inventor has surprisingly found that the AAV vectors of
the present invention allow for the regulatable expression of a
molecule of interest, e.g., a therapeutically active peptide or
protein or a therapeutically active oligo- or polynucleotide, with
very low or even zero expression in the non-induced state ("zero
background expression"). Such AAV vectors will allow gene
therapeutic approaches that are more safe and have less unwanted
side effects than current AAV vector-based approaches.
[0108] The present invention is further illustrated by the
following examples, which are not to be construed as limiting the
scope of the invention.
Examples
[0109] Several different configurations of a one-vector GS-GDNF
layout were designed, which were packaged into AAV-5 viral capsids
and tested in the rat brain for GDNF production in absence or
presence of Mfp. FIG. 2 depicts some of the constructs that were
finally assessed, Table 1 shows GDNF levels obtained from rat
brains before and after induction with Mfp.
TABLE-US-00001 TABLE 1 GFNF levels as determined by ELISA in rat
brains, injected with AAV-5 viruses at specified titres. Mfp was
given at 3 weeks after vector injection, tissues were prepared at 1
week after Mfp application. Vector injected into GDNF - Mfp (pg/mg
GDNF + Mfp rat brain Titre (vg) tissue) (pg/mg tissue) no vector --
5 5 # 17 3 .times. 10e9 650 1100 # 20 3 .times. 10e9 175 900 # 21 3
.times. 10e9 9 1900 # 21 1 .times. 10e9 5 1300
[0110] The results shown in Table 1 demonstrate that the layout of
vector #17 (see FIG. 2) made it almost useless for regulated gene
therapy, as in the non-induced state it produced GDNF levels
130-fold over background, while Mfp induction only increased GDNF
levels 2-fold further. Insertion of a small synthetic
polynucleotide containing insulator sequences (TB=transcription
pause and polyadenylation sites; see vector #20) significantly
reduced non-induced GDNF levels and improved the rate of induction,
but non-induced GDNF levels were still 35-fold higher than normal
rat GDNF brain levels.
[0111] Vector #21 contains the same elements as vector #20, except
that the expression cassette for GS is inverted, i.e., the two
expression cassettes are arranged in a tail-to-head configuration
(see FIG. 2 and Table 2). This feature provided dramatically
improved performance of the vector, in that non-induced GDNF levels
were only 2-fold increased as compared to normal background GDNF
levels in the rat brain, while induced GDNF levels were 380-fold
over background. Reducing the injected virus titre from
3.times.10e9 vector genomes to 1.times.10e9 vector genomes resulted
in non-induced GDNF levels indistinguishable from normal rat brain
levels, with still 260-fold induction. At four weeks after Mfp
induction, GDNF levels in rats injected with AAV-5 #21 at
1.times.10e9 vg had returned to background levels again. To the
inventor's knowledge, this is the first description of any
regulated gene transfer system in AAV vectors with zero background
expression in the non-induced state, and the first functional
one-vector genome layout of the gene switch system in AAV
vectors.
[0112] The vector genome of the invention (see, for example, SEQ ID
NOs: 1 and 2) contains unique restriction sites at strategic
positions, allowing easy further manipulation of its layout, for
example taking away splice sites for reduced expression levels of
the GS fusion protein as well as exchange of promoter elements
and/or the transgene/molecule of interest.
TABLE-US-00002 TABLE 2 Annotated sequence of an exemplary vector
genome of the present invention (vector #21; SEQ ID NO: 1). In the
recombinant virus, i.e., in the complete AAV vector, this sequence
was flanked by the AAV-2 inverted terminal repeats. BglII MluI
BamHI HindIII PstI ApaI ------ ------ ------ ------ ------- ------
1 agatctagga tcacgcgtaa aggatccaaa aaaaagctta aactagactg cagagggccc
tgcgtatgag >>......'HSYN-Promoter.......> 71 tgcaagtggg
ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact
>..............................'HSYN-Promoter.........................-
.....> 141 ggacaagcac ccaaccccca ttccccaaat tgcgcatccc
ctatcagaga gggggagggg aaacaggatg
>..............................'HSYN-Promoter.........................-
.....> 211 cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga
cagtgccttc gcccccgcct ggcggcgcgc
>..............................'HSYN-Promoter.........................-
.....> 281 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact
cgccggtccc ccgcaaactc cccttcccgg
>..............................'HSYN-Promoter.........................-
.....> 351 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga
ccgcaccacg cgaggcgcga gatagggggg
>..............................'HSYN-Promoter.........................-
.....> 421 cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
cgctgcctca gtctgcggtg ggcagcggag
>..............................'HSYN-Promoter.........................-
.....> NheI AgeI NcoI ------- ------- ------ 491 gagtcgtgtc
gtgcctgaga gcgcagtcga aagctgctag caaccatcca ccggtcgcca ccatggatag
>........'HSYN-Promoter.........>> >>.....> 561
ccagcagccc gatctgaaat tgctgtcctc tattgaacag gcttgtgata tttgcaggct
taaaaaactc
>.................................pSwitch.............................-
.....> 631 aaatgttcca aagaaaaacc aaagtgtgct aaatgtctga
agaacaactg ggaatgccgc tactccccca
>.................................pSwitch.............................-
.....> 701 agaccaagcg ttctccactt actcgcgctc acctgacaga
ggtagaaagt aggctggaac gcctagaaca
>.................................pSwitch.............................-
.....> EcoRV ------ 771 gttgtttttg ttgatattcc caagagaaga
cctggacatg attcttaaga tggatagcct gcaagatatc
>.................................pSwitch.............................-
.....> 841 aaggcgctcc tggagtttcc aggcgtcgat cagaaaaagt
ttaacaaggt ccgagtcgtc cgagccctag
>.................................pSwitch.............................-
.....> 911 acgccgttgc cctccctcaa cccgtgggcg tgcctaatga
aagccaggct ctctcacagc ggtttacttt
>.................................pSwitch.............................-
.....> 981 cagcccaggg caggatatac agcttatacc tcccctgata
aatttattga tgagtatcga gccggacgtg
>.................................pSwitch.............................-
.....> 1051 atttacgcag ggcatgataa cactaagcca gacacatctt
cttctctcct gaccagccta aaccaactgg
>.................................pSwitch.............................-
.....> 1121 gtgaacggca gcttctgtca gtcgtgaagt ggagcaaatc
cctccctgga tttagaaacc tgcacataga
>.................................pSwitch.............................-
.....> 1191 tgaccaaata acacttattc aatactcctg gatgagctta
atggtgtttg gtctcggatg gcggtcatat
>.................................pSwitch.............................-
.....> 1261 aagcacgtta gcggccagat gctctacttt gcccctgatt
tgattctgaa cgaacaaagg atgaaggaga
>.................................pSwitch.............................-
.....> 1331 gctccttcta tagcctttgt ctgacgatgt ggcaaatccc
gcaggagttt gtaaaactgc aagtgagtca
>.................................pSwitch.............................-
.....> 1401 ggaggagttc ctgtgtatga aagttctact gctgctcaat
acgatcccct tggaagggct cagatcacag
>.................................pSwitch.............................-
.....> 1471 acgcaattcg aagagatgag gagctcttat attagagagc
taattaaggc tattggtctg aggcaaaagg
>.................................pSwitch.............................-
.....> 1541 gtgtcgtgtc cagcagccag agattttacc aacttactaa
actactggac aacctacacg acctagtcaa
>.................................pSwitch.............................-
.....> 1611 gcagctccat ctctactgcc tgaacacctt cattcaatcc
agagccttat ctgtggaatt tccggaaatg
>.................................pSwitch.............................-
.....> NcoI ------- 1681 atgagtgagg tcattgcggg gtcaactccc
atggaatttc agtatctgcc agacaccgat gacaggcacc
>.................................pSwitch.............................-
.....> 1751 gcatcgaaga gaaacggaaa cggacatacg agaccttcaa
gtccattatg aaaaagagtc ccttttctgg
>.................................pSwitch.............................-
.....> 1821 ccctaccgac cccagacccc ctccaaggag aatcgcagtg
ccttccagga gtagtgcatc agttccgaaa
>.................................pSwitch.............................-
.....> NcoI ------- 1891 ccggccccac agccatatcc ttttaccagt
tctctgagta ccattaacta cgacgaattt cctaccatgg
>.................................pSwitch.............................-
.....> 1961 ttttcccctc ggggcaaatt agccaggcgt ccgcgctggc
gcccgcgccc ccccaggtcc ttccgcaggc
>.................................pSwitch.............................-
.....> 2031 tccagcccct gctccagccc ccgcaatggt ttctgccctg
gcacaagccc ccgcacccgt gcctgtgttg
>.................................pSwitch.............................-
.....> 2101 gcaccaggcc cgccacaggc cgtggcacct ccggctccta
aacctactca ggccggagag ggcaccctta
>.................................pSwitch.............................-
.....> 2171 gtgaggccct gttacagctt caatttgacg acgaggacct
cggggctctc cttggcaatt cgacagaccc
>.................................pSwitch.............................-
.....> 2241 cgctgtgttt acagacctgg cttcggtaga caattctgag
tttcagcaac ttctcaacca gggcatcccc
>.................................pSwitch.............................-
.....> BstEII -------- 2311 gtagcccctc atacaacaga gcccatgtta
atggagtacc cagaggctat cacaaggctg gtaaccggcg
>.................................pSwitch.............................-
.....> 2381 cccaaagacc accagatcct gcaccagcac cactgggagc
tcctggttta cccaatggat tattatcagg
>.................................pSwitch.............................-
.....> SpeI ------ 2451 agatgaggat ttcagttcca ttgccgatat
ggacttcagc gcgcttcttt ctcagatcag ctcttgaact
>...............................pSwitch...............................-
...> BlnI ------- StuI ------- 2521 agtaaaaggc ctaggtaagt
atcaaggtta caagacaggt ttaaggagac cacatagaaa ctgggcttgt
<...............................'INTRON.........................-
.....> >> 2591 cgagacagag aagactcttg cgtttctgat aggcacctat
tggtcttact gacatccact ttgcctttct
>.................................'INTRON.............................-
.....> >> StuI PsiI ------ ------ 2661 ctccacaggt
gtaggccttt cgagcaactt gtttattgca gcttataatg gttacaaata aagcaatagc
>.'INTRON.>> >>...>>
>>..........................SV40-pA.>>>>>>>>-
;>>>>>>>>>>>>>>>>>>&-
gt; 2731 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
tttgtccaaa ctcatcaatg
>.................................SV40-pA.............................-
.....> >> XbaI ------ 2801 tatcttatca tgtctggatc
gtctagcatc gaagatccac tagatgcata aatctagaca ataaaatatc
>.............SV40-pA.............>>
>>...TB'...> ClaI ------ 2871 tttattttca ttacatctgt
gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa
>..................TB'.....................>>
>>............'TB.............> 2941 aacaaaacga aacaaaacaa
actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc
>...................................'TB...............................-
.....> ApaI ------- 3011 tctattaggg cccaagcgga gtactgtcct
ccgagtggag tactgtcctc cgagcggagt actgtcctcc >>>
>>.....GAL4....>> >>.....GAL4 ....>>
>>.....GAL4.....> 3081 gagtcgaggg tcgaagcgga gtactgtcct
ccgagtggag tactgtcctc cgagcggagt actgtcctcc >
>>.....GAL4....>> >>.....GAL4 ....>>
>>.....GAL4.....> EcoRV ...... XhoI ------- 3151
gagtcgacta gagggtatat aatggatctc gagatatcgg agctcgttta gtgaaccgtc
agatcgcctg > >>...TATA....>>
<<.............................> > +1 of 1.91E
transcript 3221 gagacgccat ccacgctgtt ttgacctcca tagaagacac
cgggaccgat ccagcctccg cggccgggaa >....................HCMV IE 3
from TATA (mRNA 5end).....................> PacI --------- 3291
cggtgcattg gaacgcgcat tccccgtgtt aattaacagg taagtgtctt cctcctgttt
ccttcccctg >...........................>>
>>IVS8'..........................> PstI NheI -------
------- 3361 ctattctgct caaccttcct atcagaaact gcagtatctg tatttttgct
agcagtaata ctaacggttc
>..................................IVS8'..............................-
......> KpnI ------- MfeI AgeI NcoI ------ ------- ------- 3431
tttttttctc ttcacaggcc accaattggt accgagctac cggtcgccac catgggaaag
ttatgggatg >.....IVS8'.....>>
>>......GDNF.......> 3501 tcgtggctgt ctgcctggtg ttgctccaca
ccgcgtctgc cttcccgctg cccgccggta agaggcttct
>.................................GDNF................................-
.....> 3571 cgaagcgccc gccgaagacc actccctcgg ccaccgccgc
gtgcccttcg cgctgaccag tgactccaat
>.................................GDNF................................-
.....> 3641 atgcccgaag attatcctga ccagtttgat gacgtcatgg
attttattca agccaccatc aaaagactga
>.................................GDNF................................-
.....> BstEII PstI ------- ------- 3711 aaaggtcacc agataaacaa
gcggcggcac ttcctcgaag agagaggaac cggcaagctg cagctgccag
>.................................GDNF................................-
.....> 3781 cccagagaac agcagaggga aaggtcgcag aggccagagg
ggcaaaaatc gggggtgcgt cttaactgca
>.................................GDNF................................-
.....> 3851 atacacttaa atgtcactga cttgggtttg ggctacgaaa
ccaaggagga actgatcttt cgatattgta
>.................................GDNF................................-
.....> 3921 gcggttcctg tgaagcggcc gagacaatgt acgacaaaat
actaaaaaat ctgtctcgaa gtagaaggct
>.................................GDNF................................-
.....> 3991 aacaagtgac aaggtaggcc aggcatgttg caggccggtc
gccttcgacg acgacctgtc gtttttagac
>.................................GDNF................................-
.....> NotI -------- 4061 gacagcctgg tttaccatat cctaagaaag
cattccgcta aacggtgtgg atgtatctga gcggccgcac
>-----------------------------GDNF.............................>>-
; BclI ------- 4131 cgtcgactag agctcgctga tcagcctcga ctgtgccttc
tagttgccag ccatctgttg tttgcccctc
>>.........................bGH
pA..........................> 4201 ccccgtgcct tccttgaccc
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
>..................................bGH
pA..................................> 4271 tcgcattgtc tgagtaggtg
tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
>..................................bGH
pA..................................> SphI BglII ------- ------
4341 gggaagacaa tagcagggca tgctggggag agatct >....bGH
pA....>>
Sequence CWU 1
1
214376DNAArtificial SequenceVector #21 1agatctagga tcacgcgtaa
aggatccaaa aaaaagctta aactagactg cagagggccc 60tgcgtatgag tgcaagtggg
ttttaggacc aggatgaggc ggggtggggg tgcctacctg 120acgaccgacc
ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc
180ctatcagaga gggggagggg aaacaggatg cggcgaggcg cgtgcgcact
gccagcttca 240gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
gccaccgccg cctcagcact 300gaaggcgcgc tgacgtcact cgccggtccc
ccgcaaactc cccttcccgg ccaccttggt 360cgcgtccgcg ccgccgccgg
cccagccgga ccgcaccacg cgaggcgcga gatagggggg 420cacgggcgcg
accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg
480ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga aagctgctag
caaccatcca 540ccggtcgcca ccatggatag ccagcagccc gatctgaaat
tgctgtcctc tattgaacag 600gcttgtgata tttgcaggct taaaaaactc
aaatgttcca aagaaaaacc aaagtgtgct 660aaatgtctga agaacaactg
ggaatgccgc tactccccca agaccaagcg ttctccactt 720actcgcgctc
acctgacaga ggtagaaagt aggctggaac gcctagaaca gttgtttttg
780ttgatattcc caagagaaga cctggacatg attcttaaga tggatagcct
gcaagatatc 840aaggcgctcc tggagtttcc aggcgtcgat cagaaaaagt
ttaacaaggt ccgagtcgtc 900cgagccctag acgccgttgc cctccctcaa
cccgtgggcg tgcctaatga aagccaggct 960ctctcacagc ggtttacttt
cagcccaggg caggatatac agcttatacc tcccctgata 1020aatttattga
tgagtatcga gccggacgtg atttacgcag ggcatgataa cactaagcca
1080gacacatctt cttctctcct gaccagccta aaccaactgg gtgaacggca
gcttctgtca 1140gtcgtgaagt ggagcaaatc cctccctgga tttagaaacc
tgcacataga tgaccaaata 1200acacttattc aatactcctg gatgagctta
atggtgtttg gtctcggatg gcggtcatat 1260aagcacgtta gcggccagat
gctctacttt gcccctgatt tgattctgaa cgaacaaagg 1320atgaaggaga
gctccttcta tagcctttgt ctgacgatgt ggcaaatccc gcaggagttt
1380gtaaaactgc aagtgagtca ggaggagttc ctgtgtatga aagttctact
gctgctcaat 1440acgatcccct tggaagggct cagatcacag acgcaattcg
aagagatgag gagctcttat 1500attagagagc taattaaggc tattggtctg
aggcaaaagg gtgtcgtgtc cagcagccag 1560agattttacc aacttactaa
actactggac aacctacacg acctagtcaa gcagctccat 1620ctctactgcc
tgaacacctt cattcaatcc agagccttat ctgtggaatt tccggaaatg
1680atgagtgagg tcattgcggg gtcaactccc atggaatttc agtatctgcc
agacaccgat 1740gacaggcacc gcatcgaaga gaaacggaaa cggacatacg
agaccttcaa gtccattatg 1800aaaaagagtc ccttttctgg ccctaccgac
cccagacccc ctccaaggag aatcgcagtg 1860ccttccagga gtagtgcatc
agttccgaaa ccggccccac agccatatcc ttttaccagt 1920tctctgagta
ccattaacta cgacgaattt cctaccatgg ttttcccctc ggggcaaatt
1980agccaggcgt ccgcgctggc gcccgcgccc ccccaggtcc ttccgcaggc
tccagcccct 2040gctccagccc ccgcaatggt ttctgccctg gcacaagccc
ccgcacccgt gcctgtgttg 2100gcaccaggcc cgccacaggc cgtggcacct
ccggctccta aacctactca ggccggagag 2160ggcaccctta gtgaggccct
gttacagctt caatttgacg acgaggacct cggggctctc 2220cttggcaatt
cgacagaccc cgctgtgttt acagacctgg cttcggtaga caattctgag
2280tttcagcaac ttctcaacca gggcatcccc gtagcccctc atacaacaga
gcccatgtta 2340atggagtacc cagaggctat cacaaggctg gtaaccggcg
cccaaagacc accagatcct 2400gcaccagcac cactgggagc tcctggttta
cccaatggat tattatcagg agatgaggat 2460ttcagttcca ttgccgatat
ggacttcagc gcgcttcttt ctcagatcag ctcttgaact 2520agtaaaaggc
ctaggtaagt atcaaggtta caagacaggt ttaaggagac cacatagaaa
2580ctgggcttgt cgagacagag aagactcttg cgtttctgat aggcacctat
tggtcttact 2640gacatccact ttgcctttct ctccacaggt gtaggccttt
cgagcaactt gtttattgca 2700gcttataatg gttacaaata aagcaatagc
atcacaaatt tcacaaataa agcatttttt 2760tcactgcatt ctagttgtgg
tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 2820gtctagcatc
gaagatccac tagatgcata aatctagaca ataaaatatc tttattttca
2880ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc
tctccatcaa 2940aacaaaacga aacaaaacaa actagcaaaa taggctgtcc
ccagtgcaag tgcaggtgcc 3000agaacatttc tctattaggg cccaagcgga
gtactgtcct ccgagtggag tactgtcctc 3060cgagcggagt actgtcctcc
gagtcgaggg tcgaagcgga gtactgtcct ccgagtggag 3120tactgtcctc
cgagcggagt actgtcctcc gagtcgacta gagggtatat aatggatctc
3180gagatatcgg agctcgttta gtgaaccgtc agatcgcctg gagacgccat
ccacgctgtt 3240ttgacctcca tagaagacac cgggaccgat ccagcctccg
cggccgggaa cggtgcattg 3300gaacgcgcat tccccgtgtt aattaacagg
taagtgtctt cctcctgttt ccttcccctg 3360ctattctgct caaccttcct
atcagaaact gcagtatctg tatttttgct agcagtaata 3420ctaacggttc
tttttttctc ttcacaggcc accaattggt accgagctac cggtcgccac
3480catgggaaag ttatgggatg tcgtggctgt ctgcctggtg ttgctccaca
ccgcgtctgc 3540cttcccgctg cccgccggta agaggcttct cgaagcgccc
gccgaagacc actccctcgg 3600ccaccgccgc gtgcccttcg cgctgaccag
tgactccaat atgcccgaag attatcctga 3660ccagtttgat gacgtcatgg
attttattca agccaccatc aaaagactga aaaggtcacc 3720agataaacaa
gcggcggcac ttcctcgaag agagaggaac cggcaagctg cagctgccag
3780cccagagaac agcagaggga aaggtcgcag aggccagagg ggcaaaaatc
gggggtgcgt 3840cttaactgca atacacttaa atgtcactga cttgggtttg
ggctacgaaa ccaaggagga 3900actgatcttt cgatattgta gcggttcctg
tgaagcggcc gagacaatgt acgacaaaat 3960actaaaaaat ctgtctcgaa
gtagaaggct aacaagtgac aaggtaggcc aggcatgttg 4020caggccggtc
gccttcgacg acgacctgtc gtttttagac gacagcctgg tttaccatat
4080cctaagaaag cattccgcta aacggtgtgg atgtatctga gcggccgcac
cgtcgactag 4140agctcgctga tcagcctcga ctgtgccttc tagttgccag
ccatctgttg tttgcccctc 4200ccccgtgcct tccttgaccc tggaaggtgc
cactcccact gtcctttcct aataaaatga 4260ggaaattgca tcgcattgtc
tgagtaggtg tcattctatt ctggggggtg gggtggggca 4320ggacagcaag
ggggaggatt gggaagacaa tagcagggca tgctggggag agatct
437623737DNAArtificial SequenceEmpty vector #21 2agatctagga
tcacgcgtaa aggatccaaa aaaaagctta aactagactg cagagggccc 60tgcgtatgag
tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg
120acgaccgacc ccgacccact ggacaagcac ccaaccccca ttccccaaat
tgcgcatccc 180ctatcagaga gggggagggg aaacaggatg cggcgaggcg
cgtgcgcact gccagcttca 240gcaccgcgga cagtgccttc gcccccgcct
ggcggcgcgc gccaccgccg cctcagcact 300gaaggcgcgc tgacgtcact
cgccggtccc ccgcaaactc cccttcccgg ccaccttggt 360cgcgtccgcg
ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg
420cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca
gtctgcggtg 480ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga
aagctgctag caaccatcca 540ccggtcgcca ccatggatag ccagcagccc
gatctgaaat tgctgtcctc tattgaacag 600gcttgtgata tttgcaggct
taaaaaactc aaatgttcca aagaaaaacc aaagtgtgct 660aaatgtctga
agaacaactg ggaatgccgc tactccccca agaccaagcg ttctccactt
720actcgcgctc acctgacaga ggtagaaagt aggctggaac gcctagaaca
gttgtttttg 780ttgatattcc caagagaaga cctggacatg attcttaaga
tggatagcct gcaagatatc 840aaggcgctcc tggagtttcc aggcgtcgat
cagaaaaagt ttaacaaggt ccgagtcgtc 900cgagccctag acgccgttgc
cctccctcaa cccgtgggcg tgcctaatga aagccaggct 960ctctcacagc
ggtttacttt cagcccaggg caggatatac agcttatacc tcccctgata
1020aatttattga tgagtatcga gccggacgtg atttacgcag ggcatgataa
cactaagcca 1080gacacatctt cttctctcct gaccagccta aaccaactgg
gtgaacggca gcttctgtca 1140gtcgtgaagt ggagcaaatc cctccctgga
tttagaaacc tgcacataga tgaccaaata 1200acacttattc aatactcctg
gatgagctta atggtgtttg gtctcggatg gcggtcatat 1260aagcacgtta
gcggccagat gctctacttt gcccctgatt tgattctgaa cgaacaaagg
1320atgaaggaga gctccttcta tagcctttgt ctgacgatgt ggcaaatccc
gcaggagttt 1380gtaaaactgc aagtgagtca ggaggagttc ctgtgtatga
aagttctact gctgctcaat 1440acgatcccct tggaagggct cagatcacag
acgcaattcg aagagatgag gagctcttat 1500attagagagc taattaaggc
tattggtctg aggcaaaagg gtgtcgtgtc cagcagccag 1560agattttacc
aacttactaa actactggac aacctacacg acctagtcaa gcagctccat
1620ctctactgcc tgaacacctt cattcaatcc agagccttat ctgtggaatt
tccggaaatg 1680atgagtgagg tcattgcggg gtcaactccc atggaatttc
agtatctgcc agacaccgat 1740gacaggcacc gcatcgaaga gaaacggaaa
cggacatacg agaccttcaa gtccattatg 1800aaaaagagtc ccttttctgg
ccctaccgac cccagacccc ctccaaggag aatcgcagtg 1860ccttccagga
gtagtgcatc agttccgaaa ccggccccac agccatatcc ttttaccagt
1920tctctgagta ccattaacta cgacgaattt cctaccatgg ttttcccctc
ggggcaaatt 1980agccaggcgt ccgcgctggc gcccgcgccc ccccaggtcc
ttccgcaggc tccagcccct 2040gctccagccc ccgcaatggt ttctgccctg
gcacaagccc ccgcacccgt gcctgtgttg 2100gcaccaggcc cgccacaggc
cgtggcacct ccggctccta aacctactca ggccggagag 2160ggcaccctta
gtgaggccct gttacagctt caatttgacg acgaggacct cggggctctc
2220cttggcaatt cgacagaccc cgctgtgttt acagacctgg cttcggtaga
caattctgag 2280tttcagcaac ttctcaacca gggcatcccc gtagcccctc
atacaacaga gcccatgtta 2340atggagtacc cagaggctat cacaaggctg
gtaaccggcg cccaaagacc accagatcct 2400gcaccagcac cactgggagc
tcctggttta cccaatggat tattatcagg agatgaggat 2460ttcagttcca
ttgccgatat ggacttcagc gcgcttcttt ctcagatcag ctcttgaact
2520agtaaaaggc ctaggtaagt atcaaggtta caagacaggt ttaaggagac
cacatagaaa 2580ctgggcttgt cgagacagag aagactcttg cgtttctgat
aggcacctat tggtcttact 2640gacatccact ttgcctttct ctccacaggt
gtaggccttt cgagcaactt gtttattgca 2700gcttataatg gttacaaata
aagcaatagc atcacaaatt tcacaaataa agcatttttt 2760tcactgcatt
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
2820gtctagcatc gaagatccac tagatgcata aatctagaca ataaaatatc
tttattttca 2880ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac
taacatacgc tctccatcaa 2940aacaaaacga aacaaaacaa actagcaaaa
taggctgtcc ccagtgcaag tgcaggtgcc 3000agaacatttc tctattaggg
cccaagcgga gtactgtcct ccgagtggag tactgtcctc 3060cgagcggagt
actgtcctcc gagtcgaggg tcgaagcgga gtactgtcct ccgagtggag
3120tactgtcctc cgagcggagt actgtcctcc gagtcgacta gagggtatat
aatggatctc 3180gagatatcgg agctcgttta gtgaaccgtc agatcgcctg
gagacgccat ccacgctgtt 3240ttgacctcca tagaagacac cgggaccgat
ccagcctccg cggccgggaa cggtgcattg 3300gaacgcgcat tccccgtgtt
aattaacagg taagtgtctt cctcctgttt ccttcccctg 3360ctattctgct
caaccttcct atcagaaact gcagtatctg tatttttgct agcagtaata
3420ctaacggttc tttttttctc ttcacaggcc accaattggt accgagctac
cggtcgccac 3480cgcggccgca ccgtcgacta gagctcgctg atcagcctcg
actgtgcctt ctagttgcca 3540gccatctgtt gtttgcccct cccccgtgcc
ttccttgacc ctggaaggtg ccactcccac 3600tgtcctttcc taataaaatg
aggaaattgc atcgcattgt ctgagtaggt gtcattctat 3660tctggggggt
ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcagggc
3720atgctgggga gagatct 3737
* * * * *
References