U.S. patent application number 10/440799 was filed with the patent office on 2003-12-25 for materials and methods involving conditional retention domains.
This patent application is currently assigned to ARIAD Gene Therapeutics, Inc.. Invention is credited to Clackson, Timothy P., Rivera, Victor, Rothman, James E..
Application Number | 20030235889 10/440799 |
Document ID | / |
Family ID | 27379806 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235889 |
Kind Code |
A1 |
Rivera, Victor ; et
al. |
December 25, 2003 |
Materials and methods involving conditional retention domains
Abstract
Materials and methods involving conditional retention domains
(CRDs) are disclosed. Also disclosed are fusion proteins containing
CRDs and cells expressing such fusion proteins. In addition, the
invention provides novel methods for producing target proteins in
vivo using fusion proteins containing conditional retention domains
and methods for identifying novel CRDs.
Inventors: |
Rivera, Victor; (Arlington,
MA) ; Clackson, Timothy P.; (Arlington, MA) ;
Rothman, James E.; (New York, NY) |
Correspondence
Address: |
ARIAD Gene Therapeutics, Inc.
26 Landsdowne Street
Cambridge
MA
02139-4234
US
|
Assignee: |
ARIAD Gene Therapeutics,
Inc.
|
Family ID: |
27379806 |
Appl. No.: |
10/440799 |
Filed: |
May 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10440799 |
May 19, 2003 |
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09420819 |
Oct 19, 1999 |
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6566073 |
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10440799 |
May 19, 2003 |
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09174799 |
Oct 19, 1998 |
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60104743 |
Oct 19, 1998 |
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60137787 |
Jun 2, 1999 |
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Current U.S.
Class: |
435/69.7 ;
435/320.1; 435/325; 435/326; 530/303; 530/387.1; 530/399 |
Current CPC
Class: |
C07K 14/62 20130101;
C07K 2319/60 20130101; C07K 2319/03 20130101; C07K 2319/75
20130101; C12N 15/625 20130101; C12N 2799/022 20130101; C07K
2319/715 20130101; C07K 2319/04 20130101; C07K 2319/71 20130101;
C07K 14/47 20130101; C07K 14/811 20130101; C07K 2319/09 20130101;
A01K 2217/05 20130101; C07K 2319/32 20130101; C07K 2319/81
20130101; C07K 14/4702 20130101; C07K 2319/50 20130101; C07K
2319/00 20130101 |
Class at
Publication: |
435/69.7 ;
435/320.1; 435/325; 530/303; 530/387.1; 530/399; 435/326 |
International
Class: |
C12P 021/04; C12N
005/06; C07K 014/675; C07K 014/62; C07K 014/635; C07K 016/18 |
Claims
What is claimed:
1. A cell containing a recombinant nucleic acid encoding a fusion
protein comprising at least one conditional retention domain
("CRD") and at least one additional domain that is heterologous
thereto.
2. A cell of claim 1 wherein the fusion protein contains more than
one CRD.
3. A cell of claim 1 wherein the fusion protein molecules form
aggregates with one another in the absence of a ligand which binds
to the CRD.
4. A cell of claim 1 wherein the CRD is derived from retinol
binding protein, FKBP, IgM or alpha1-antitrypsin.
5. A cell of claim 4 wherein the CRD comprises an FKBP domain with
an amino acid replacement at F36 or W59.
6. A cell of claim 5 wherein the CRD comprises an FKBP domain
containing the mutation F36M or W59V.
7. A cell of claim 1 wherein the heterologous domain of the fusion
protein comprises the polypeptide sequence of a protein of
interest.
8. A cell of claim 7 wherein the protein of interest is a hormone,
an endorphin, an antibody or an immunogen.
9. A cell of claim 8 wherein the protein of interest is selected
from the group consisting of insulin, parathyroid hormone and
beta-endorphin.
10. A cell of claim 1 wherein the fusion protein comprises an
enzymatic cleavage site.
11. A cell of claim 10 wherein the cleavage site is a furin
cleavage site.
12. A cell of claim 11 wherein the furin cleavage site comprises
the amino acid sequence SARNRQKR (SEQ ID NO. 1).
13. A cell of claim 1 wherein the fusion protein further comprises
a secretory signal sequence.
14. A cell of claim 13 wherein the secretory signal sequence is the
signal sequence from the human growth hormone gene.
15. A cell of claim 1 wherein the fusion protein comprises a
secretory signal sequence, at least one conditional retention
domain, a furin cleavage site, and a polypeptide sequence of
interest.
16. A cell of claim 15 wherein the fusion protein comprises a
secretory signal sequence from human growth hormone, three F36M
FKBP domains, a human stromelysin-3 furin cleavage site, and a
polypeptide sequence of interest.
17. A cell of claim 1 wherein the fusion protein comprises a
lysosomal targeting signal.
18. A cell of claim 1 wherein the cells are mammalian cells.
19. A cell of claim 1 wherein the cells are of human origin.
20. A cell of claim 1 wherein the cells are primary cells.
21. A cell of claim 1 wherein the cells are in an animal.
Description
BACKGROUND OF THE INVENTION
[0001] A number of important applications, including for example,
gene therapy, production of biological materials and materials and
methods for biological research, depend on the ability to induce
cells to produce proteins of therapeutic, commercial, or
experimental value. A variety of regulatable expression systems
have been developed, including systems involving allostery-based
switches triggered by tetracycline, RU486 or ecdysone, as well as
dimerization-based switches triggered by dimerizing agents such as
rapamycin, coumermycin, dimers of FK506, synthetic FKBP-binders
and/or CsA, or analogs thereof. See e.g. Clackson, "Controlling
mammalian gene expression with small molecules" Current Opinion in
Chemical Biology, 1:210-218, 1997. In these expression systems,
protein production is regulated at the transcriptional level. An
inherent limitation of all such systems is the inability to achieve
fine temporal control over secretion of the target protein. For
example, secretion of maximal, therapeutic levels of the protein is
delayed by many hours or even days until the transcribed mRNA
accumulates to levels high enough to produce significant amounts of
secreted protein. Likewise, secretion cannot return to low baseline
levels following removal of the inducing drug until the mRNA is
completely degraded, which may also take many hours or days. For
many applications this level of control is not sufficient; in these
instances, it would be desirable to induce protein production on a
much more rapid time scale than that achievable using
transcription-based methods.
SUMMARY OF THE INVENTION
[0002] This invention takes a unique approach to the regulated
production of a target protein, based not on regulated
transcription, but on regulated release or secretion of the target
protein. Compositions and methods of this invention are useful in
biological research and in gene therapy applications.
[0003] Key features of the invention include conditional retention
domains ("CRDs"), fusion proteins containing them, ligands which
bind to the CRDs and permit release or secretion of the fusion
proteins, recombinant nucleic acids encoding such fusion proteins,
vectors containing such recombinant nucleic acids, cells transduced
with these vectors and other materials and important methods
involving such. Key fusion proteins of the invention contain at
least two mutually heterologous domains, one of which being a
CRD.
[0004] More specifically, the fusion proteins of this invention are
designed to contain at least one conditional retention domain (CRD)
and at least one additional domain that is heterologous thereto,
usually with a secretory signal sequence. Proteins containing a
secretory signal sequence are translated in the endoplasmic
reticulum (ER) and then pass through other secretory compartments
such as the cis, medial and trans Golgi on their way to being
secreted. However, proteins containing one or more CRDs are, as a
rule, retained in the secretory machinery except in the presence of
a ligand which binds to the protein. Illustrative examples of CRDs
include retinol binding proteins and human FKBP 12 mutants such as
F36M hFKBP12, as are discussed in detail below. Concatenation of
multiple CRDs may allow the user to modulate the degree of
aggregation or retention.
[0005] Typically the fusion protein also contains a secretory
signal sequence to target the fusion protein to a secretory
compartment such as the ER or any part of the Golgi apparatus. Many
secretory signal sequences are known. Human growth hormone, for
example, is the source of a secretory signal sequence suitable for
use in this invention.
[0006] Additionally, it is preferred in many embodiments that the
fusion protein further contain an enzymatic cleavage site such that
a portion of the fusion protein containing the CRD can be cleaved
from a portion of the fusion protein containing a peptide sequence
heterologous to the CRD. Preferably the enzymatic cleavage site
comprises a peptide sequence recognized by a trans-Golgi specific
endoprotease such as furin. For instance, a cleavage site for furin
is provided by the peptide sequence SARNRQKR (SEQ ID NO. 1).
[0007] The portion of the fusion protein which is heterologous to
the CRD may comprise any protein or protein domain of interest to
the practitioner. For instance, the heterologous portion may
comprise a target protein such as insulin, parathyroid hormone or
beta-endorphin.
[0008] To illustrate this further, one typical fusion protein of
the invention comprises a signal sequence, a conditional retention
domain, a furin cleavage site, and a polypeptide sequence
comprising a selected target protein sequence. An example of such a
fusion protein comprises, in N-terminal to C-terminal order, a
signal sequence from human growth hormone, three F36M hFKBP 12
domains, a human stromelysin-3 furin cleavage site, and a selected
target protein sequence. Fusion proteins may also contain several
target proteins each separated by an enzymatic cleavage site. For
example, such a fusion protein might contain a signal sequence from
human growth hormone, one or more copies of a CRD such as F36M
hFKBP 1{overscore (2)}, a furin cleavage site, a target protein,
another furin cleavage site and another target protein. This type
of construct allows for simultaneous release of more than one
target protein.
[0009] In addition, the fusion proteins of this invention may
optionally comprise a lysosomal targeting signal or other
polypeptide sequence targeting it for degradation. By locating such
a peptide sequence together with the CRD(s) on one side of the
cleavage site and the selected target polypeptide on the other side
of the cleavage site, one can help assure cellular removal of the
CRD-containing portion of the fusion protein.
[0010] One object of the invention is thus the fusion proteins
described herein.
[0011] Another object of the invention is the recombinant nucleic
acids encoding such fusion proteins. Those recombinant nucleic
acids may be operably linked to an expression control sequence
permitting their expression in host cells into which they have been
transduced, or which otherwise contain them. Any promoter may be
used to drive expression of these fusion proteins, including strong
promoters like the CMV enhancer, other viral promoters such as the
RSV promoter or tissue specific promoters like the MCK
enhancer.
[0012] Another object is a vector containing a recombinant nucleic
acid of the invention, generally operably linked to an expression
control sequence. Such vectors include "viral" vectors which
contain part or all of a viral genome in addition to the
recombinant nucleic acid encoding the fusion protein of this
invention. Viral vectors can be designed and used for the
production of recombinant viruses harboring a recombinant nucleic
acid of this invention. A wide variety of such viral systems are
known in the art and may be adapted to the practice of this
invention, including e.g. adenovirus, AAV, retrovirus, hybrid
adeno-AAV, lentivirus and others.
[0013] Recombinant nucleic acids of this invention may be
transduced into host cells by any available means e.g. in order to
render those cells capable of regulated secretion of a target
protein. The cells are preferably eukaryotic cells, generally are
animal cells, and in many embodiments are mammalian, whether human
or non-human. The cells may be transduced in situ within their host
organism, or they may be transduced while being maintained in
vitro. The cells may be primary cells or may be from a cell
line.
[0014] The invention thus provides methods for rendering a cell
capable of regulated secretion of a target protein which involves
introducing into the cell a recombinant nucleic acid of this
invention to yield engineered cells which can express the encoded
fusion protein. The recombinant nucleic acid may be introduced in
viral or other form into cells maintained in vitro or into cells
present within an organism. The resultant engineered cells and
their progeny containing one or more of these recombinant nucleic
acids may be used in a variety of important applications discussed
elsewhere, including human gene therapy, analogous veterinary
applications, the creation of cellular or animal models (including
transgenic applications), assay applications, and the production of
a desired protein in vitro, e.g. for recovery and use. Such cells
are useful, for example, in methods involving the addition of a
ligand, preferably a cell permeant ligand, to the cells (or
administration of the ligand to an organism containing the cells)
to regulate secretion of a target protein. Particularly important
animal models include rodent (especially mouse and rat) and
non-human primate models. In human gene therapy applications, the
cells will generally be human and the peptide sequence of each of
the various domains present in the fusion proteins will preferably
be, or be derived from, a peptide sequence of human origin, to the
extent possible.
[0015] The invention also provides methods for identifying novel
CRDs. CRDs may be identified by two hybrid type methods, in which a
genetically engineered host cell is provided which comprises (a) a
reporter gene linked to a regulatable expression control element,
and (b) a recombinant nucleic acid comprising a polylinker linked
to two recombinant nucleic acid sequences, the first recombinant
nucleic acid sequence encoding a DNA binding domain and the second
recombinant nucleic acid sequence encoding a transcription
activation domain, wherein association of the DNA binding domain
with the transcription activation domain activates expression of
the reporter gene. As described herein, the construct contains a
single polylinker linked to two independent translational
cassettes. This allows for expression of two fusion proteins, one
with a DNA binding domain and the other with a transcription
activation domain, each linked to an identical CRD candidate. In
addition, genetically engineered host cells are provided which
comprise (a) a reporter gene linked to a regulatable expression
control element, (b) a first recombinant nucleic acid encoding a
fusion protein comprising a transcription activation domain linked
to a candidate conditional retention domain, (c) a second
recombinant nucleic acid encoding a fusion protein containing a DNA
binding domain linked to the candidate conditional retention domain
wherein association of the fusion proteins activates expression of
the reporter gene.
[0016] The invention further provides methods for identifying a
ligand capable of binding to a conditional retention domain. See,
"Methods for identifying CRDs", part 3, page 46 et seq, below. One
such method uses cells genetically engineered to express a reporter
gene when CRD-containing aggregates are disaggregated by an
appropriate ligand. The method involves the following steps: (a)
contact the genetically engineered cells with candidate ligands
under suitable conditions permitting gene expression, (b) observe
the presence and/or amount of expression of the reporter gene, and
(c) correlate the presence and/or amount of reporter gene
expression with contact of cells with one or more candidate
ligands.
[0017] The invention also provides methods for screening directly
for CRDs which enable ligand-dependent secretion of a target
protein or ligand-dependent localization of a membrane protein. For
these screening assays, fusion proteins are expressed which encode
members of a library of candidate CRDs linked to a signal sequence
and an enzymatic cleavage site. These domains are further linked to
either a secreted target protein or the extracellular and membrane
domain of a membrane protein. The fusion proteins are expressed
under conditions permitting secretion of the target protein or
localization of the membrane protein. Cells containing the fusion
proteins are treated with a ligand that binds the CRD, and then the
ligand-dependent presence of the secreted protein or membrane
protein is assessed. Secretion of the target protein and/or
localization of the membrane protein is then correlated with one or
more individual members of the CRD library.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1: General design of fusion proteins for use in this
invention, containing, from amino- to carboxy-terminus, a secretion
signal sequence, a "conditional retention domain", a protease
cleavage site, and the secreted target protein of interest.
[0019] FIG. 2: Constructs used to make CRD-containing fusion
proteins. FIG. 2A: F36M-EGFP fusion proteins; FIG. 2B: F36M-hGH
fusion proteins; FIG. 2C: EGFP-F36M-hGH fusion proteins; FIG. 2D:
F36M-insulin fusion proteins; FIG. 2E: LNGFR-F36M fusion
proteins.
[0020] FIG. 3: Ligand dependent secretion of hGH. Levels of hGH
secreted into the culture medium of transiently transfected (FIG.
3A) or stably transfected (FIG. 3B) HT1080 cells in the absence and
presence of ligand.
[0021] FIG. 4: Immunoblots of cell lysates and supernatants
prepared from the HT88 cells incubated in the presence or absence
of ligand for 2 hours. The samples were immunoblotted with anti-hGH
and anti-FKBP antibodies.
[0022] FIG. 5: Dose-dependence of hGH secretion from HT88 cells in
response to ligand (FIG. 5A). Time course of accumulation of
secreted hGH in the culture medium (FIG. 5B).
[0023] FIG. 6: Kinetics of secretion in response to ligand. FIG. 6A
Group A: the constitutive rate of secretion from the cells. Group
B: secretion from cells not previously exposed to ligand. Group C:
cells exposed to ligand following a large bolus release of hGH.
FIG. 6B shows the amount of hGH released by incubation with maximal
concentration of ligand. FIG. 6c shows the amount of hGH secreted
following addition of sub-maximal concentrations of ligand.
[0024] FIG. 7: Effect of varying the number of CRDs on hGH
secretion. hGH secretion was measured following addition of ligand
in cell lines expressing fusion proteins containing varying numbers
of CRDs.
[0025] FIG. 8: Regulated secretion of insulin. Levels of insulin
secretion were measured in transiently transfected HT1080 cells
treated with varying concentrations of AP21998.
[0026] FIG. 9: Regulated expression of a membrane tethered protein.
3, 4, or 6 copies of F(36M) were fused to the extracellular and
transmembrane portions of the low-affinity nerve growth factor
receptor (LNGFR; FIG. 3E). Surface expression was assessed by FACS
analysis using anti-LNGFR antibodies.
[0027] FIG. 10: Constructs useful for screening for novel CRDs. A.
Candidate DNA sequences may be cloned into the polylinker for
identifying CRDs that induce ligand-dependent secretion of hGH. B.
Candidate DNA sequences may be cloned into the polylinker for
identifying CRDs that induced ligand-dependent localization of p75.
C. Construct used for "two hybrid" style assay, in which fusion
proteins containing CRDs cause association of the DNA binding
domain and transcription activation domain to induce
transcription.
[0028] FIG. 11: Ligand-mediated regulation of insulin and glucose
levels in vivo. (A) Insulin and glucose levels were measured in
mice implanted with FKBP(F36M)-insulin-containing constructs before
and after administration of AP22542. (B) Levels of serum glucose
were measured in mice implanted with FKBP(F36M)-insulin-containing
constructs at various time points following administration of
AP22542.
DETAILED DESCRIPTION
[0029] Definitions:
[0030] For convenience, the intended meaning of certain terms and
phrases used herein are provided below.
[0031] "Capable of selectively hybridizing" means that two DNA
molecules are susceptible to detectable hybridization with one
another, despite the presence of other DNA molecules, under
hybridization conditions which can be chosen or readily determined
empirically by the practitioner of ordinary skill in this art. Such
treatments include conditions of high stringency such as washing
extensively with buffers containing 0.2 to 6.times.SSC, and/or
containing 0.1% to 1% SDS, at temperatures ranging from room
temperature to 65-75.degree. C. See for example F. M. Ausubel et
al., Eds, Short Protocols in Molecular Biology, Units 6.3 and 6.4
(John Wiley and Sons, New York, 3d Edition, 1995).
[0032] "Cells", "host cells" or "recombinant host cells" refer not
only to the particular cells under discussion, but also to their
progeny. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0033] "Cell line" refers to a population of cells capable of
continuous or prolonged growth and division in vitro. Often, cell
lines are clonal populations derived from a single progenitor cell.
It is further known in the art that spontaneous or induced changes
can occur in karyotype during storage or transfer of such clonal
populations. Therefore, cells derived from a given cell line may
not be precisely identical to the ancestral cells or cultures, and
the cell line referred to includes such variants.
[0034] "Composite", "fusion", and "recombinant" denote a material
such as a nucleic acid, nucleic acid sequence or polypeptide which
contains at least two constituent portions which are mutually
heterologous in the sense that they are not otherwise found
directly (covalently) linked in nature, i.e., are not found in the
same continuous polypeptide or gene in nature, at least not in the
same order or orientation or with the same spacing present in the
composite, fusion or recombinant product. Typically, such materials
contain components derived from at least two different proteins or
genes or from at least two non-adjacent portions of the same
protein or gene. In general, "composite" refers to portions of
different proteins or nucleic acids which are joined together to
form a single functional unit, while "fusion" generally refers to
two or more functional units which are linked together.
"Recombinant" is generally used in the context of nucleic acids or
nucleic acid sequences.
[0035] A "coding sequence" or a sequence which "encodes" a
particular polypeptide or RNA, is a nucleic acid sequence 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 an appropriate expression control sequence. The
boundaries of the coding sequence are generally determined by a
start codon at the 5' (amino) terminus and a translation stop codon
at the 3' (carboxy) terminus. A coding sequence can include, but is
not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic
DNA sequences from procaryotic or eukaryotic DNA, and synthetic DNA
sequences. A transcription termination sequence will usually be
located 3' to the coding sequence.
[0036] A "construct", e.g., a "nucleic acid construct" or "DNA
construct", refers to a nucleic acid or nucleic acid sequence.
[0037] "Derived from" denotes a peptide or nucleotide sequence
selected from within a given sequence. A peptide or nucleotide
sequence derived from a named sequence may further contain a small
number of modifications relative to the parent sequence, in most
cases representing deletion, replacement or insertion of less than
about 15%, preferably less than about 10%, and in many cases less
than about 5%, of amino acid residues or bases present in the
parent sequence. In the case of DNAs, one DNA molecule is also
considered to be derived from another if the two are capable of
selectively hybridizing to one another. Polypeptides or polypeptide
sequences are also considered to be derived from a reference
polypeptide or polypeptide sequence if any DNAs encoding the two
polypeptides or sequences are capable of selectively hybridizing to
one another. Typically, a derived peptide sequence will differ from
a parent sequence by the replacement of up to 5 amino acids, in
many cases up to 3 amino acids, and very often by 0 or 1 amino
acids. A derived nucleic acid sequence will differ from a parent
sequence by the replacement of up to 15 bases, in many cases up to
9 bases, and very often by 0-3 bases. In some cases the amino
acid(s) or base(s) is/are added or deleted rather than
replaced.
[0038] "Domain" refers to a portion of a protein or polypeptide. In
the art, the term "domain" may refer to a portion of a protein
having a discrete secondary structure. However, as will be apparent
from the context used herein, the term "domain" as used in this
document does not necessarily connote a given secondary structure.
Rather, a peptide sequence is referred to herein as a "domain"
simply to denote a polypeptide sequence from a defined source, or
having or conferring an intended or observed activity. Domains can
be derived from naturally occurring proteins or may comprise
non-naturally-occurring sequence.
[0039] "Expression control element", or simply "control element",
refers to DNA sequences, such as initiation signals, enhancers,
promoters and silencers, which induce or control transcription of
DNA sequences with which they are operably linked. Control elements
of a gene may be located in introns, exons, coding regions, and 3'
flanking sequences. Some control elements are "tissue specific",
i.e., affect expression of the selected DNA sequence preferentially
in specific cells (e.g., cells of a specific tissue), while others
are active in many or most cell types. Gene expression occurs
preferentially in a specific cell if expression in this cell type
is observably higher than expression in other cell types. Control
elements include so-called "leaky" promoters, which regulate
expression of a selected DNA primarily in one tissue, but cause
expression in other tissues as well. Furthermore, a control element
can act constitutively or inducibly. An inducible promoter, for
example, is demonstrably more active in response to a stimulus than
in the absence of that stimulus. A stimulus can comprise a hormone,
cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), retinoic
acid or derivative thereof, etc. A nucleotide sequence containing
one or more expression control elements may be referred to as an
"expression control sequence".
[0040] "Gene" refers to a nucleic acid molecule or sequence
comprising an open reading frame and including at least one exon
and (optionally) one or more intron sequences.
[0041] "Genetically engineered cells" denotes cells which have been
modified by the introduction of recombinant or heterologous nucleic
acids (e.g. one or more DNA constructs or their RNA counterparts)
and further includes the progeny of such cells which retain part or
all of such genetic modification.
[0042] "Heterologous", as it relates to nucleic acid or peptide
sequences, denotes sequences that are not normally joined together,
and/or are not normally associated with a particular cell. Thus, a
"heterologous" region of a nucleic acid construct is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a construct could include a
coding sequence flanked by sequences not found in association with
the coding sequence in nature. Another example of a heterologous
coding sequence is a construct where the coding sequence itself is
not found in nature (e.g., synthetic sequences having codons
different from the native gene). Similarly, in the case of a cell
transduced with a nucleic acid construct which is not normally
present in the cell, the cell and the construct would be considered
mutually heterologous for purposes of this invention.
[0043] "Interact" refers to directly or indirectly detectable
interactions between molecules, such as can be detected using, for
example, a yeast two hybrid assay or by immunoprecipitation. The
term "interact" encompasses "binding" interactions between
molecules. Interactions may be, for example, protein-protein,
protein-nucleic acid, protein-small molecule or small
molecule-nucleic acid in nature.
[0044] "Nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic
acid (RNA). The term should also be understood to include
derivatives, variants and analogs of either RNA or DNA made from
nucleotide analogs, and, as applicable to the embodiment being
described, single (sense or antisense) and double-stranded
polynucleotides.
[0045] A "polylinker", also sometimes referred to as a "multiple
cloning site" is a region within a vector which contains multiple
sites for restriction enzyme cleavage, thus rendering the vector
suitable for cloning of exogenous genes.
[0046] "Protein", "polypeptide" and "peptide" are used
interchangeably.
[0047] A "recombinant virus" is a virus particle in which the
packaged nucleic acid contains a heterologous portion.
[0048] The "secretory machinery" (also called secretory apparatus)
of the cell refers to the cellular compartments to which secreted
and membrane proteins are targeted and processed. These
compartments include the endoplasmic reticulum (ER) and the cis,
medial and trans Golgi. In this document, the term ER is often used
generically to mean "secretory compartment."
[0049] A "target protein" is a protein of interest, the secretion
of which is modulated according to the methods of the invention.
The target protein can be, for example, a hormone, an endorphin,
etc.
[0050] "Transfection" means the introduction of a naked nucleic
acid molecule into a recipient cell. "Infection" refers to the
process wherein a nucleic acid is introduced into a cell by a virus
containing that nucleic acid. A "productive infection" refers to
the process wherein a virus enters the cell, is replicated, and is
then released from the cell (sometimes referred to as a "lytic"
infection). "Transduction" encompasses the introduction of nucleic
acid into cells by any means.
[0051] "Transgene" refers to a nucleic acid sequence which has been
introduced into a cell. Daughter cells deriving from a cell in
which a transgene has been introduced are also said to contain the
transgene (unless it has been deleted). The polypeptide or RNA
encoded by a transgene may be partly or entirely heterologous,
i.e., foreign, with respect to the animal or cell into which it is
introduced. Alternatively, the transgene can be homologous to an
endogenous gene of the transgenic animal or cell into which it is
introduced, but is designed to be inserted, or is inserted, into
the animal's genome in such a way as to alter the genome of the
cell into which it is inserted (e.g., it is inserted at a location
which differs from that of the natural gene). A transgene can also
be present in an episome. A transgene can include one or more
expression control elements and any other nucleic acid, (e.g.
intron), that may be necessary or desirable for optimal expression
of a selected coding sequence.
[0052] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is an episome, i.e., a nucleic acid capable of
extra-chromosomal replication. Often vectors are used which are
capable of autonomous replication and/or expression of nucleic
acids to which they are linked. Vectors capable of directing the
expression of an included gene operatively linked to an expression
control sequence can be referred to as "expression vectors".
Expression vectors are typically in the form of "plasmids" which
refer generally to circular double stranded DNA loops which, in
their vector form are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of vectors which
serve equivalent functions and which are or become known in the
art. Viral vectors are nucleic acid molecules containing viral
sequences which can be packaged into viral particles.
[0053] Conditional Retention Domains:
[0054] A conditional retention domain is any domain which is
retained in the ER or other secretory compartment in the absence of
ligand and is released from the secretory machinery when ligand is
bound, i.e. in the presence of ligand. The use of CRDs is
considered to take advantage of the phenomenon of ER "quality
control", whereby proteins that are incorrectly folded or
aggregated are retained in the ER rather than traveling to the
Golgi. Eventually, most misfolded proteins are degraded, but others
have been observed to accumulate in substantial steady-state
amounts (eg. the VSV-G protein: A. M. de Silva et al. (1990) J.
Cell Biol. 111, 857-866. See also, Kopito, R. R. (1997) Cell 88,
427-430). Several types of domains can function as conditional
retention domains:
[0055] 1) The CRD can be a Natural Example of a Protein that is
Retained in the Secretory Machinery in the Absence of a Particular
Small Molecule.
[0056] An example of this type of conditional retention domain is
or is derived from retinol binding protein (RBP). Retinol binding
protein is a serum protein of approximately 20 kD that is a
specific carrier for retinol (Vitamin A) (Melhus, H. et al. (1992)
J Biol. Chem. 267, 12036-12041). It is retained in the ER in
complex with another protein, transerythrin. Upon binding of
retinol to RBP, the complex is released from its molecular
chaperone and is free to enter the Golgi apparatus. Thus, the
retinol binding protein acts as a CRD which is retained in the ER
in the absence of ligand and secreted in its presence. Although
retinol binding protein is expressed primarily in hepatocytes, it
is generally useful as a CRD, since several groups have shown that
retinol-mediated secretion of RBP is cell-type independent and
requires no hepatocyte specific cofactors (see, e.g. Melhus et al.,
J. Biol. Chem. 267:12036-12041, 1992.)
[0057] Another example of a protein that is retained in the ER in
the absence of a small molecule ligand is IgM. Retention of soluble
.mu. chains in the ER is dependent on a single unpaired cysteine
residue. Although secretion of IgM normally requires binding of
light chains to the .mu. heavy chain, secretion of IgM
intermediates can be induced by addition of 2-mercaptoethanol or
other reducing agents (Alberini et al., Nature 347:485-487, 1990).
Thus, soluble .mu. chains can function as CRDs which are secreted
in the presence of a thiol-reactive small molecule.
[0058] 2) The CRD can be an Engineered Mutant of a Natural Protein,
Chosen because it has the Property of being Selectively Retained in
the Absence of a Given Small Molecule.
[0059] It is known that mutations that destabilize proteins can
lead to ER retention. Without wishing to be bound to any one
theory, including that theory, we have observed that some mutations
at human FKBP Phe36 lead to proteins that are poorly expressed (eg.
F36A), probably due to instability. Such proteins are thought to be
retained to some extent in the secretory apparatus. Using a high
affinity ligand that binds to the protein to permit ER exit.
[0060] 3) The CRD can be a Protein that Self-aggregates in a Small
Molecule-reversible Manner.
[0061] It is known that large protein aggregates are retained in
the ER. In such cases, ER retention occurs because of formation of
aggregates rather than due to misfolding of proteins. A naturally
occurring example of aggregation-dependent ER retention is found in
the Z mutation of .alpha..sub.1-antitrypsin. In the secreted M form
of this plasma protease, a glutamic acid residue is located at
position 342 in the reactive center loop of the molecule. In the
mutant Z form, this glutamic acid is substituted by lysine; this
substitution allows the reactive loop to insert itself into the
A-sheet of an adjacent .alpha..sub.1-antitrypsi- n molecule,
forming linear, transport-incompetent aggregates. The aggregates
accumulate in the ER, but can be released by addition of a peptide
which inserts into the A-sheet and prevents polymerization (Hammond
and Helenius, Current Opinion in Cell Biology 7:523-529, 1995;
Lomas et al., Nature 357:605-607, Jun. 18, 1992).
[0062] The mutant form of .alpha.-galactosidase A that is found in
Fabry lymphoblasts provides an additional example of small-molecule
dependent release of aggregates from the ER. Whereas the wild-type
form of the enzyme is efficiently routed through the secretory
pathway, the mutant protein aggregates in the endoplasmic
reticulum, contributing, at least in part, to enzyme deficiency in
Fabry patients. Recently, Fan et al reported that addition of
1-deoxy-galactonojirimycin (DGJ), a competitive inhibitor of
.alpha.-galactosidase A, enhances .alpha.-galactosidase A activity
in Fabry lymphoblasts by acting as a "chemical chaperone", thus
accelerating transport and processing of the mutant enzyme (Fan et
al., Nature Medicine 5:112-115, 1999).
[0063] In a preferred embodiment, the CRD is derived from human
FKBP12. In particular, the FKBP mutant F36M functions as a
conditional retention domain when fused to a signal sequence and
heterologous target sequence in mammalian cells. In the absence of
ligand, fusion proteins containing FKBP F36M and a signal sequence
self-aggregate and accumulate in the endoplasmic reticulum. Upon
addition of ligand, the fusion protein disaggregates and transits
through the ER, resulting in secretion of the fusion protein or
cleavage products thereof. Another FKBP mutant which functions as a
CRD is FKBP W59V.
[0064] Ligands for CRDs:
[0065] A wide variety of ligands, including both naturally
occurring and synthetic substances, can be used in this invention
to effect disaggregation and/or secretion of the fusion protein
molecules from the secretory machinery. Criteria for selecting a
ligand are: (A) physiologic acceptability of the ligand (i.e., the
ligand lacks undue toxicity towards the cell or animal for which it
is to be used), (B) reasonable therapeutic dosage range, (C)
suitability for oral administration (i.e., suitable stability in
the gastrointestinal system and absorption into the vascular
system), for applications in whole animals, including gene therapy
applications, (D) ability to cross cellular and other membranes, as
necessary, (E) reasonable binding affinity for the CRD (for the
desired application), and (F) efficacy in stimulating transit of
the fusion protein. Preferably the compound is relatively
physiologically inert, but for its affinity for the CRD. The less
the ligand binds to native proteins or other materials within the
cells to be targeted, the better the response will normally be.
Preferably the ligand will be other than a peptide or nucleic acid,
and will preferably have a molecular weight of less than about 5000
Daltons, more preferably less than about 1200 Daltons.
[0066] In various embodiments where a ligand binding domain for a
candidate ligand is endogenous to the cells to be engineered, it is
often desirable to alter the peptide sequence of the ligand binding
domain and to use a ligand which discriminates between the
endogenous and engineered ligand binding domains. Such a ligand
should bind preferentially to the engineered ligand binding domain
relative to a naturally occurring peptide sequence, e.g., from
which the modified domain was derived. This approach can avoid
untoward intrinsic activities of the ligand. Significant guidance
and illustrative examples toward that end are provided in the
various references cited herein.
[0067] Substantial structural modification of a ligand for a ligand
binding domain is permitted, so long as the modified compound still
functions as a ligand for the ligand binding domain of interest,
i.e., so long as the compound possesses sufficient binding affinity
and specificity to function as disclosed herein. Some of the
compounds will be macrocyclics, e.g. macrolides, although linear
and branched compounds may be preferred in specific embodiments.
Suitable binding affinities will be reflected in Kd values well
below 10.sup.-4, preferably below 10.sup.-6, more preferably below
about 10.sup.-7, although binding affinities below 10.sup.-9 or
10.sup.-10 are possible, and in some cases will be most
desirable.
[0068] Illustrative examples of ligand binding domain/ligand pairs
include retinol binding protein or variants thereof and retinol or
derivatives thereof; cyclophilin or variants thereof and
cyclosporin or analogs thereof; FKBP or variants thereof and FK506,
FK520, rapamycin, analogs thereof or synthetic FKBP ligands. In the
case of a ligand binding domain comprising or derived from an
immunophilin or cyclophilin, the complex of the ligand with the
ligand binding domain will desirably not bind specifically to
calcineurin or FRAP. A wide variety of FK506 derivatives and
synthetic FKBP ligands are known which do not have observable
immunosuppressive activity. Likewise, a variety of rapamycin
analogs are known which bind to FKBP but are not immunosuppressive.
See e.g. WO 98/02441 for non-immunosuppressive rapalogs. Those and
other ligands can be used as well, depending on the choice of CRD.
Numerous assays are known in the art for identifying ligands which
bind to CRDs that are identified through screening, as described
below.
[0069] Ligand binding domain/ligand pairs are illustrated by FKBP
domains, e.g. F36M FKBP, and FKBP ligands. In general, it is
preferred that the ligand bind preferentially to a mutated (i.e.,
having a peptide sequence not naturally occurring in the cells to
be engineered) FKBP relative to wild-type FKBP. Ligands for FKBP
proteins, including F36M FKBP, can comprise or be derived from a
naturally occurring FKBP ligand such as rapamycin, FK506 or FK520,
or a synthetic FKBP ligand, e.g. as disclosed in PCT/US95/10559;
Holt, et al., J. Amer. Chem. Soc., 1993, 115, 9925-9938; Holt, et
al., Biomed. Chem. Lett., 1993, 4, 315-320; Luengo, et al., Biomed.
Chem. Lett., 1993, 4, 321-324; Yamashita, et al., Biomed. Chem.
Lett., 1993, 4, 325-328; PCT/US94/08008. See also EP 0 455 427 A1;
EP 0 465 426 A1; US 5,023,26; WO 92/00278; WO 94/18317; WO
97/31898; WO 96/41865; and Van Duyne et al (1991) Science 252,
839.
[0070] Illustrative types of ligands for FKBP-derived ligand
binding domains include the following Genus I: 1
[0071] where
[0072] n=1 or 2;
[0073] X=O, S, NH or CH.sub.2;
[0074] B.sup.1 and B.sup.2 are independently H or aliphatic,
heteroaliphatic, aryl or heteroaryl as those terms are defined
below, usually containing one to about 12 carbon atoms (not
counting carbon atoms of optional substituents);
[0075] Y=O, S, NH, --NH(C.dbd.O)--, --NH(C.dbd.O)--O--,
--NH(SO.sub.2)-- or NR.sup.3, or represents a direct, i.e.
covalent, bond from R.sup.2 to carbon 9;
[0076] R.sup.1, R.sup.2, and R.sup.3 are aliphatic,
heteroaliphatic, aryl or heteroaryl, usually containing one to
about 36 carbon atoms (not counting carbon atoms of optional
substituents);
[0077] two or more of B.sup.1, B.sup.2 and R.sup.2 may be
covalently linked to form a C3-C7 cyclic or heterocyclic moiety;
and,
[0078] The term "aliphatic" as used herein includes both saturated
and unsaturated straight chain, branched, cyclic, or polycyclic
aliphatic hydrocarbons, which are optionally substituted with one
or more substituents.
[0079] The term "substituents" includes aliphatic, aryl, heteroaryl
and heterocyclic moietites, which may themselves be substituted, as
well as functional groups such as R.sup.8, --OR.sup.8, --SR.sup.8,
--CN,--CHO, .dbd.O, --COOH, --COR.sup.8, OS(O).sub.2 R.sup.8,
--SO.sub.2--NHR.sup.8, --NHSO.sub.2 R.sup.8, sulfate, sulfonate,
(or ester, carbamate, urea, oxime or carbonate thereof), --NH.sub.2
(or substituted amine, amide, urea, carbamate or guanidino
derivative therof), halo, trihaloalkyl, --SO.sub.2--CF.sub.3, and
--OSO.sub.2F, where R.sup.8 may be H, aliphatic, aryl, heteroaryl
or heteroaliphatic. Aliphatic, heteraliphatic, aryl and
heterocyclic substituents may themselves be substituted or
unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl;
methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or
-phenyl-C(Me).sub.2--CH.sub.2--O--CO--[C3-C6] alkyl or alkylamino).
Additional examples of substituents are illustrated by the specific
embodiments shown in the Examples which follow. (Unless otherwise
specified, the alkyl, other aliphatic, alkoxy and acyl groups
preferably contain 1-8, and in many cases 1-6, contiguous aliphatic
carbon atoms).
[0080] The term "aliphatic" is thus intended to include alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl
moieties.
[0081] As used herein, the term "alkyl" includes both straight and
branched alkyl groups. An analogous convention applies to other
generic terms such as "alkenyl", "alkynyl" and the like.
Furthermore, as used herein, the language "alkyl", "alkenyl",
"alkynyl" and the like encompasses both substituted and
unsubstituted groups.
[0082] The term "alkyl" refers to groups usually having one to
eight, preferably one to six carbon atoms. For example, "alkyl" may
refer to methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl,
sec-butyl, tert-butyl, pentyl, isopentyl tert-pentyl, hexyl,
isohexyl, and the like. Suitable substituted alkyls include, but
are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl,
2-fluoroethyl, 3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl,
3-hydroxypropyl, and the like.
[0083] The term "alkenyl" refers to groups usually having two to
eight, preferably two to six carbon atoms. For example, "alkenyl"
may refer to prop-2-enyl, but-2-enyl, but-3-enyl,
2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl,
2,3-dimethylbut-2-enyl, and the like. The language "alkynyl," which
also refers to groups having two to eight, preferably two to six
carbons, includes, but is not limited to, prop-2-ynyl, but-2-ynyl,
but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl,
hex-5-ynyl, and the like.
[0084] The term "cycloalkyl" as used herein refers to groups having
three to seven, preferably three to six carbon atoms. Suitable
cycloalkyls include, but are not limited to cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
[0085] The term "heteroaliphatic" as used herein refers to
aliphatic moieties which contain one or more oxygen, sulfur, or
nitrogen atoms, e.g., in place of carbon atoms.
[0086] The term "heterocycle" as used herein refers to cyclic
aliphatic groups having one or more heteroatoms, and preferably
three to seven ring atoms total, includes, but is not limited to
oxetane, tetrahydrofuranyl, tetrahydropyranyl, aziridine,
azetidine, pyrrolidine, piperidine, morpholine, piperazine and the
like.
[0087] The terms "aryl" and "heteroaryl" as used herein refer to
stable mono- or polycyclic, heterocyclic, polycyclic, and
polyheterocyclic unsaturated moieties having 3-14 carbon atom which
may be substituted or unsubstituted. Non-limiting examples of
useful aryl ring groups include phenyl, halophenyl, alkoxypheriyl,
dialkoxyphenyl, trialkoxyphenyl, alkylenedioxyphenyl, naphthyl,
phenanthryl, anthryl, phenanthro and the like. Examples of typical
heteroaryl rings include 5-membered monocyclic ring groups such as
thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl, isothiazolyl,
furazanyl, isoxazolyl, thiazolyl and the like; 6-membered
monocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl,
pyridazinyl, triazinyl and the like; and polycyclic heterocyclic
ring groups such as benzo[b]thienyl, naphtho[2,3-b]thienyl,
thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl,
phenoxathienyl, indolizinyl, isoindolyl, indolyl, indazolyl,
purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,
quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole,
tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl,
beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,
phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,
phenoxazinyl, and the like(see e.g. Katritzky, Handbook of
Heterocyclic Chemistry). The aryl or heteroaryl moieties may be
substituted with one to five members selected from the group
consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched or
straight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino,
nitro, halo, trihalomethyl, cyano, and carboxyl.
[0088] A "halo" substituent according to the present invention may
be a fluoro, chloro, bromo or iodo substituent.
[0089] As discussed above, R.sup.1 may be aliphatic,
heteroaliphatic, aryl or heteroaryl and usually comprises one to
about 36 carbon atoms, exclusive of optional substituents.
[0090] In certain embodiments, R.sup.1 is optionally be joined,
i.e., covalently linked, to R.sup.2, B.sup.1 or B.sup.2, forming a
macrocyclic structure.
[0091] In certain embodiments --XR.sup.1 is a moiety of the formula
2
[0092] where R.sup.4 is a H, aliphatic, heteroaliphatic, aryl or
heteroaryl. The aliphatic moieties may be branched, unbranched,
cyclic, saturated or unsaturated, substituted or unsubstituted and
include, e.g, methyl, ethyl, isopropyl, t-butyl, cyclopentyl,
cyclohexyl, etc. Heteroaliphatic moieties may be branched,
unbranched or cyclic and include heterocycles such as morpholino,
pyrrolidinyl, etc. Illustrative ortho-, meta- or para-,
substitutents for a phenyl group at this position include one or
more of the following: halo, e.g. chloro or flouro; hydroxyl,
amino, --SO.sub.2NH.sub.2, --SO.sub.2NH(aliphatic),
--SO.sub.2N(aliphatic).sub.2, --O-aliphatic-COOH,
--O-aliphatic-NH.sub.2 (which may contain one or two N-aliphatic or
N-acyl substituents), C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy,
e.g. methoxy, ethoxy, methylenedioxy, ethylenedioxy, etc.
Heteroaryl groups are as discussed previously, including indolyl,
pyridyl, pyrrolyl, etc. Particular R.sup.4 moieties include the
following: 3
[0093] R.sup.5 is a branched, unbranched or cyclic aliphatic moiety
of 1 to 8 carbon atoms, which may be optionally substituted,
including for example, --CH--, --CHCH2--, --CH.sub.2CH--,
--CHCH.sub.2CH.sub.2---, --CH.sub.2CHCH.sub.2--,
--CH(CH.sub.3)--CH.sub.2--CH, --CH(CH.sub.2CH.sub.3)--CH.sub.2--CH,
--CH.sub.2CH.sub.2CH--, --C(CH.sub.3)CH.sub.2--, and the like;
[0094] R.sup.6 is an aliphatic, heteroaliphatic, heterocylic, aryl
or heteroaryl moiety, which may be substituted or unsubstituted.
Typical substituents for R.sup.6 include branched, unbranched or
cyclic, C1-C8, aliphatic or heteroaliphatic groups, including
unsaturated groups such as substitute or unsubstituted alkenes,
heterocycles, phenyl, etc.
[0095] R.sup.7 is H or a substituent such as, in certain
embodiments, --(CH.sub.2).sub.z--CH.dbd.CH.sub.2,
--(CH.sub.2).sub.z--COOH, --(CH.sub.2).sub.z--CHO,
--(CH.sub.2).sub.z--OH, --(CH.sub.2).sub.z--NH.s- ub.2,
--(CH.sub.2).sub.z--NH--alkyl, --(CH.sub.2).sub.z--SH, or an amino
group which may be substituted or unsubstituted (preferably a
tertiary amine), etc. In embodiments where R.sup.6 is aryl, R.sup.7
may be present in the o, m, or p position. z is an integer from 0
through 4.
[0096] As discussed above, B.sup.1, B.sup.2 and R.sup.2 may be
aliphatic, heteroaliphatic, aryl or heteroaryl. Typical groups
include a branched, unbranched or cyclic, saturated or unsaturated,
aliphatic moiety, preferably of 1 to about 12 carbon atoms
(including for example methyl, ethyl, n-propyl, isopropyl,
cyclopropyl, --CH.sub.2-cyclopropyl, allyl, n-butyl, sec-butyl,
isobutyl, tert-butyl, cyclobutyl, --CH.sub.2-cyclobutyl, n-pentyl,
sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,
--CH.sub.2--cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,
--CH.sub.2-cyclohexyl and the like), which aliphatic moiety may
optionally be substituted with an --OH, --C.dbd.O, --COOH, CHO,
allyl, NH.sub.2 (or substituted amine, amide, urea or carbamate),
ether (or thio-ether, in either case, aliphatic or aromatic), aryl,
or heteroaryl moiety, and may optionally contain a heteroatom in
place of one or more CH.sub.2 or CH units; or a substituted or
unsubstituted aryl (e.g. mono-, di- and tri-alkoxyphenyl;
methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or
-phenyl-C(Me).sub.2--CH.sub.2--O--CO--[C3-C6] alkyl or alkylamino)
or heteroaromatic moiety. In such embodiments, where YR.sup.2 is
-OPhenyl and B.sup.1 is H, B.sup.2 is preferably not cyclopentyl.
In other embodiments, Y is NH and the moiety
--(C.dbd.O)--CH(B.sup.1)NHR.sup- .2 comprises among other groups,
D- or L-forms of naturally occurring or synthetic alpha amino acids
as well as N-alkyl, N-acyl, N-aryl and N-aroyl derivatives thereof.
Particular XR.sup.1, G, B.sup.1, B.sup.2 and YR.sup.2 groups for
the various foregoing structures further include those illustrated
in compounds described in the examples, tables of monomers and
dimers and other disclosure in WO 96/06097, WO 97/31899 and WO
97/31898.
[0097] One preferred class of compounds are those compounds of
Genus I in which n is 2.
[0098] Another preferred class of compounds are those compounds of
Genus I in which B.sup.1 is H; B.sup.2 is branched, unbranched or
cyclic, saturated or unsaturated, aliphatic moiety, preferably of 1
to 8, more preferably 1 to 6, carbon atoms (including for example
methyl, ethyl, n-propyl, isopropyl, cyclopropyl,
--CH.sub.2-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,
tert-butyl, cyclobutyl, --CH.sub.2-cyclobutyl, n-pentyl,
sec-pentyl, isopentyl, tert-pentyl, cyclopentyl,
--CH.sub.2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,
--CH.sub.2-cyclohexyl and the like), which aliphatic moiety may
optionally be substituted, e.g. with an --OH, --C.dbd.O, --COOH,
CHO, allyl, NH.sub.2 (or substituted amine, amide, urea or
carbamate), or ether (or thio-ether, in either case, aliphatic or
aromatic), and may optionally contain a heteroatom in place of one
or more CH.sub.2 or CH units; and YR.sup.2 is aryl, heteroaryl and
may be optionally substituted (YR.sup.2, for instance, includes
moieties such as o-, m-, or p-alkoxyphenyl; 3,5-, 2,3-, 2,4-, 2,5-,
3,4- or 3,5-dialkoxyphenyl, or 3,4,5-trialkoxyphenyl, e.g. where
the alkoxy groups are independently selected from methoxy and
ethoxy (one or more of which may bear a hydroxy or amino
moiety).
[0099] Another preferred class of compounds are those compounds of
Genus I in which B.sup.1, B.sup.2 and YR.sup.2 are the same or
different lower aliphatic moieties.
[0100] Another preferred class of compounds are those compounds of
Genus I which contain a moiety --NB.sup.1R.sup.2 in which B.sup.1
is H and R.sup.2 is lower aliphatic.
[0101] Another preferred class of compound are those compounds of
Genus I in which G is an alicyclic or heterocyclic group bearing
optional substituents.
[0102] Another preferred class of compounds are those compounds of
Genus I in which X is oxygen and R.sup.1 comprises
R.sup.4R.sup.5R.sup.6R.sup.7 where R.sup.4 is aliphatic, alicyclic,
aryl, heteroaryl, or heterocyclic, optionally substituted; R.sup.5
is a branched or unbranched lower aliphatic group; R.sup.6 is
aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl or
heteroaryl, optionally substituted.
[0103] Another preferred class of compounds are those compounds of
Genus I in which R1 comprises R.sup.4R.sup.5R.sup.6R.sup.7 as
described in the immediately preceding paragraph and
YR.sup.2comprises a substituted or unsubstituted aryl or
heteroaryl, including phenyl; o-, m- or p- substituted phenyl where
the substituent is halo such as chloro, lower alkyl, or alkoxy,
such as methoxy or ethoxy; disubstituted phenyl, e.g.
dialkoxyphenyl such as 2,4-, 3,4- or 3.5-dimethoxy or diethoxy
phenyl or such as methylenedioxyphenyl, or
3-methoxy-5-ethoxyphenyl; or trisubstituted phenyl, such as
trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl),
3,5-dimethoxy-4-chloro-phenyl, etc.).
[0104] In addition, such compounds may comprise a substituted
proline and pipecolic acid derivative, numerous examples of which
have been described in the literature. Using synthetic procedures
similar to those described in the patent documents and scientific
literature cited herein, substituted prolines and pipecolates can
be utilized to prepare ligands with substituents at positions C-2
to C-6 (with reference to the FK506 numbering of most of the
references cited below), as exemplified in the patent applications
cited herein.
[0105] For representative examples of substituted prolines and
pipecolic acids see: Chung, et al., J. Org. Chem., 1990, 55, 270;
Shuman, et al., J. Org. Chem., 1990, 55, 738; Hanson, et al.,
Tetrahedron Lett., 1989, 30, 5751; Bailey, et al., Tetrahedron
Lett., 1989, 30, 6781.
[0106] For a variety of guidance on chemical transformations,
synthesis, formulation and delivery of a variety of compounds,
including additional information relating to FKBP ligands and/or to
ligands for other ligand binding domains, see e.g., WO 94/18317 and
Belshaw et al, 1996, PNAS 93:4604-4607) (for methods and materials
based on ligands for an immunophilin such as FKBP, a cyclophilin,
and/or FRB domain); WO 96/06097 and WO 97/31898 (more ligands for
FKBP and variants thereof); WO 93/33052, WO 96/41865 and Rivera et
al, "A humanized system for pharmacologic control of gene
expression", Nature Medicine 2(9):1028-1032 (1997)) (rapamycin
analogs); WO 94/18317 (cyclophilin/cyclosporin); Licitra et al,
1996, Proc. Natl. Acad. Sci. USA 93:12817-12821
(DHFR/methotrexate); and Farrar et al, 1996, Nature 383:178-181
(DNA gyrase/coumermycin). Numerous variations and modifications to
ligands and ligand binding domains, as well as methodologies for
designing, selecting and/or characterizing them, which may be
adapted to the present invention are disclosed in the cited
references.
[0107] Cleavage Enzymes:
[0108] It is often preferable in the design of fusion proteins of
this invention to have an enzymatic cleavage site located between
the CRD and the target protein. When the fusion protein exits the
ER following addition of ligand, the enzymatic cleavage site allows
the target protein to be released from the CRD and secreted.
Ideally, the cleavage site should be specific to an enzyme which
resides in a cellular compartment between the ER and the plasma
membrane, e.g. the Golgi apparatus. An exemplary cleavage enzyme is
furin, also known as PACE. Furin is a member of the KEX2/subtilisin
family of pro-protein convertases, which convert pro-proteins and
pro-hormones to their active forms (Kazuhisa Nakayama, Biochem J.
(1997) 327:625-635). It is a protein which resides in the
trans-golgi, although like many golgi proteins such as TGN38, it
constitutively cycles between the cell surface and the TGN
(trans-golgi network). Furin has a ubiquitous tissue distribution
and its substrates are numerous and varied. However, nearly all
share the consensus cleavage sequence RX(K/R)R. Proteins which are
substrates for furin include: human pro-neurotrophin-3 (MSMRVRR),
human pro-insulin like growth factor I (KPAKSAR), human
pro-parathyroid hormone (KSVKKR), human stromelysin-3 (ARNRQKR).
Furin is also capable of cleaving membrane bound substrates, such
as human insulin pro-receptor (RPSRKRR) and human hepatocyte growth
factor pro-receptor (TEKRKKR). A cleavage site from any furin
substrate can be used in the fusion proteins of the invention. In
some cases, the site will be be a non-naturally occurring peptide
sequence containing the consensus furin cleavage sequence. One
particular advantage of having furin as the cleavage enzyme is that
its recognition sequence is located exclusively N-terminal to the
cleavage site. This allows the portion of the protein that encodes
the target protein to be released from the cell unaltered by the
presence of additional amino acids.
[0109] The furin family contains other members which may also be
useful in the practice of this invention. Many of these proteins
have a unique tissue distribution. For example, PC1/PC3 and PC2 are
only found in neuroendocrine tissues like pancreatic islets,
pituitary and brain and PC4 is expressed primarily within
testicular-germ cells. PACE4, as well as PC5/PC6 and
LPC/PC7/PC8/SPC7 are expressed ubiquitously (Nakayama, 1997).
Cleavage sites for these enzymes may also be used in the practice
of this invention, provided the fusion proteins are expressed in
the appropriate cell type.
[0110] In addition, any mammalian protease with a specific cleavage
sequence, such as subtilisin, could be used to cleave the fusion
proteins of this invention, if it were targeted to the desired
location in the cell. For example, subtilisin could be targeted to
the TGN by fusing it to a localization sequence from a resident
golgi protein such as TGN38. Alternatively, the motifs which are
known to target furin to the TGN, including YKGL and the
Ser-containing cluster SDSEEDE, may suffice to target a cytoplasmic
protease to the Golgi. Cells may also be engineered to express an
enzyme tailored to cut a sequence found only in the CRD containing
fusion protein. For example, Ballinger et al. describe mutant forms
of sub tilisin in which the enzyme has been engineered to acquire
the specificity of furin (Ballinger Md., et al. Biochemistry. Oct
22, 1996;35(42):13579-85. Ballinger Md., et al. Biochemistry. Oct
17, 1995;34(41):13312
[0111] Secretory Signal Sequences:
[0112] When secretory proteins are translated on the ribosome, an
amino acid sequence of 16-30 residues, known as the signal
sequence, directs the ribosome to the ER membrane. This sequence
then initiates a signal which transports the nascent chain into the
ER, across the ER membrane. Generally, such sequences are found at
the N-terminus of a protein and contain one or more positively
charged amino acids followed by a stretch of 6-12 hydrophobic
residues. Numerous signal sequences are known, and any signal
sequence which normally directs the translocation of a secretory or
transmembrane protein to the ER may be used in the fusion proteins
of this invention. Exemplary signal sequences are those from
preproalbumin, prelysozyme, human growth hormone, proinsulin,
acetylcholine receptor or IgG light chain. For use in this
invention, a signal sequence is encoded at the N-terminus of the
protein to be regulatably secreted. This signal sequence then
directs the ribosome to the ER, where the translated protein
containing the CRD aggregates until ligand is added to the
cell.
[0113] Target Proteins:
[0114] Fusion proteins of this invention may contain any target
protein which one may want to secrete or translocate rapidly and
efficiently. Preferably, the target protein will be a therapeutic
protein. The target protein can provide a desired phenotype. It can
be a membrane-bound or membrane-spanning protein, a secreted
protein, or a cytoplasmic protein. The proteins which are
expressed, singly or in combination, can involve homing,
cytotoxicity, proliferation, differentiation, immune response,
inflammatory response, clotting, thrombolysis, hormonal regulation,
angiogenesis, etc. The polypeptide may be of naturally occurring or
non-naturally occurring peptide sequence.
[0115] Various secreted products include hormones, such as insulin,
human growth hormone, glucagon, pituitary releasing factor, ACTH,
melanotropin, relaxin, leptin, etc.; growth factors, such as EGF,
IGF-1, TGF-alpha, -beta, PDGF, G-CSF, M-CSF, GM-CSF, members of the
FGF family, erythropoietin, thrombopoietin, megakaryocytic growth
factors, nerve growth factors, etc.; proteins which stimulate or
inhibit angiogenesis such as angiostatin, endostatin and VEGF and
variants thereof; interleukins, such as IL-1 to -15; TNF-alpha and
-beta; interferons -alpha, -beta and -gamma; and enzymes and other
factors, such as tissue plasminogen activator, members of the
complement cascade, perforins, superoxide dismutase;
coagulation-related factors such as antithrombin-III, Factor V,
Factor VII, Factor VIIIc, vWF, Factor IX, alpha-anti-trypsin,
protein C, and protein S; endorphins, dynorphin, bone morphogenetic
protein, CFTR, etc.
[0116] The protein may be a naturally-occurring surface membrane
protein or a protein made so by introduction of an appropriate
signal peptide and transmembrane sequence. Various such proteins
include homing receptors, e.g. L-selectin (Mel-14), hematopoietic
cell markers, e.g. CD3, CD4, CD8, B cell receptor, TCR subunits
alpha, beta, gamma or delta, CD10, CD19, CD28, CD33, CD38, CD41,
etc., receptors, such as the interleukin receptors IL-2R, IL-4R,
etc.; receptors for other ligands including the various hormones,
growth factors, etc.; receptor antagonists for such receptors and
soluble forms of such receptors; channel proteins, for influx or
efflux of ions, e.g. H.sup.+, Ca.sup.+2, K.sup.+, Na.sup.+,
Cl.sup.-, etc., and the like; CFTR, tyrosine activation motif,
zap-70, etc.
[0117] The target protein can be an intracellular protein such as a
protein involved in a metabolic pathway, or a regulatory protein,
steroid receptor, transcription factor, etc.,
[0118] By way of further illustration, in T-cells, one may wish to
introduce genes encoding one or both chains of a T-cell receptor.
For B-cells, one could provide the heavy and light chains for an
immunoglobulin for secretion. For cutaneous cells, e.g.
keratinocytes, particularly keratinocyte stem cells, one could
provide for protection against infection, by secreting alpha, beta
or gamma interferon, antichemotactic factors, proteases specific
for bacterial cell wall proteins, various anti-viral proteins,
etc.
[0119] In various situations, one may wish to direct a cell to a
particular site. The site can include anatomical sites, such as
lymph nodes, mucosal tissue, skin, synovium, lung or other internal
organs or functional sites, such as clots, injured sites, sites of
surgical manipulation, inflammation, infection, etc. Regulated
expression of a membrane protein which recognizes or binds to the
particular site of interest, for example, provides a method for
directing the engineered cells to that site. Thus one can achieve a
localized concentration of a secreted product or effect cell-based
healing, scavenging, protection from infection, anti-tumor
activity, etc. Proteins of interest include homing receptors, e.g.
L-selectin, GMP140, CLAM-1, etc., or addressins, e.g. ELAM-1, PNAd,
LNAd, etc., clot binding proteins, or cell surface proteins that
respond to localized gradients of chemotactic factors.
[0120] In one embodiment of this invention, binding of a ligand to
a CRD regulates transcription of a target gene. In this embodiment,
the target gene may encode any protein, including those described
above.
[0121] Disposal Targeting Sequences:
[0122] In many embodiments of the invention, it would be desirable
to dispose of the CRD following its cleavage from the target
protein. Disposal of the CRD would prevent its secretion from the
cell and its accumulation in the bloodstream. One way to achieve
this goal is to target the CRD to a lysosomal compartment, where it
would be degraded. During normal cellular trafficking, lysosomal
proteins are sorted from the trans-golgi network, where they are
directed to the endosomal pathway, and subsequently, to lysosomes.
Resident soluble lysosomal enzymes such as cathepsin D are marked
for targeting to the lysosomal pathway by attachment of a phosphate
group on carbon 6 of one or more mannose residues on a particular
N-linked oligosaccharide, which are then recognized by the
mannose-6-phosphate receptor in the lysozyme. The
phosphotransferase recognition sequence of cathepsin D consists of
two discontinuous sequences: amino acids 188-230, including a
critical lysine residue at position 203, and amino acids 265-292
(Baranski et al., Cell 1990, 63:281-291.) Baranski et al. have
demonstrated that splicing of these sequences into the appropriate
location on pepsinogen, a secretory protein, resulted in
phosphorylation of the sugars on the chimeric molecule (Baranski et
al., supra). Other groups have shown that fusion of the entire
cathepsin B sequence onto MyoD resulted in targeting of the complex
to the lysosome (Li et al., J. Cell Biol., 135:1043-1057, November
1996.) Similarly, chimeric proteins consisting of soluble CD4,
procathepsin D and the C-terminal tails of three lysosomal membrane
proteins were able to direct the HIV glycoprotein gp1 60 to the
lysosome for degradation (Lin et al., FASEB J., 7:1070-1080, August
1993.) Lysosomal membrane proteins such as lamp-1 and LAP are
directed to the lysosome via a tyrosine-based targeting motif in
their C-terminal tails (Williams et al., J. Cell Biol.,
111:955-966, 1990; Klionsky et al., J. Biol. Chem., 265:5349-5352,
1990.) Fusion of these tails onto the extracellular and
transmembrane domains of resident plasma membrane proteins is
sufficient to target those proteins to the lysosome.
[0123] Either of the aforementioned lysosomal targeting signals may
be used to target CRDs of this invention for disposal. For soluble
proteins, the preferred method is to fuse a resident lysosomal
protein containing a mannose-6-phosphate signal to the CRD.
Examples of such proteins are the cysteine proteases of the
cathepsin family: cathepsins B, D, H, L, S, C and K. Other
lysosomal enzymes which may be used include the carboxypeptidases
prolylcarboxypeptidase and deamidase (cathepsin A). For membrane
bound CRDs, the preferred targeting sequence would be one found in
lysosomal membrane proteins, e.g. a tyrosine-based internalization
motif. These motifs are short, linear stretches of amino acids
within the cytoplasmic region of the protein to be targeted.
Tyrosine-based motifs center on a critical tyrosine residue within
the sequence NPXY or YXX.O slashed., where X is any amino acid and
.O slashed. is an amino acid with a bulky hydrophobic group. In
many proteins, a glycine preceding the tyrosine in a YXX.O
slashed.-type signal enhances targeting of these proteins to the
lysosome. Sequences for use in some applications of this invention
may be derived from proteins such as Lamp-1, LAP (lysosomal acid
phosphatase), CD63, Lamp-2 or CD3-gamma, all of which are normally
targeted to the lysosome. For additional information on
tyrosine-based sorting motifs, see, for example the review by Marks
et al., Trends in Cell Biology, 7:124-128, 1997.
[0124] Design and Assembly of the DNA Constructs
[0125] Constructs may be designed in accordance with the
principles, illustrative examples and materials and methods
disclosed in the patent documents and scientific literature cited
herein, with modifications and further exemplification as
described. Components of the constructs can be prepared in
conventional ways, where the coding sequences and regulatory
regions may be isolated, as appropriate, ligated, cloned in an
appropriate cloning host, analyzed by restriction or sequencing, or
other convenient means. Particularly, using PCR, individual
fragments including all or portions of a functional unit may be
isolated, where one or more mutations may be introduced using
"primer repair", ligation, in vitro mutagenesis, etc. as
appropriate. In the case of DNA constructs encoding fusion
proteins, DNA sequences encoding individual domains and sub-domains
are joined such that they constitute a single open reading frame
encoding a fusion protein capable of being translated in cells or
cell lysates into a single polypeptide harboring all component
domains. The DNA construct encoding the fusion protein may then be
placed into a vector for transducing host cells and permitting the
expression of the protein. For biochemical analysis of the encoded
chimera, it may be desirable to construct plasmids that direct the
expression of the protein in bacteria or in reticulocyte-lysate
systems. For use in the production of proteins in mammalian cells,
the protein-encoding sequence is introduced into an expression
vector that directs expression in these cells. Expression vectors
suitable for such uses are well known in the art. Various sorts of
such vectors are commercially available.
[0126] Promoters
[0127] The fusion proteins described herein may be used in
combination with any promoter that will direct their expression in
mammalian cells. The promoter may be a strong promoter, such as the
human CMV promoter, or a weaker promoter, such as a promoter for an
endogenous human gene. Other promoters which may be used include,
but are not limited to, the Rous Sarcoma Virus (RSV) promoter, the
retroviral LTR from Murine Moloney Leukemia Virus (MMLV), the
muscle creatine kinase (MCK) enhancer, the SV40 promoter, and the
CMV enhancer from the major immediate early gene. Genbank accession
numbers for the above promoters are given in the table below.
1 Promoter Genbank Accession Number CMV AF067197 RSV M83237 MMLV
LTR M77239 SV40 U47120 CMV enhancer for MIE gene K03104 MCK
enhancer X67536
[0128] In many cases, the selection of promoter will depend upon
the configuration of the fusion protein used in a particular
application. Thus, if the practitioner desired the CRD-containing
fusion protein to be expressed at high levels, a stronger promoter,
such as CMV, would be used. Alternatively, for tissue specific
expression, a tissue specific promoter like the MCK enhancer (for
expression in muscle) would be selected.
[0129] Introduction of Constructs into Cells
[0130] This invention is particularly useful for the engineering of
animal cells and in applications involving the use of such
engineered animal cells. The animal cells may be, among others,
insect, worm or mammalian cells. While various mammalian cells may
be used, including, by way of example, equine, bovine, ovine,
canine, feline, murine, and non-human primate cells, human and
mouse cells are of particular interest. Across the various species,
various types of cells may be used, such as hematopoietic, neural,
glial, mesenchymal, cutaneous, mucosal, stromal, muscle (including
smooth muscle cells), spleen, reticuloendothelal, epithelial,
endothelial, hepatic, kidney, gastrointestinal, pulmonary,
fibroblast, and other cell types. Of particular interest are muscle
cells (including skeletal, cardiac and other muscle cells), cells
of the central and peripheral nervous systems, and hematopoietic
cells, which may include any of the nucleated cells which may be
involved with the erythroid, lymphoid or myelomonocytic lineages,
as well as myoblasts and fibroblasts. Also of interest are stem and
progenitor cells, such as hematopoietic, neural, stromal, muscle,
hepatic, pulmonary, gastrointestinal and mesenchymal stem cells
[0131] The cells may be autologous cells, syngeneic cells,
allogeneic cells and even in some cases, xenogeneic cells with
respect to an intended host organism. The cells may be modified by
changing the major histocompatibility complex ("MHC") profile, by
inactivating .beta.2-microglobulin to prevent the formation of
functional Class I MHC molecules, inactivation of Class II
molecules, providing for expression of one or more MHC molecules,
enhancing or inactivating cytotoxic capabilities by enhancing or
inhibiting the expression of genes associated with the cytotoxic
activity, and the like.
[0132] In some instances specific clones or oligoclonal cells may
be of interest, where the cells have a particular specificity, such
as T cells and B cells having a specific antigen specificity or
homing target site specificity.
[0133] Constructs encoding the fusion proteins and comprising
target genes of this invention can be introduced into the cells as
one or more nucleic acid molecules or constructs, in many cases in
association with one or more markers to allow for selection of host
cells which contain the construct(s). The constructs can be
prepared in conventional ways, where the coding sequences and
regulatory regions may be isolated, as appropriate, ligated, cloned
in an appropriate cloning host, analyzed by restriction or
sequencing, or other convenient means. Particularly, using PCR,
individual fragments including all or portions of a functional
domain may be isolated, where one or more mutations may be
introduced using "primer repair", ligation, in vitro mutagenesis,
etc. as appropriate.
[0134] The construct(s) once completed and demonstrated to have the
appropriate sequences may then be introduced into a host cell by
any convenient means. The constructs may be incorporated into
vectors capable of episomal replication (e.g. BPV or EBV vectors)
or into vectors designed for integration into the host cells'
chromosomes. The constructs may be integrated and packaged into
non-replicating, defective viral genomes like Adenovirus,
Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or
others, including retroviral vectors, for infection or transduction
into cells. Alternatively, the construct may be introduced by
protoplast fusion, electroporation, biolistics, calcium phosphate
transfection, lipofection, microinjection of DNA or the like. The
host cells will in some cases be grown and expanded in culture
before introduction of the construct(s), followed by the
appropriate treatment for introduction of the construct(s) and
integration of the construct(s). The cells may then be expanded
and/or screened by virtue of a marker present in the constructs.
Various markers which may be used successfully include hprt,
neomycin resistance, thymidine kinase, hygromycin resistance, etc.,
and various cell-surface markers such as Tac, CD8, CD3, Thy1 and
the NGF receptor.
[0135] In some instances, one may have a target site for homologous
recombination, where it is desired that a construct be integrated
at a particular locus. For example, one can delete and/or replace
an endogenous gene (at the same locus or elsewhere) with a
recombinant target construct of this invention. For homologous
recombination, one may generally use either .OMEGA. or O-vectors.
See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512;
Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al.,
Nature (1989) 338, 153-156.
[0136] The constructs may be introduced as a single DNA molecule
encoding all of the genes, or different DNA molecules having one or
more genes. The constructs may be introduced simultaneously or
consecutively, each with the same or different markers.
[0137] Vectors containing useful elements such as bacterial or
yeast origins of replication, selectable and/or amplifiable
markers, promoter/enhancer elements for expression in prokaryotes
or eukaryotes, and mammalian expression control elements, etc.
which may be used to prepare stocks of construct DNAs and for
carrying out transfections are well known in the art, and many are
commercially available.
[0138] Introduction of Constructs into Animals
[0139] Any means for the introduction of genetically engineered
cells or heterologous DNA into animals, preferably mammals, human
or non-human, may be adapted to the practice of this invention for
the delivery of the various DNA constructs into the intended
recipient. For the purpose of this discussion, the various DNA
constructs described herein may together be referred to as the
transgene.
[0140] By ex vivo Genetic Engineering
[0141] Cells which have been transduced ex vivo or in vitro with
the DNA constructs may be grown in culture under selective
conditions and cells which are selected as having the desired
construct(s) may then be expanded and further analyzed, using, for
example, the polymerase chain reaction for determining the presence
of the construct in the host cells and/or assays for the production
of the desired gene product(s). After being transduced with the
heterologous genetic constructs, the modified host cells may be
identified, selected, grown, characterized, etc. as desired, and
then may be used as planned, e.g. grown in culture or introduced
into a host organism.
[0142] Depending upon the nature of the cells, the cells may be
introduced into a host organism, e.g. a mammal, in a wide variety
of ways, generally by injection or implantation into the desired
tissue or compartment, or a tissue or compartment permitting
migration of the cells to their intended destination. Illustrative
sites for injection or implantation include the vascular system,
bone marrow, muscle, liver, cranium or spinal cord, peritoneum, and
skin. Hematopoietic cells, for example, may be administered by
injection into the vascular system, there being usually at least
about 10.sup.4 cells and generally not more than about 10.sup.10
cells. The number of cells which are employed will depend upon the
circumstances, the purpose for the introduction, the lifetime of
the cells, the protocol to be used, for example, the number of
administrations, the ability of the cells to multiply, the
stability of the therapeutic agent, the physiologic need for the
therapeutic agent, and the like. Generally, for myoblasts or
fibroblasts for example, the number of cells will be at least about
10.sup.4 and not more than about 10.sup.9 and may be applied as a
dispersion, generally being injected at or near the site of
interest. The cells will usually be in a physiologically-acceptable
medium.
[0143] Cells engineered in accordance with this invention may also
be encapsulated, e.g. using conventional biocompatible materials
and methods, prior to implantation into the host organism or
patient for the production of a therapeutic protein. See e.g.
Hguyen et al, Tissue Implant Systems and Methods for Sustaining
viable High Cell Densities within a Host, U.S. Pat. No. 5,314,471
(Baxter International, Inc.); Uludag and Sefton, 1993, J Biomed.
Mater. Res. 27(10):1213-24 (HepG2 cells/hydroxyethyl
methacrylate-methyl methacrylate membranes); Chang et al, 1993, Hum
Gene Ther 4(4):433-40 (mouse Ltk- cells expressing
hGH/immunoprotective perm-selective alginate microcapsules; Reddy
et al, 1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun,
1993, FASEB J 7(11):1061-9 (mouse fibroblasts expressing
hGH/alginate-poly-L-lysine-alg- inate membrane); Ao et al, 1995,
Transplantation Proc. 27(6):3349, 3350 (alginate); Rajotte et al,
1995, Transplantation Proc. 27(6):3389 (alginate); Lakey et al,
1995, Transplantation Proc. 27(6):3266 (alginate); Korbutt et al,
1995, Transplantation Proc. 27(6):3212 (alginate); Dorian et al,
U.S. Pat. No. 5,429,821 (alginate); Emerich et al, 1993, Exp Neurol
122(1):37-47 (polymer-encapsulated PC12 cells); Sagen et al, 1993,
J Neurosci 13(6):2415-23 (bovine chromaffin cells encapsulated in
semipermeable polymer membrane and implanted into rat spinal
subarachnoid space); Aebischer et al, 1994, Exp Neurol 126(2):151-8
(polymer-encapsulated rat PC12 cells implanted into monkeys; see
also Aebischer, WO 92/19595); Savelkoul et al, 1994, J Immunol
Methods 170(2):185-96 (encapsulated hybridomas producing
antibodies; encapsulated transfected cell lines expressing various
cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8 (engineered BHK
cells expressing human nerve growth factor encapsulated in an
immunoisolation polymeric device and transplanted into rats);
Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry
18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);
Kordower et al, 1994, PNAS USA 91(23):10898-902
(polymer-encapsulated engineered BHK cells expressing hNGF
implanted into monkeys) and Butler et al WO 95/04521 (encapsulated
device). The cells may then be introduced in encapsulated form into
an animal host, preferably a mammal and more preferably a human
subject in need thereof. Preferably the encapsulating material is
semipermeable, permitting release into the host of secreted
proteins produced by the encapsulated cells. In many embodiments
the semipermeable encapsulation renders the encapsulated cells
immunologically isolated from the host organism in which the
encapsulated cells are introduced. In those embodiments the cells
to be encapsulated may express one or more fusion proteins
containing component domains derived from proteins of the host
species and/or from viral proteins or proteins from species other
than the host species. The cells may be derived from one or more
individuals other than the recipient and may be derived from a
species other than that of the recipient organism or patient.
[0144] By in vivo Genetic Engineering
[0145] Instead of ex vivo modification of the cells, in many
situations one may wish to modify cells in vivo. A variety of
techniques have been developed for genetic engineering of target
tissue and cells in vivo, including viral and non-viral
systems.
[0146] In one approach, the DNA constructs are delivered to cells
by transfection, i.e., by delivery to cells of "naked DNA",
lipid-complexed or liposome-formulated DNA, or otherwise formulated
DNA. Prior to formulation of DNA, e.g., with lipid, or as in other
approaches, prior to incorporation in a final expression vector, a
plasmid containing a transgene bearing the desired DNA constructs
may first be experimentally optimized for expression (e.g.,
inclusion of an intron in the 5' untranslated region and
elimination of unnecessary sequences (Felgner, et al., Ann NY Acad
Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or
liposome materials, may then be effected using known methods and
materials and delivered to the recipient mammal. See, e.g.,
Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994 (in vivo
transfer of an aerosolized recombinant human alphal-antitrypsin
gene complexed to cationic liposomes to the lungs of rabbits); Tsan
et al, Am J Physiol 268 (Lung Cell Mol Physiol 12): L1052-L1056,
1995 (transfer of genes to rat lungs via tracheal insufflation of
plasmid DNA alone or complexed with cationic liposomes); Alton et
al., Nat Genet. 5:135-142, 1993 (gene transfer to mouse airways by
nebulized delivery of cDNA-liposome complexes). In either case,
delivery of vectors or naked or formulated DNA can be carried out
by instillation via bronchoscopy, after transfer of viral particles
to Ringer's, phosphate buffered saline, or other similar vehicle,
or by nebulization.
[0147] Viral systems include those based on viruses such as
adenovirus, adeno-associated virus, hybrid adeno-AAV, lentivirus
and retroviruses, which allow for transduction by infection, and in
some cases, integration of the virus or transgene into the host
genome. See, for example, Dubensky et al. (1984) Proc. Natl. Acad.
Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243,375-378;
Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598;
Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 and Ferry,
et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. The virus
may be administered by injection (e.g. intravascularly or
intramuscularly), inhalation, or other parenteral mode. Non-viral
delivery methods such as administration of the DNA via complexes
with liposomes or by injection, catheter or biolistics may also be
used. See e.g. WO 96/41865, PCT/US97/22454 and U.S. Ser. No.
60/084819, for example, for additional guidance on formulation and
delivery of recombinant nucleic acids to cells and to
organisms.
[0148] By employing an attenuated or modified retrovirus carrying a
target transcriptional initiation region, if desired, one can
activate the virus using one of the subject transcription factor
constructs, so that the virus may be produced and transduce
adjacent cells.
[0149] The use of recombinant viruses to deliver the nucleic acid
constructs are of particular interest. The transgene(s) may be
incorporated into any of a variety of viruses useful in gene
therapy.
[0150] In clinical settings, the gene delivery systems (i.e., the
recombinant nucleic acids in vectors, virus, lipid formulation or
other form) can be introduced into a patient, e.g., by any of a
number of known methods. For instance, a pharmaceutical preparation
of the gene delivery system can be introduced systemically, e.g. by
intravenous injection, inhalation, etc. In some systems, the means
of delivery provides for specific or selective transduction of the
construct into desired target cells. This can be achieved by
regional or local administration (see U.S. Pat. No. 5,328,470) or
by stereotactic injection, e.g. Chen et al., (1994) PNAS USA 91:
3054-3057 or by determinants of the delivery means. For instance,
some viral systems have a tissue or cell-type specificity for
infection. In some systems cell-type or tissue-type expression is
achieved by the use of cell-type or tissue-specific expression
control elements controlling expression of the gene.
[0151] In preferred embodiments of the invention, the subject
expression constructs are derived by incorporation of the genetic
construct(s) of interest into viral delivery systems including a
recombinant retrovirus, adenovirus, adeno-associated virus (AAV),
hybrid adenovirus/AAV, herpes virus or lentivirus (although other
applications may be carried out using recombinant bacterial or
eukaryotic plasmids). While various viral vectors may be used in
the practice of this invention, AAV- and adenovirus-based
approaches are of particular interest for the transfer of exogenous
genes in vivo, particularly into humans and other mammals. The
following additional guidance on the choice and use of viral
vectors may be helpful to the practitioner, especially with respect
to applications involving whole animals (including both human gene
therapy and the development and use of animal model systems),
whether ex vivo or in vivo.
[0152] Viral Vectors:
[0153] Adenoviral Vectors
[0154] A viral gene delivery system useful in the present invention
utilizes adenovirus-derived vectors. Knowledge of the genetic
organization of adenovirus, a 36 kb, linear and double-stranded DNA
virus, allows substitution of a large piece of adenoviral DNA with
foreign sequences up to 8 kb. In contrast to retrovirus, the
infection of adenoviral DNA into host cells does not result in
chromosomal integration because adenoviral DNA can replicate in an
episomal manner without potential genotoxicity. Also, adenoviruses
are structurally stable, and no genome rearrangement has been
detected after extensive amplification. Adenovirus can infect
virtually all epithelial cells regardless of their cell cycle
stage. So far, adenoviral infection appears to be linked only to
mild disease such as acute respiratory disease in the human.
[0155] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target-cell range, and high
infectivity. Both ends of the viral genome contain 100-200 base
pair (bp) inverted terminal repeats (ITR), which are cis elements
necessary for viral DNA replication and packaging. The early (E)
and late (L) regions of the genome contain different transcription
domains that are divided by the onset of viral DNA replication. The
E1 region (E1A and E1B) encodes proteins responsible for the
regulation of transcription of the viral genome and a few cellular
genes. The expression of the E2 region (E2A and E2B) results in the
synthesis of the proteins for viral DNA replication. These proteins
are involved in DNA replication, late gene expression, and host
cell shut off (Renan (1990) Radiotherap. Oncol. 19:197). The
products of the late genes, including the majority of the viral
capsid proteins, are expressed only after significant processing of
a single primary transcript issued by the major late promoter
(MLP). The MLP (located at 16.8 m.u.) is particularly efficient
during the late phase of infection, and all the mRNAs issued from
this promoter possess a 5' tripartite leader (TL) sequence which
makes them preferred mRNAs for translation.
[0156] The genome of an adenovirus can be manipulated such that it
encodes a gene product of interest, but is inactivated in terms of
its ability to replicate in a normal lytic viral life cycle (see,
for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld
et al., (1991) Science 252:431-434; and Rosenfeld et al., (1992)
Cell 68:143-155). Suitable adenoviral vectors derived from the
adenovirus strain Ad type 5 dl324 or other strains of adenovirus
(e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the
art. Recombinant adenoviruses can be advantageous in certain
circumstances in that they are not capable of infecting nondividing
cells and can be used to infect a wide variety of cell types,
including airway epithelium (Rosenfeld et al., (1992) cited supra),
endothelial cells (Lemarchand et al., (1992) PNAS USA
89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA
90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA
89:2581-2584). Adenovirus vectors have also been used in vaccine
development (Grunhaus and Horwitz (1992) Seminar in Virology 3:237;
Graham and Prevec (1992) Biotechnology 20:363). Experiments in
administering recombinant adenovirus to different tissues include
trachea instillation (Rosenfeld et al. (1991); Rosenfeld et al.
(1992) Cell 68:143), muscle injection (Ragot et al. (1993) Nature
361:647), peripheral intravenous injection (Herz and Gerard (1993)
Proc. Natl. Acad. Sci. U.S.A. 90:2812), and stereotactic
inoculation into the brain (Le Gal La Salle et al. (1993) Science
254:988).
[0157] Furthermore, the virus particle is relatively stable and
amenable to purification and concentration, and as above, can be
modified so as to affect the spectrum of infectivity. Additionally,
adenovirus is easy to grow and manipulate and exhibits broad host
range in vitro and in vivo. This group of viruses can be obtained
in high titers, e.g., 10.sup.9-10.sup.11 plaque-forming unit
(PFU)/ml, and they are highly infective. The life cycle of
adenovirus does not require integration into the host cell genome.
The foreign genes delivered by adenovirus vectors are episomal, and
therefore, have low genotoxicity to host cells. No side effects
have been reported in studies of vaccination with wild-type
adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating
their safety and therapeutic potential as in vivo gene transfer
vectors. Moreover, the carrying capacity of the adenoviral genome
for foreign DNA is large (up to 8 kilobases) relative to other gene
delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham
(1986) J. Virol. 57:267). Most replication-defective adenoviral
vectors currently in use and therefore favored by the present
invention are deleted for all or parts of the viral E.sup.1 and E3
genes but retain as much as 80% of the adenoviral genetic material
(see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al.,
supra; and Graham et al., in Methods in Molecular Biology, E. J.
Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).
Expression of the inserted gene can be under control of, for
example, the E1A promoter, the major late promoter (MLP) and
associated leader sequences, the viral E3 promoter, or exogenously
added promoter sequences.
[0158] Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be of
any of the 42 different known serotypes or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the method of the present invention. This is
because Adenovirus type 5 is a human adenovirus about which a great
deal of biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector. As stated above, the typical vector according to the
present invention is replication defective and will not have an
adenovirus E1 region. Thus, it will be most convenient to introduce
the nucleic acid of interest at the position from which the E1
coding sequences have been removed. However, the position of
insertion of the nucleic acid of interest in a region within the
adenovirus sequences is not critical to the present invention. For
example, the nucleic acid of interest may also be inserted in lieu
of the deleted E3 region in E3 replacement vectors as described
previously by Karlsson et. al. (1986) or in the E4 region where a
helper cell line or helper virus complements the E4 defect.
[0159] A preferred helper cell line is 293 (ATCC Accession No.
CRL1573). This helper cell line, also termed a "packaging cell
line" was developed by Frank Graham (Graham et al. (1987) J. Gen.
Virol. 36:59-72 and Graham (1977) J.General Virology 68:937-940)
and provides E1A and E1B in trans. However, helper cell lines may
also be derived from human cells such as human embryonic kidney
cells, muscle cells, hematopoietic cells or other human embryonic
mesenchymal or epithelial cells. Alternatively, the helper cells
may be derived from the cells of other mammalian species that are
permissive for human adenovirus. Such cells include, e.g., Vero
cells or other monkey embryonic mesenchymal or epithelial
cells.
[0160] Various adenovirus vectors have been shown to be of use in
the transfer of genes to mammals, including humans.
Replication-deficient adenovirus vectors have been used to express
marker proteins and CFTR in the pulmonary epithelium. Because of
their ability to efficiently infect dividing cells, their tropism
for the lung, and the relative ease of generation of high titer
stocks, adenoviral vectors have been the subject of much research
in the last few years, and various vectors have been used to
deliver genes to the lungs of human subjects (Zabner et al., Cell
75:207-216, 1993; Crystal, et al., Nat Genet. 8:42-51, 1994;
Boucher, et al., Hum Gene Ther 5:615-639, 1994). The first
generation E1a deleted adenovirus vectors have been improved upon
with a second generation that includes a temperature-sensitive E2a
viral protein, designed to express less viral protein and thereby
make the virally infected cell less of a target for the immune
system (Goldman et al., Human Gene Therapy 6:839-851,1995). More
recently, a viral vector deleted of all viral open reading frames
has been reported (Fisher et al., Virology 217:11-22, 1996).
Moreover, it has been shown that expression of viral IL-10 inhibits
the immune response to adenoviral antigen (Qin et al., Human Gene
Therapy 8:1365-1374, 1997).
[0161] Adenoviruses can also be cell type specific, i.e., infect
only restricted types of cells and/or express a transgene only in
restricted types of cells. For example, the viruses comprise a gene
under the transcriptional control of a transcription initiation
region specifically regulated by target host cells, as described
e.g., in U.S. Pat. No. 5,698,443, by Henderson and Schuur, issued
Dec. 16, 1997. Thus, replication competent adenoviruses can be
restricted to certain cells by, e.g., inserting a cell specific
response element to regulate a synthesis of a protein necessary for
replication, e.g., E1A or E1B.
[0162] DNA sequences of a number of adenovirus types are available
from Genbank. For example, human adenovirus type 5 has GenBank
Accession No.M73260. The adenovirus DNA sequences may be obtained
from any of the 42 human adenovirus types currently identified.
Various adenovirus strains are available from the American Type
Culture Collection, Rockville, Md., or by request from a number of
commercial and academic sources. A transgene as described herein
may be incorporated into any adenoviral vector and delivery
protocol, by the same methods (restriction digest, linker ligation
or filling in of ends, and ligation) used to insert the CFTR or
other genes into the vectors.
[0163] Adenovirus producer cell lines can include one or more of
the adenoviral genes E1, E2a, and E4 DNA sequence, for packaging
adenovirus vectors in which one or more of these genes have been
mutated or deleted are described, e.g., in PCT/US95/15947 (WO
96/18418) by Kadan et al.; PCT/US95/07341 (WO 95/346671) by Kovesdi
et al.; PCT/FR94/00624 (WO94/28152) by Imler et al.; PCT/FR94/00851
(WO 95/02697) by Perrocaudet et al., PCT/US95/14793 (WO96/14061) by
Wang et al.
[0164] AAV Vectors
[0165] Another viral vector system useful for delivery of DNA is
the adeno-associated virus (AAV). Adeno-associated virus is a
naturally occurring defective virus that requires another virus,
such as an adenovirus or a herpes virus, as a helper virus for
efficient replication and a productive life cycle. (For a review,
see Muzyczka et al., Curr. Topics in Micro. and Immunol. (1992)
158:97-129).
[0166] AAV has not been associated with the cause of any disease.
AAV is not a transforming or oncogenic virus. AAV integration into
chromosomes of human cell lines does not cause any significant
alteration in the growth properties or morphological
characteristics of the cells. These properties of AAV also
recommend it as a potentially useful human gene therapy vector.
[0167] AAV is also one of the few viruses that may integrate its
DNA into non-dividing cells, e.g., pulmonary epithelial cells or
muscle cells, and exhibits a high frequency of stable integration
(see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol.
Biol. 7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828;
and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate. Space for exogenous DNA is limited to about 4.5 kb.
An AAV vector such as that described in Tratschin et al., (1985)
Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into
cells. A variety of nucleic acids have been introduced into
different cell types using AAV vectors (see for example Hermonat et
al., (1984) PNAS USA 81:6466-6470; Tratschin et al., (1985) Mol.
Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol.
2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte
et al., (1993) J. Biol. Chem. 268:3781-3790).
[0168] The AAV-based expression vector to be used typically
includes the 145 nucleotide AAV inverted terminal repeats (ITRs)
flanking a restriction site that can be used for subcloning of the
transgene, either directly using the restriction site available, or
by excision of the transgene with restriction enzymes followed by
blunting of the ends, ligation of appropriate DNA linkers,
restriction digestion, and ligation into the site between the ITRs.
The capacity of AAV vectors is about 4.4 kb. The following proteins
have been expressed using various AAV-based vectors, and a variety
of promoter/enhancers: neomycin phosphotransferase, chloramphenicol
acetyl transferase, Fanconi's anemia gene, cystic fibrosis
transmembrane conductance regulator, and granulocyte macrophage
colony-stimulating factor (Kotin, R. M., Human Gene Therapy
5:793-801, 1994, Table I). A transgene incorporating the various
DNA constructs of this invention can similarly be included in an
AAV-based vector. As an alternative to inclusion of a constitutive
promoter such as CMV to drive expression of the recombinant DNA
encoding the fusion protein(s), e.g. fusion proteins comprising an
activation domain or DNA-binding domain, an AAV promoter can be
used (ITR itself or AAV p5 (Flotte, et al. J. Biol.Chem.
268:3781-3790, 1993)).
[0169] Such a vector can be packaged into AAV virions by reported
methods. For example, a human cell line such as 293 can be
co-transfected with the AAV-based expression vector and another
plasmid containing open reading frames encoding AAV rep and cap
(which are obligatory for replication and packaging of the
recombinant viral construct) under the control of endogenous AAV
promoters or a heterologous promoter. In the absence of helper
virus, the rep proteins Rep68 and Rep78 prevent accumulation of the
replicative form, but upon superinfection with adenovirus or herpes
virus, these proteins permit replication from the ITRs (present
only in the construct containing the transgene) and expression of
the viral capsid proteins. This system results in packaging of the
transgene DNA into AAV virions (Carter, B. J., Current Opinion in
Biotechnology 3:533-539, 1992; Kotin, R. M, Human Gene Therapy
5:793-801, 1994)). Typically, three days after transfection,
recombinant AAV is harvested from the cells along with adenovirus
and the contaminating adenovirus is then inactivated by heat
treatment.
[0170] Methods to improve the titer of AAV can also be used to
express the transgene in an AAV virion. Such strategies include,
but are not limited to: stable expression of the ITR-flanked
transgene in a cell line followed by transfection with a second
plasmid to direct viral packaging; use of a cell line that
expresses AAV proteins inducibly, such as temperature-sensitive
inducible expression or pharmacologically inducible expression.
Alternatively, a cell can be transformed with a first AAV vector
including a 5' ITR, a 3' ITR flanking a heterologous gene, and a
second AAV vector which includes an inducible origin of
replication, e.g., SV40 origin of replication, which is capable of
being induced by an agent, such as the SV40 T antigen and which
includes DNA sequences encoding the AAV rep and cap proteins. Upon
induction by an agent, the second AAV vector may replicate to a
high copy number, and thereby increased numbers of infectious AAV
particles may be generated (see, e.g, U.S. Pat. No. 5,693,531 by
Chiorini et al., issued Dec. 2, 1997. In yet another method for
producing large amounts of recombinant AAV, a plasmid is used which
incorporate the Epstein Barr Nuclear Antigen (EBNA) gene, the
latent origin of replication of Epstein Barr virus (oriP) and an
AAV genome. These plasmids are maintained as a multicopy
extra-chromosomal elements in cells, such as in 293 cells. Upon
addition of wild-type helper functions, these cells will produce
high amounts of recombinant AAV (U.S. Pat. No. 5,691,176 by
Lebkowski et al., issued Nov. 25, 1997). In another system, an AAV
packaging plasmid is provided that allows expression of the rep
gene, wherein the p5 promoter, which normally controls rep
expression, is replaced with a heterologous promoter (U.S. Pat. No.
5,658,776, by Flotte et al., issued Aug. 19, 1997). Additionally,
one may increase the efficiency of AAV transduction by treating the
cells with an agent that facilitates the conversion of the single
stranded form to the double stranded form, as described in Wilson
et al., WO96/39530.
[0171] AAV stocks can be produced as described in Hermonat and
Muzyczka (1984) PNAS 81:6466, modified by using the pAAV/Ad
described by Samulski et al. (1989) J. Virol. 63:3822.
Concentration and purification of the virus can be achieved by
reported methods such as banding in cesium chloride gradients, as
was used for the initial report of AAV vector expression in vivo
(Flotte, et al. J.Biol. Chem. 268:3781-3790, 1993) or
chromatographic purification, as described in O'Riordan et al.,
WO97/08298.
[0172] Methods for in vitro packaging AAV vectors are also
available and have the advantage that there is no size limitation
of the DNA packaged into the particles (see, U.S. Pat. No.
5,688,676, by Zhou et al., issued Nov. 18, 1997). This procedure
involves the preparation of cell free packaging extracts.
[0173] For additional detailed guidance on AAV technology which may
be useful in the practice of the subject invention, including
methods and materials for the incorporation of a transgene, the
propagation and purification of the recombinant AAV vector
containing the transgene, and its use in transfecting cells and
mammals, see e.g. Carter et al, U.S. Pat. No. 4,797,368 (Jan. 10,
1989); Muzyczka et al, U.S. Pat. No. 5,139,941 (Aug. 18, 1992);
Lebkowski et al, U.S. Pat. No. 5,173,414 (Dec. 22, 1992);
Srivastava, U.S. Pat. No. 5,252,479 (Oct. 12, 1993); Lebkowski et
al, U.S. Pat. No. 5,354,678 (Oct. 11, 1994); Shenk et al, U.S. Pat.
No. 5,436,146(Jul. 25, 1995); Chatterjee et al, U.S. Pat. No.
5,454,935 (Dec. 12, 1995), Carter et al WO 93/24641 (published Dec.
9, 1993), and Natsoulis, U.S. Pat. No. 5,622,856 (Apr. 22, 1997).
Further information regarding AAVs and the adenovirns or herpes
helper functions required can be found in the following articles.
Berns and Bohensky (1987), "Adeno-Associated Viruses: An Update",
Advanced in Virus Research, Academic Press, 33:243-306. The genome
of AAV is described in Laughlin et al. (1983) "Cloning of
infectious adeno-associated virus genomes in bacterial plasmids",
Gene, 23: 65-73. Expression of AAV is described in Beaton et al.
(1989) "Expression from the Adeno-associated virus p5 and p19
promoters is negatively regulated in trans by the rep protein", J.
Virol., 63:4450-4454. Construction of rAAV is described in a number
of publications: Tratschin et al. (1984) "Adeno-associated virus
vector for high frequency integration, expression and rescue of
genes in mammalian cells", Mol. Cell. Biol., 4:2072-2081; Hermonat
and Muzyczka (1984) "Use of adeno-associated virus as a mammalian
DNA cloning vector: Transduction of neomycin resistance into
mammalian tissue culture cells", Proc. Natl. Acad. Sci. USA,
81:6466-6470; McLaughlin et al. (1988) "Adeno-associated virus
general transduction vectors: Analysis of Proviral Structures", J.
Virol., 62:1963-1973; and Samulski et al. (1989) "Helper-free
stocks of recombinant adeno-associated viruses: normal integration
doqutre viral gene expression", J. Virol., 63:3822-3828. Cell lines
that can be transformed by rAAV are those described in Lebkowski et
al. (1988) "Adeno-associated virus: a vector system for efficient
introduction and integration of DNA into a variety of mammalian
cell types", Mol. Cell. Biol., 8:3988-3996. "Producer" or
"packaging" cell lines used in manufacturing recombinant
retroviruses are described in Dougherty et al. (1989) J. Virol.,
63:3209-3212; and Markowitz et al. (1988) J. Virol.,
62:1120-1124.
[0174] Hybrid Adenovirus-AAV Vectors
[0175] Hybrid Adenovirus-AAV vectors represented by an adenovirus
capsid containing a nucleic acid comprising a portion of an
adenovirus, and 5' and 3' ITR sequences from an AAV which flank a
selected transgene under the control of a promoter. See e.g. Wilson
et al, International Patent Application Publication No. WO
96/13598. This hybrid vector is characterized by high titer
transgene delivery to a host cell and the ability to stably
integrate the transgene into the host cell chromosome in the
presence of the rep gene. This virus is capable of infecting
virtually all cell types (conferred by its adenovirus sequences)
and stable long term transgene integration into the host cell
genome (conferred by its AAV sequences).
[0176] The adenovirus nucleic acid sequences employed in the this
vector can range from a minimum sequence amount, which requires the
use of a helper virus to produce the hybrid virus particle, to only
selected deletions of adenovirus genes, which deleted gene products
can be supplied in the hybrid viral process by a packaging cell.
For example, a hybrid virus can comprise the 5' and 3' inverted
terminal repeat (ITR) sequences of an adenovirus (which function as
origins of replication). The left terminal sequence (5') sequence
of the Ad5 genome that can be used spans bp 1 to about 360 of the
conventional adenovirus genome (also referred to as map units 0-1)
and includes the 5' ITR and the packaging/enhancer domain. The 3'
adenovirus sequences of the hybrid virus include the right terminal
3' ITR sequence which is about 580 nucleotides (about bp 35,353-
end of the adenovirus, referred to as about map units 98.4-100.
[0177] The AAV sequences useful in the hybrid vector are viral
sequences from which the rep and cap polypeptide encoding sequences
are deleted and are usually the cis acting 5' and 3' ITR sequences.
Thus, the AAV ITR sequences are flanked by the selected adenovirus
sequences and the AAV ITR sequences themselves flank a selected
transgene. The preparation of the hybrid vector is further
described in detail in published PCT application entitled "Hybrid
Adenovirus-AAV Virus and Method of Use Thereof", WO 96/13598 by
Wilson et al.
[0178] For additional detailed guidance on adenovirus and hybrid
adenovirus-AAV technology which may be useful in the practice of
the subject invention, including methods and materials for the
incorporation of a transgene, the propagation and purification of
recombinant virus containing the transgene, and its use in
transfecting cells and mammals, see also Wilson et al, WO 94/28938,
WO 96/13597 and WO 96/26285, and references cited therein.
[0179] Retroviruses
[0180] The retroviruses are a group of single-stranded RNA viruses
characterized by an ability to convert their RNA to double-stranded
DNA in infected cells by a process of reverse-transcription (Coffin
(1990) Retroviridae and their Replication" In Fields, Knipe ed.
Virology. New York: Raven Press). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes, gag, pol,
and env that code for capsidal proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene, termed psi, functions as a signal for packaging of
the genome into virions. Two long terminal repeat (LTR) sequences
are present at the 5' and 3' ends of the viral genome. These
contain strong promoter and enhancer sequences and are also
required for integration in the host cell genome (Coffin (1990),
supra).
[0181] In order to construct a retroviral vector, a nucleic acid of
interest is inserted into the viral genome in the place of certain
viral sequences to produce a virus that is replication-defective.
In order to produce virions, a packaging cell line containing the
gag, pol, and env genes but without the LTR and psi components is
constructed (Mann et al. (1983) Cell 33:153). When a recombinant
plasmid containing a human cDNA, together with the retroviral LTR
and psi sequences is introduced into this cell line (by calcium
phosphate precipitation for example), the psi sequence allows the
RNA transcript of the recombinant plasmid to be packaged into viral
particles, which are then secreted into the culture media (Nicolas
and Rubenstein (1988) "Retroviral Vectors", In: Rodriguez and
Denhardt ed. Vectors: A Survey of Molecular Cloning Vectors and
their Uses. Stoneham:Butterworth; Temin, (1986) "Retrovirus Vectors
for Gene Transfer: Efficient Integration into and Expression of
Exogenous DNA in Vertebrate Cell Genome", In: Kucherlapati ed. Gene
Transfer. New York: Plenum Press; Mann et al., 1983, supra). The
media containing the recombinant retroviruses is then collected,
optionally concentrated, and used for gene transfer. Retroviral
vectors are able to infect a broad variety of cell types. However,
integration and stable expression require the division of host
cells (Paskind et al. (1975) Virology 67:242).
[0182] A major prerequisite for the use of retroviruses is to
ensure the safety of their use, particularly with regard to the
possibility of the spread of wild-type virus in the cell
population. The development of specialized cell lines (termed
"packaging cells") which produce only replication-defective
retroviruses has increased the utility of retroviruses for gene
therapy, and defective retroviruses are well characterized for use
in gene transfer for gene therapy purposes (for a review see
Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus
can be constructed in which part of the retroviral coding sequence
(gag, pol, env) has been replaced by nucleic acid encoding a fusion
protein of the present invention, rendering the retrovirus
replication defective. The replication defective retrovirus is then
packaged into virions which can be used to infect a target cell
through the use of a helper virus by standard techniques. Protocols
for producing recombinant retroviruses and for infecting cells in
vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.)
Greene Publishing Associates, (1989), Sections 9.10-9.14 and other
standard laboratory manuals. Examples of suitable retroviruses
include pLJ, pZIP, pWE and pEM which are well known to those
skilled in the art. A preferred retroviral vector is a pSR MSVtkNeo
(Muller et al. (1991) Mol. Cell Biol. 11:1785 and pSR MSV(XbaI)
(Sawyers et al. (1995) J. Exp. Med. 181:307) and derivatives
thereof. For example, the unique BamHI sites in both of these
vectors can be removed by digesting the vectors with BamHI, filling
in with Klenow and religating to produce pSMTN2 and pSMTX2,
respectively, as described in PCT/US96/09948 by Clackson et al.
Examples of suitable packaging virus lines for preparing both
ecotropic and amphotropic retroviral systems include Crip, Cre, 2
and Am.
[0183] Retroviruses have been used to introduce a variety of genes
into many different cell types, including neural cells, epithelial
cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone
marrow cells, in vitro and/or in vivo (see for example Eglitis et
al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) PNAS
USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018;
Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al.,
(1991) PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA
88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van
Beusechem et al., (1992) PNAS USA 89:7640-7644; Kay et al., (1992)
Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA
89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573).
[0184] Furthermore, it has been shown that it is possible to limit
the infection spectrum of retroviruses and consequently of
retroviral-based vectors, by modifying the viral packaging proteins
on the surface of the viral particle (see, for example PCT
publications WO93/25234, WO94/06920, and WO94/11524). For instance,
strategies for the modification of the infection spectrum of
retroviral vectors include: coupling antibodies specific for cell
surface antigens to the viral env protein (Roux et al., (1989) PNAS
USA 86:9079-9083; Julan et al., (1992) J. Gen Virol 73:3251-3255;
and Goud et al., (1983) Virology 163:251-254); or coupling cell
surface ligands to the viral env proteins (Neda et al., (1991) J.
Biol. Chem. 266:14143-14146). Coupling can be in the form of the
chemical cross-linking with a protein or other variety (e.g.
lactose to convert the env protein to an asialoglycoprotein), as
well as by generating fusion proteins (e.g. single-chain
antibody/env fusion proteins). This technique, while useful to
limit or otherwise direct the infection to certain tissue types,
and can also be used to convert an ecotropic vector in to an
amphotropic vector.
[0185] Other Viral Systems
[0186] Other viral vector systems that may have application in gene
therapy have been derived from herpes virus, e.g., Herpes Simplex
Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued May 20, 1997),
vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression
vectors," In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of
molecular cloning vectors and their uses. Stoneham: Butterworth,;
Baichwal and Sugden (1986) "Vectors for gene transfer derived from
animal DNA viruses: Transient and stable expression of transferred
genes," In: Kucherlapati R, ed. Gene transfer. New York: Plenum
Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA
viruses. Preferred viruses include an alphavirus, a poxvirus, an
arena virus, a vaccinia virus, a polio virus, and the like. In
particular, herpes virus vectors may provide a unique strategy for
persistence of the recombinant gene in cells of the central nervous
system and ocular tissue (Pepose et al., (1994) Invest Ophthalmol
Vis Sci 35:2662-2666). They offer several attractive features for
various mammalian cells (Friedmann (1989) Science, 244:1275-1281
Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et
al., 1988; Horwich et al.(1990) J.Virol., 64:642-650).
[0187] With the recent recognition of defective hepatitis B
viruses, new insight was gained into the structure-function
relationship of different viral sequences. In vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990, supra). This suggested
that large portions of the genome could be replaced with foreign
genetic material. The hepatotropism and persistence (integration)
were particularly attractive properties for liver-directed gene
transfer. Chang et al. recently introduced the chloramphenicol
acetyltransferase (CAT) gene into duck hepatitis B virus genome in
the place of the polymerase, surface, and pre-surface coding
sequences. It was cotransfected with wild-type virus into an avian
hepatoma cell line. Culture media containing high titers of the
recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al. (1991) Hepatology, 14:124A).
[0188] Administration of Viral Vectors
[0189] Generally the viral particles are transferred to a
biologically compatible solution or pharmaceutically acceptable
delivery vehicle, such as sterile saline, or other aqueous or
non-aqueous isotonic sterile injection solutions or suspensions,
numerous examples of which are well known in the art, including
Ringer's, phosphate buffered saline, or other similar vehicles.
Delivery of the recombinant viral vector can be carried out via any
of several routes of administration, including intramuscular
injection, intravenous administration, subcutaneous injection,
intrahepatic administration, catheterization (including cardiac
catheterization), intracranial injection, nebulization/inhalation
or by instillation via bronchoscopy.
[0190] Preferably, the DNA or recombinant virus is administered in
sufficient amounts to transfect cells within the recipient's target
cells, including without limitation, muscle cells, liver cells,
various airway epithelial cells and smooth muscle cells, neurons,
cardiac muscle cells, etc. and provide sufficient levels of
transgene expression to provide for observable ligand-responsive
secretion of a target protein, preferably at a level providing
therapeutic benefit without undue adverse effects.
[0191] Optimal dosages of DNA or virus depends on a variety of
factors, as discussed previously, and may thus vary somewhat from
patient to patient. Again, therapeutically effective doses of
viruses are considered to be in the range of about 20 to about 50
ml of saline solution containing concentrations of from about
1.times.10.sup.7 to about 1.times.10.sup.10 pfu of virus/ml, e.g.
from 1.times.10.sup.8 to 1.times.10.sup.9 pfu of virus/ml.
[0192] Uses
[0193] In one application, cells engineered in accordance with the
invention are used to produce a target protein in vitro. In such
applications, the cells are cultured or otherwise maintained until
production of the target protein is desired. At that time, the
appropriate ligand is added to the culture medium, in an amount
sufficient to cause the desired level of target protein production.
The protein so produced may be recovered from the medium or from
the cells, and may be purified from other components of the cells
or medium as desired.
[0194] Proteins for commercial and investigational purposes are
often produced using mammalian cell lines engineered to express the
protein. The use of mammalian cells, rather than bacteria, insect
or yeast cells, is indicated where the proper function of the
protein requires post-translational modifications not generally
performed by non-mammalian cells. Examples of proteins produced
commercially this way include, among others, erythropoietin, BMP-2,
tissue plasminogen activator, Factor VIII:c, Factor IX, and
antibodies.
[0195] In other applications, cells within an animal host or human
subject are engineered in accordance with the invention, or cells
so engineered are introduced into the animal or human subject, in
either case, to prepare the recipient for ligand-mediated
regulation of secretion of a therapeutic protein. In the case of
non-human animals, this can be done as part of veterinary treatment
of the animal or to create an animal model for a variety of
research purposes. In the case of human subjects, this can be done
as part of a therapeutic or prophylactic treatment program.
[0196] This invention is applicable to a variety of treatment
approaches. For example, the target protein, e.g. a therapeutic
protein, to be regulated can be an endogenous protein or a
heterologous protein, and its secretion may be activated by
addition of ligand.
[0197] In some cases the target protein is a factor necessary for
the proliferation and/or differentiation of one or more cell types
of interest. For example, it may be desirable to stimulate the
secretion of growth factors and lymphokines in a subject in which
at least some of the blood cells have been destroyed, e.g., by
radiotherapy or chemotherapy. For example, secretion of
erythropoietin stimulates the production of red blood cells,
secretion of G-CSF stimulates the production of granulocytes,
secretion of GM-CSF stimulates the production of various white
blood cells, etc. Similarly in diseases or conditions in which one
or more specific cell types are destroyed by the disease process,
e.g., in autoimmune diseases, the specific cells can be replenished
by stimulating secretion of one or more factors stimulating
proliferation of these cells. The method of the invention can also
be used to increase the number of lymphocytes in a subject having
AIDS, such as by stimulating secretion of lymphokines, e.g., IL-4,
which stimulates proliferation of certain T helper (Th) cells.
[0198] In other cases, the target protein is a hormone or endorphin
which must be delivered rapidly and efficiently to its site of
action. For example, patients with insulin-dependent diabetes
mellitus (IDDM) must artificially maintain physiological levels of
insulin in the bloodstream. It would be highly desirable to replace
frequent insulin injections with a regulated expression system in
which the patient could rapidly produce his/her own insulin when
needed. Current regulated expression systems rely on
transcriptional mechanisms, in which protein levels increase about
12-16 hours after addition of ligand. In contrast, the present
invention would allow delivery of insulin to the appropriate site
within 20-30 minutes after ligand binding. As in the case of
insulin, the invention described herein could be used to treat any
condition which would benefit from rapid delivery of a therapeutic
protein. For example, this invention would be useful for delivery
of any protein whose biology requires pulsatile or diurnal
delivery. Such proteins include, among others, parathyroid hormone
or growth hormone. Other uses include delivery of proteins for
inflammatory, flaring-type diseases, such as rheumatoid arthritis,
inflammatory bowel disease, etc. Examples of such therapeutics
would be antibodies to TNF, soluble TNFR, and IL-1RA. More
generally, patients would benefit from regulated secretion of any
"on-demand" or self-medicating scenario, like insulin (see above)
or other agents for managing blood glucose; anti-pain peptides;
inflammation (see above); leptin; contraception e.g., antibodies to
LHRH.
[0199] Methods for Identifying CRDs
[0200] Methods are disclosed below for the identification,
validation and improvement of CRD candidates of each in each of the
three classes described earlier.
[0201] 1. CRDs Comprising Natural Examples of Proteins Retained in
Secretory Compartments in a Small-molecule Reversible Manner
[0202] Candidate CRDs of this class include any naturally secreted
protein or subdomain thereof. Such proteins can typically be
identified by the presence of a secretion signal sequence at the
start of their coding sequence. The characteristics of such signal
sequences are well known and computational algorithms are available
to assist in their identification. Using these methods, secreted
proteins can be identified from searches of sequence databases. A
preferred subset of secreted proteins are those that are known to
bind small molecules, or are predicted to do so by their homology
to other small molecule-binding proteins. The small molecule may be
a ligand or substrate that is transiently bound to the protein
during its normal function, or it may be a cofactor that normally
remains permanently bound. In either case, these small molecules
provides a starting point for identifying ligands for the candidate
CRD. In some cases (an example is rat RBP), small molecule-mediated
release of the protein from secretory compartments may already be
documented in the scientific literature.
[0203] To test whether a candidate protein can function as a CRD,
DNA encoding the candidate polypeptide is amplified by PCR or
RT-PCR using standard methods from an appropriate source, such as
genomic DNA or total or poly A+RNA isolated from an appropriate
cellular source, or a cDNA or genomic DNA library. PCR primers are
engineered to include restriction sites allowing insertion into a
vector for expression in mammalian cells, or other eukaryotic cells
of interest. Alternatively the sequence of interest can be isolated
as a restriction fragment. The PCR or restriction fragment is then
cloned in frame into the polylinker of an expression vector. A
preferred vector is of the form shown in FIG. 10A, where hCMV
indicates the human CMV immediate early promoter and enhancer, SS
indicates a signal sequence, poly is a polylinker region, FCS is a
furin cleavage site, and hGH is a cDNA for human growth hormone.
Components of this vector can be substituted as appropriate: for
example, FCS can be replaced with alternative TGN protease cleavage
sites, and hGH can be replaced with other secreted proteins that
can be easily detected and are therefore useful as reporter
proteins, such as secreted alkaline phosphatase (SEAP) or
erythropoietin (EPO). Optionally, an epitope tag allowing
immunochemical detection of the protein (for example the FLAG
sequence: IBI/Kodak) can be included in the vector sequence or
incorporated via either PCR primer.
[0204] To determine whether the candidate polypeptide acts as a
CRD, the expression vector is introduced into cells in culture
using standard techniques, for example lipofection. After 24 hours,
an aliquot of culture medium is removed and assayed for presence of
hGH using standard techniques (Rivera et al., 1996). Then, new
medium containing various concentrations of candidate CRD ligand
are added. After a further period of 2-24 hours, medium is again
sampled for presence of hGH. CRD-like activity of the candidate
polypeptide is indicated by a low level of hGH in the culture
medium in the absence of compound, and increased amounts in the
presence of compound. Suitable candidate CRD ligands to investigate
include compounds that are known ligands of the protein under study
(for example retinol for RBP), and chemically related molecules
that may have usefully different properties, such as cell
permeability or effects on ER retention of the protein (for example
diverse retinoids for RBP). Suitable concentrations of these
ligands to investigate are in the range 1 pM to 1 mM.
[0205] An important approach for optimizing the effectiveness of
CRD candidates is the reiteration of those domains in multiple
copies, to attempt to amplify any conditional retention effect. It
is anticipated, for example, that some proteins will be `retarded`
in the secretory pathway in the absence of ligand, but not
completely retained--that is, retention will be "leaky". In some
applications of the invention this will be desirable; in others,
tightly repressed protein production in the absence of drug will be
needed. In these cases, reiterating the CRD may augment the ability
to cause retention of the heterologous protein. Thus, the
experiments described above will optionally be repeated on
constructs that harbor different numbers of concatenated candidate
CRDs: typically between one and eight.
[0206] Additional controls that can be performed to verify the
activity of a CRD discovered through the above methods include
immunochemical detection of the CRD and hGH domains inside cells
treated or not treated with the CRD ligand, to confirm that the
proteins are retained inside the secretory apparatus. These
experiments use standard cell fixing procedures followed by
immunofluorescence. Also, the secreted hGH can be checked for
correct processing from the fusion protein by size analysis using
SDS-PAGE followed by immunoblot with anti-hGH antibodies. For a
more exact check, the hGH can be purified (eg. on an hGH binding
protein affinity column), and then analyzed for molecular weight by
mass spectrometry and for correct processing by immobilization on
PVDF followed by N-terminal sequence analysis.
[0207] Although the search for CRDs will typically focus on those
proteins that are naturally secreted, and further on that subset of
secreted proteins with known small molecule-binding activities, any
polypeptide can be tested using the methods described above. Thus a
protein that is not naturally secreted, but that has a known small
molecule binding activity, can be cloned into the FCS-hGH
expression vector and tested for CRD behavior that can be reversed
by that small molecule (or related molecules). Most generally, any
polypeptide--including one that is apparently not normally
secreted, and that has no known small molecule binding
activity--can be tested. In these cases, the candidate CRD-FCS-hGH
expression construct can be first tested for retention of hGH. If
retention is observed, cells containing the construct can be
challenged in separate experiments with a diverse set of candidate
small molecules in order to identify a molecule that can promote
secretion of the retained fusion proteins. Suitable sets of
molecules include collections of natural products, and the members
of synthetic or semi-synthetic combinatorial libraries. Screening
may be expedited by arraying cells in 96- or 384-well plates to
enable robotic high-throughput set-up and analysis of
experiments.
[0208] 2. CRDs that are Mutants of a Natural Protein, Chosen for
the Property of being Selectively Retained in the Absence of a
Given Small Molecule
[0209] Screening methods for such CRDs follow on naturally from the
methods described above. A polypeptide of interest is cloned into
the FCS-hGH fusion expression vector described above. Again,
preferred polypeptides are those with known small molecule-binding
activities. Individual mutants of the candidate CRD are engineered
by standard methods. These mutant constructs are then iteratively
assayed for (i) the retention of hGH and (ii) the secretion of hGH
upon addition of a small molecule. Choice of small molecules to
test, and their concentrations, are as described above. Assays on
many mutants can be performed simultaneously by using multi-well
plate assays.
[0210] Mutations can be chosen to optimize the likelihood of
inducing a change in the properties of the protein that results in
conditional retention. Mutations of particular interest are those
anticipated to disrupt the efficient folding of the protein: such
proteins might be subject to retention via the ER quality control
system. Example mutations include gain-of-size mutations of side
chains that constitute the hydrophobic core of the protein; and
alterations of other residues of critical importance in secondary
or tertiary structural features, such as glycine residues at
beta-turn motifs. Other amino acids of interest are those that
form, or are close to, the small molecule binding site. Mutants
with reduced folding efficiency are preferred because such changes
are most likely to be stabilized by binding of a small molecule,
providing a mechanism for selective small molecule-mediated release
of retained proteins. Thus, knowledge of the three-dimensional
structure of a candidate CRD can be of great use in focusing
mutagenesis to key positions.
[0211] Both singly and multiply mutated proteins can be engineered
and tested. Often, the best variant protein will be altered at
several positions. Identifying the best combination of changes at
multiple residues by iterative screening of mutants can be tedious
and time-consuming. An alternative is the use of selection
procedures, in which a large set of mutants is created and then
subjected en masse to a selection step to identify the best mutants
directly. See Clackson and Wells (Trends Biotech 1994 12: 173). To
provide a means to directly select for proteins that act as CRDs,
the expression vector described above is altered by exchanging the
hGH coding sequence for DNA encoding a cell surface marker, such as
CD2 or the p75 low affinity nerve growth factor receptor. The
extracellular and transmembrane domains of cell surface marker are
included, but most of the intracellular domain is preferably
deleted to remove the potential for signaling through the receptor.
A suitable expression vector using p75 is shown in FIG. 10B, where
ECD and TM are respectively the extracellular and transmembrane
domains of p75.
[0212] To select CRDs from a large set of candidates, genes
encoding the candidates are ligated into the polylinker to create a
library. The library is introduced into mammalian cells by
established methods. Methods should ideally be chosen that (i) lead
to a low number of variants being introduced into each cell, so
that the properties of variants can be tested individually, and
(ii) provide stable introduction of the vector so that cells can be
propagated and selected through multiple rounds. A preferred
approach is therefore to construct the library in a retroviral
vector followed by retroviral infection of cells, since this
results single- or low-copy stable integration of the vector.
[0213] Selection of CRDs can be performed directly or indirectly.
Direct screening is performed using a fluorescence-activated cell
sorter, in two stages. In the first stage, cells harboring the
library of CRD candidates are grown in culture and then incubated
with a fluorescently-labeled antibody to the p75 ECD. Cells
containing a clone for an active CRD will not bind, as p75 will be
retained in the secretory apparatus. However cells harboring
ineffective CRDs will bind as the protein will not be retained. The
labeled cells are sorted by FACS and cells that are not stained are
gated and retrieved, and allowed to grow again in culture. The sort
can optionally be repeated several times with a progressively
higher gate, in order to isolate the cells expressing lowest levels
of p75. In the second stage, a candidate CRD ligand (chosen as
described above) is added and then the labeling process repeated.
Now the cells with effective CRDs will be labeled, since the
retained p75 will be released by the CRD ligand. The cells are
sorted by FACS and the labeled cells are isolated. Again, the
selection step can be repeated if desired. Once a suitable
population of cells has been isolated, the variants that are
conferring the CRD activity can be identified by isolating the
genomic DNA of the cells followed by PCR amplification with primers
located each side of the vector polylinker. The PCR products can
then be cloned and sequenced. The ability of the identified
variants to act as CRDs can be confirmed by cloning them
individually into the hGH expression vector followed by testing as
described earlier. Indirect screening may be accomplished by
determining whether the CRD directs surface localization of a
membrane protein which can then activate a signaling pathway.
[0214] The mutants introduced can be targeted to the residues of
interest indicated earlier, or can randomly incorporated. Several
suitable methods for engineering sets of multiple mutants have been
described, including alanine-scanning mutagenesis (Cunningham and
Wells (1989) Science 244 1081-1085), degenerate primer-mediated
`Kunkel` mutagenesis (See eg. Lowman and Wells 1993 J Mol Biol 234:
563-578), PCR misincorporation mutagenesis (see eg. Cadwell and
Joyce (1992) PCR Meth. Applic. 2, 28-33), and DNA shuffling
(Stemmer (1994) Nature 370 389-391).
[0215] 3. CRDs that are Proteins that Self-aggregate in a Small
Molecule-reversible Manner.
[0216] Methods to identify proteins that interact with one another
are well known. A commonly used technique is the two-hybrid system,
in which one partner is fused to a DNA binding domain and the other
to an transcriptional activation domain. Interaction of the
partners reconstitutes the transcription factor, activating
transcription of a reporter gene that can be identified by
screening (eg. production of beta-galactosidase or SEAP) and/or
that leads to cell survival and therefore provides a means for
selecting for interacting partners (eg. his gene transcription in a
his- strain of yeast). Two-hybrid assays can be performed in yeast
or mammalian cells and methods are well known in the art.
[0217] A preferred embodiment is based on the vectors and cells
described by Rivera et al. (Nature Med 1996 2, 1028-1032). Two
expression vectors are constructed for chimeric transcription
factors in which the candidate CRD is fused to the hybrid DNA
domain ZFHD1 (in one case) and to an activiation domain of NF-kB
p65 subunit, such as amino acids 361-550 (in the other). These
vectors are transiently or stably transfected into mammalian cells,
for example HT1080 cells, together with a SEAP reporter gene under
the control of ZFHD1 binding sites. Aggregation of the candidate
CRDs results in reconstitution of an active transcription factor
and therefore prediction of SEAP. Once a self-aggregating protein
has been identified in this way, addition of candidate CRD ligand
can be used to examine whether the aggregates can be dissociated
with ligand. Reduction in the production of SEAP upon addition of
ligand would indicate this activity. Any polypeptide can be chosen
for testing in this way for CRD activity, but preferred proteins to
try are those that already have known small molecule binding
activity. In these cases the known binding ligands provide a
starting point for choosing compounds that might disaggregate bound
protein.
[0218] As before, an important additional configuration to explore
is the concatenation of candidate CRDs. Presence of more than one
aggregating domain may increase the apparent affinity of the
aggregative interaction by virtue of the avidity effect.
[0219] Either natural or mutated proteins can be tested for CRD
activity. Mutants of natural proteins are likely to provide good
sources of CRDs as examples are known on the literature of
aggregative activity induced by point mutations: for example
sickle-cell hemoglobin, or alpha-1 antitrypsin as described
earlier. Thus, large sets of mutants of a candidate protein can be
cloned into two-hybrid vectors as described above, and tested for
aggregative activity that can be reduced by addition of a small
molecule. The criteria that dictate choice of positions to mutate
will largely be the same as those described above for screening for
CRDs directly in a secretion system (2 above); in addition, mutants
that aggregate might be provided by converting polar surface
residues to less polar amino ones. Single or multiple mutants can
be engineered, using methods as described above.
[0220] Selection schemes for CRDs can also be devised. In these
cases, libraries of mutant proteins are cloned into two hybrid
vectors and analyzed en masse for CRD activity. These experiments
are most easily performed in yeast and methods for two-hybrid
selections are well known in the art. For example, expression
vectors for mutants of candidate CRDs, fused to GAL4 DNA binding
domain or activation domain vectors, and transformed into a
his-deficient yeast reporter strain harboring a his gene under the
control of GAL4 binding sites. Plating the library on his-deficient
medium will result in growth only of cells that contain interacting
CRDs on the two chimeric transcription factors. These positives can
then be replica plated onto plates containing increasing amounts of
candidate CRD ligands, to identify those CRDs whose interactions
can be disrupted by small molecules. Such proteins are candidates
for use as CRDs.
[0221] A complication with the above selection scheme is the desire
to have the same mutant fused to both the DNA binding and
activation domains, in order to identify proteins that
self-aggregate. To achieve this, the expression vector for the
chimeric proteins can be modified to allow a mutant gene to be
joined to both transcription domains at the level of splicing. The
domains of interest are encoded in separate exons. An outline of a
suitable vector is shown in FIG. 10C. CRD and is the candidate CRD:
a library of candidates (eg mutant proteins) is inserted here. DBD
and AD are the DNA binding and activation domains of a
transcription factor. A and D indicate donor and acceptor splice
sites. stop indicates a translational stop codon. By equipping the
DBD with a suboptimal splice acceptor site, the CRD exon will be
spliced to both DBD and AD exons. Thus, in each cell fusion
proteins will be expressed in which the AD and DBD are both fused
to an identical CRD candidate.
[0222] An alternative format for selection of self-aggregating
proteins is the lambda repressor fusion system in E.coli (Hu et al.
1990 Science 250:1400-1403; for review see Hu 1995 Structure 3:
431-433). This strategy utilizes the fact that bacteriophage lambda
repressor cI binds to DNA as a homodimer and that binding of such
homodimers to operator DNA prevents transcription of phage genes
involved in the lytic pathway of the phage life cycle. Thus,
bacterial cells expressing functional lambda repressor are immune
to lysis by superinfecting lambda bacteriophage. Repressor protein
comprises an amino terminal DNA binding domain (amino acids 1-92)
joined by a 40 amino acid flexible linker to a C-terminal
dimerization domain. The isolated N-terminal domain binds very
weakly to DNA sue to inefficient dimer formation. High affinity DNA
binding can be restored by fusing the domain to a heterlogous
dimerization domain, such as the GCN4 leucine zipper. A selection
system is therefore possible in which phage immunity is used as a
selection for interacting proteins.
[0223] For example, to select CRDs from a library of candidates,
the candidates are cloned in frame with the repressor N-terminus
and the library transformed into E.coli. Genes for proteins that
aggregate are isolated from colonies that survive on plates
containing high titers of lambda phage. These colonies can then be
restreaked on to plates containing both lambda phage and candidate
CRD ligand. If the ligand dissociates the aggregates, the E.coli
will now no lolnger grow on these plates. Lambda repressor
selection has several advantages for identifying CRDs, including
the fact that the system is suitable for screening homodimers, and
the large library sizes that can be obtained through the use of
E.coli.
[0224] Another way to directly test whether a protein can act as a
CRD in living cells is to fuse its coding sequence to green
fluorescent protein (GFP) or variants thereof. Cells expressing
such a fusion protein can then be examined directly by fluorescent
microscopy to examine whether the CRD candidate appears to cause
aggregates of the GFP. Candidate CRD ligands can then be added to
determine whether the aggregates then dissociate. Once a CRD
candidate has been identified by any of these methods, it can be
tested for activity as a CRD by use of the methods outlined in
section 1.
[0225] Pharmaceutical Compositions & Their Administration to
Subjects Containing Engineered Cells
[0226] Administration
[0227] The ligand may be administered to a human or non-human
subject using pharmaceutically acceptable materials and methods of
administration. Various formulations, routes of administration,
dose and dosing schedule may be used for the administration of
ligand, depending upon factors such as the condition and
circumstances of the recipient, the response desired, the
biological half-life and bioavailability of the ligand, the
biological half-life and specific activity of the target protein
product, the number and location of engineered cells present, etc.
The drug may be administered parenterally, or more preferably
orally. For use in this invention, the most preferable route of
administration are those in which a rapid onset of response occurs;
such methods include, for example, sublingual, buccal, skin patch
and inhalation. Dosage and frequency of administration will depend
upon factors such as described above. The drug may be taken orally
as a pill, powder, or dispersion; buccally; sublingually; injected
intravascularly, intraperitoneally, subcutaneously; or the like.
The drug may be formulated using conventional methods and materials
well known in the art for the various routes of administration. The
precise dose and particular method of administration will depend
upon the above factors and be determined by the attending physician
or healthcare provider.
[0228] The particular dosage of the drug for any application may be
determined in accordance with conventional approaches and
procedures for therapeutic dosage monitoring. A dose of the drug
within a predetermined range is given and the patient's response is
monitored so that the level of therapeutic response and the
relationship of protein secretion over time may be determined.
Depending on the expression levels observed during the time period
and the therapeutic response, one may adjust the level of
subsequent dosing to alter the resultant expression level over time
or to otherwise improve the therapeutic response. This process may
be iteratively repeated until the dosage is optimized for
therapeutic response. Where the drug is to be administered
chronically, once a maintenance dosage of the drug has been
determined, one may conduct periodic follow-up monitoring to assure
that the overall therapeutic response continues to be achieved.
[0229] In the event that the activation by the drug is to be
reversed, administration of drug may be suspended so that cells
return to a basal rate of secretion. To effect a more active
reversal of therapy, an antagonist of the drug may be administered.
An antagonist is a compound which binds to the drug or drug-binding
domain to inhibit interaction of the drug with the fusion
protein(s) and thus inhibit the downstream biological event. Thus,
in the case of an adverse reaction or the desire to terminate the
therapeutic effect, an antagonist can be administered in any
convenient way, particularly intravascularly or by
inhalation/nebulization, if a rapid reversal is desired.
[0230] Compositions
[0231] Drugs (i.e., the ligands) for use in this invention can
exist in free form or, where appropriate, in salt form. The
preparation of a wide variety of pharmaceutically acceptable salts
is well-known to those of skill in the art. Pharmaceutically
acceptable salts of various compounds include the conventional
non-toxic salts or the quaternary ammonium salts of such compounds
which are formed, for example, from inorganic or organic acids of
bases. The drugs may form hydrates or solvates. It is known to
those of skill in the art that charged compounds form hydrated
species when lyophilized with water, or form solvated species when
concentrated in a solution with an appropriate organic solvent.
[0232] The drugs can also be administered as pharmaceutical
compositions comprising a therapeutically (or prophylactically)
effective amount of the drug, and a pharmaceutically acceptable
carrier or excipient. Carriers include e.g. saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof, and are discussed in greater detail below. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. The composition can
be a liquid solution, suspension, emulsion, tablet, pill, capsule,
sustained release formulation, or powder. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Formulation may involve mixing, granulating and
compressing or dissolving the ingredients as appropriate to the
desired preparation. The pharmaceutical carrier employed may be,
for example, either a solid or liquid.
[0233] Illustrative solid carriers include lactose, terra alba,
sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,
stearic acid and the like. A solid carrier can include one or more
substances which may also act as flavoring agents, lubricants,
solubilizers, suspending agents, fillers, glidants, compression
aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the carrier is a finely divided
solid which is in admixture with the finely divided active
ingredient. In tablets, the active ingredient is mixed with a
carrier having the necessary compression properties in suitable
proportions and compacted in the shape and size desired. The
powders and tablets preferably contain up to 99% of the active
ingredient. Suitable solid carriers include, for example, calcium
phosphate, magnesium stearate, talc, sugars, lactose, dextrin,
starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl
cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange
resins.
[0234] Illustrative liquid carriers include syrup, peanut oil,
olive oil, water, etc. Liquid carriers are used in preparing
solutions, suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active ingredient can be dissolved or suspended
in a pharmaceutically acceptable liquid carrier such as water, an
organic solvent, a mixture of both or pharmaceutically acceptable
oils or fats. The liquid carrier can contain other suitable
pharmaceutical additives such as solubilizers, emulsifiers,
buffers, preservatives, sweeteners, flavoring agents, suspending
agents, thickening agents, colors, viscosity regulators,
stabilizers or osmo-regulators. Suitable examples of liquid
carriers for oral and parenteral administration include water
(partially containing additives as above, e.g. cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g. glycols) and their derivatives, and oils (e.g. fractionated
coconut oil and arachis oil). For parenteral administration, the
carrier can also be an oily ester such as ethyl oleate and
isopropyl myristate. Sterile liquid carders are useful in sterile
liquid form compositions for parenteral administration. The liquid
carrier for pressurized compositions can be halogenated hydrocarbon
or other pharmaceutically acceptable propellant. Liquid
pharmaceutical compositions which are sterile solutions or
suspensions can be utilized by, for example, intramuscular,
intraperitoneal or subcutaneous injection. Sterile solutions can
also be administered intravenously. The drugs can also be
administered orally either in liquid or solid composition form.
[0235] The carrier or excipient may include time delay material
well known to the art, such as glyceryl monostearate or glyceryl
distearate along or with a wax, ethylcellulose,
hydroxypropylmethylcellulose, methylmethacrylate and the like. When
formulated for oral administration, 0.01% Tween 80 in PHOSAL PG-50
(phospholipid concentrate with 1,2-propylene glycol, A. Nattermann
& Cie. GmbH) may be used as an oral formulation for a variety
of drugs for use in the practice of this invention.
[0236] A wide variety of pharmaceutical forms can be employed. If a
solid carrier is used, the preparation can be tableted, placed in a
hard gelatin capsule in powder or pellet form or in the form of a
troche or lozenge. The amount of solid carrier will vary widely but
preferably will be from about 25 mg to about 1 g. If a liquid
carrier is used, the preparation will be in the form of a syrup,
emulsion, soft gelatin capsule, sterile injectable solution or
suspension in an ampule or vial or nonaqueous liquid
suspension.
[0237] To obtain a stable water soluble dosage form, a
pharmaceutically acceptable salt of the drug may be dissolved in an
aqueous solution of an organic or inorganic acid, such as a 0.3M
solution of succinic acid or citric acid. Alternatively, acidic
derivatives can be dissolved in suitable basic solutions. If a
soluble salt form is not available, the compound is dissolved in a
suitable cosolvent or combinations thereof. Examples of such
suitable dissolved in a suitable cosolvent or combinations thereof.
Examples of such suitable cosolvents include, but are not limited
to, alcohol, propylene glycol, polyethylene glycol 300, polysorbate
80, glycerin, polyoxyethylated fatty acids, fatty alcohols or
glycerin hydroxy fatty acids esters and the like in concentrations
ranging from 0-60% of the total volume.
[0238] Various delivery systems are known and can be used to
administer the drugs, or the various formulations thereof,
including tablets, capsules, injectable solutions, encapsulation in
liposomes, microparticles, microcapsules, etc. Preferred routes of
administration to a patient are oral, sublingual, transdermal
(patch), intranasal, pulmonary or bucal. Methods of introduction
also could include but are not limited to dermal, intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
epidural, ocular and (as is usually preferred) oral routes. The
drug may be administered by any convenient or otherwise appropriate
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local. For ex vivo applications, the drug will be
delivered as a liquid solution to the cellular composition.
[0239] In a specific embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic to
ease pain at the side of the injection. Generally, the ingredients
are supplied either separately or mixed together in unit dosage
form, for example, as a lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0240] In addition, in certain instances, it is expected that the
compound may be disposed within devices placed upon, in, or under
the skin. Such devices include patches, implants, and injections
which release the compound into the skin, by either passive or
active release mechanisms.
[0241] Materials and methods for producing the various formulations
are well known in the art and may be adapted for practicing the
subject invention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311
(tablets, capsules and other oral formulations as well as
intravenous formulations) and European Patent Application
Publication Nos. 0 649 659 (published Apr. 26, 1995; rapamycin
formulation for IV administration) and 0 648 494 (published Apr.
19, 1995; rapamycin formulation for oral administration).
[0242] The effective dose of the drug will typically be in the
range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to
about 10 mg/kg of mammalian body weight, administered in single or
multiple doses. Generally, the compound may be administered to
patients in need of such treatment in a daily dose range of about 1
to about 2000 mg per patient. In embodiments in which the compound
is rapamycin or an analog thereof with some residual
immunosuppressive effects, it is preferred that the dose
administered be below that associated with undue immunosuppressive
effects.
[0243] The amount of a given drug which will be effective in the
treatment or prevention of a particular disorder or condition will
depend in part on the severity of the disorder or condition, and
can be determined by standard clinical techniques. In addition, in
vitro or in vivo assays may optionally be employed to help identify
optimal dosage ranges. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. The precise dosage level should be determined by the
attending physician or other health care provider and will depend
upon well known factors, including route of administration, and the
age, body weight, sex and general health of the individual; the
nature, severity and clinical stage of the disease; the use (or
not) of concomitant therapies; and the nature and extent of genetic
engineering of cells in the patient.
[0244] The drugs can also be provided in a pharmaceutical pack or
kit comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions. Optionally
associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceutical or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration.
[0245] The full contents of all references cited in this document,
including references from the scientific literature, issued patents
and published patent applications, are hereby expressly
incorporated by reference.
[0246] The following examples contain important additional
information, exemplification and guidance which can be adapted to
the practice of this invention in its various embodiments and the
equivalents thereof. The examples are offered by way of
illustration only and should not be construed as limiting in any
way. As noted throughout this document, the invention is broadly
applicable and permits a wide range of design choices by the
practitioner.
[0247] The practice of this invention will employ, unless otherwise
indicated, conventional techniques of cell biology, cell culture,
molecular biology, transgenic biology, microbiology, recombinant
DNA, immunology, virology, pharmacology, chemistry, and
pharmaceutical formulation and administration which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring
Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1986).
EXAMPLES
Example 1
[0248] Generation of Domains and Vectors Used for Expression of
F(36M) Fusion Proteins.
[0249] A. Expression Vectors:
[0250] Vectors for driving expression of fusion proteins were
derived from the mammalian expression vector pCGNN (Attar and
Gilman, MCB 12:2432-2443, 1992). Inserts cloned as XbaI-BamHI
fragments into pCGNN are transcribed under the control of the human
CMV promoter and enhancer sequences (nucleotides -522 to +72
relative to the cap site), and are expressed with an N-terminal
nuclear localization sequence (NLS; from SV40 T antigen) and
epitope tag (a 16 amino acid portion of the H. influenzae
hemaglutinin gene).
[0251] pCGNN was modified by site directed mutagenesis with
oligonucleotides VR65, VR119, and VR120 to create pC4EN. The
resulting plasmid has unique restriction sites upstream of the CMV
enhancer/promoter region (MluI) and between the promoter and
protein coding region (EcoRI).
[0252] VR65: TCCCGCACCTCTTCGGCCAGCGaaTTccAGAAGCGCGTAT (SEQ ID
NO.2)
[0253] VR119: GACTCACTATAGGaCGcgTTCGAGCTCGCCCC (SEQ ID NO. 3)
[0254] VR120: CATCATTTTGGCAAAGgATTCACTCCTCAGG (SEQ ID NO. 4)
[0255] Individual components of fusion proteins were generally
produced as fragments containing an XbaI site immediately upstream
of the first codon and a SpeI site, an in-frame stop codon, and a
BamHI site immediately downstream of the last codon. Chimeric
proteins comprising multiple components were assembled by stepwise
insertion of XbaI-BamHI fragments into SpeI-BamHI-opened vectors or
by insertion of XbaI-SpeI fragments into XbaI or SpeI-opened
vectors.
[0256] B. F(36M) Domain
[0257] F(36M), in which the phenylalanine at amino acid 36 was
changed to methionine, was created by mutagenizing a single FKBP
domain, cloned into pCGNN with upstream XbaI and downstream SpeI
and BamHI sites (Rivera et al., Nat. Med 2:1028-1032, 1996) with
oligo VR1 to create pCGNN-F(36M). Two, 3, 4 and 6 tandem copies of
F(36M) were created by the stepwise insertion of XbaI-BamHI
fragments into SpeI-BamHI-opened vectors.
[0258] VR1: GATGGAAAGAAAatgGATTCCTCCCGG (SEQ ID NO. 5)
[0259] C. F(36M) Fusion Proteins: (FIG. 3)
[0260] (a) EGFP Fusions
[0261] EGFP coding sequence was amplified from pEGFP-1 (Clontech)
with oligos VR2 and VR3. The resulting fragment, with upstream XbaI
and downstream SpeI sites was inserted into pCGN, a derivative of
pCGNN that lacks the SV40 nuclear localization sequence, to create
pCGN-EGFP.
[0262] VR2: tctagaGTGAGCAAGGGCGAGGAG (SEQ ID NO. 6)
[0263] VR3: ggatccttaTTAACTAGTCTTGTACAGCTCGTCCATG (SEQ ID NO.
7)
[0264] F(36M)-EGFP fusions were created by inserting XbaI-SpeI
fragments containing 3, 4 or 6 copies of F(36M) into the XbaI site
of pCGN-EGFP to create pCGN-F(36M)3-EGFP, pCGN-F(36M)4-EGFP, and
pCGN-F(36M)6-EGFP.
[0265] (b) hGH Fusions
[0266] An hGH cDNA (506-81) was obtained by RT-PCR amplification of
RNA expressed from a cell line containing a genomic hGH gene
(Rivera et al., Nat. Med 2:1028-1032, 1996) using oligos VR109 and
VR110 to amplify the region from 40 bp upstream of the ATG to 60 bp
after the stop codon. The resulting HindIII to EcoRI fragment was
cloned into Z12I-PL-2, a derivative of ZHWTx12-IL2-SEAP (Rivera et
al., Nat. Med 2:1028-1032, 1996) in which the SEAP gene and SV40
early intron and polyadenylation signal were replaced by a
polylinker and the SV40 late polyadenylation signal.
[0267] VR109: aagcttACCACTCAGGGTCCTGTGG (SEQ ID NO. 8)
[0268] VR110: gaattcGTGGCAACTTCCA (SEQ ID NO. 9)
[0269] To construct hGH fusion proteins, Z12I-hGH-2 was mutagenized
with oligos VR185, VR186, and VR187 to create i) an EcoRI site 32
bp upstream of the ATG, ii) an XbaI site immediately after the last
amino acid of the signal sequence and iii) a Spe I site immediately
after the last amino acid of hGH.
[0270] VR185: cacaggaccctGAATTCtaagcttgtggc (SEQ ID NO. 10)
[0271] VR186: ATAAGGGAATGGTtctagaGGCACTGCCCT (SEQ ID NO. 11)
[0272] VR187: atgccacccgggactagtGAAGCCACAGCTG (SEQ ID NO. 12)
[0273] Cloning the resulting EcoRI-SpeI fragment into pC4EN
produced pC4S1-hGH which expresses hGH from the CMV enhancer. The
XbaI-BamHI fragment of pC4S1-hGH was then replaced by XbaI-SpeI
fragments containing 2, 3, 4, or 6 copies of F(36M) and a
SpeI-BamHI fragment encoding the furin cleavage site-hGH fusion to
generate pC4S1-F(36M)-FCS-hGH fusions.
[0274] A SpeI-BamHI fragment encoding an FCS-hGH fusion protein was
generated by amplification of the hGH cDNA with oligos VR4 and
VR5.
[0275] VR4;actagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGC (SEQ ID
NO. 13)
[0276] VR5: ggatcccgggCTAGAAGCCACAGCTGCCCTC (SEQ ID NO. 14)
[0277] An XbaI-BamHI fragment containing the neo resistance gene
downstream of the encephalomyocarditis virus internal ribosome
entry sequence (IRES/Neo; Amara et al PNAS 94:10618-23, 1997) was
inserted into appropriate SpeI-BamHI-opened vectors to generate
pC4S1-F(36M)-FCS-hGH/ne- o and pC4S1- hGH/neo vectors.
[0278] (c) Insulin Fusions
[0279] A human insulin cDNA was obtained by RT-PCR amplification of
human pancreas polyA+RNA (Clontech) using oligos VR220 and VR221 to
amplify the region from 9 bp upstream of ATG (EcoRI) to 13 bp after
stop codon (BamHI). The resulting EcoRI-BamHI fragment was cloned
into pC4EN to generate pC4-hIn.
[0280] VR220: cGAATTCttctgccATGGCCCTGTGGATGCGC (SEQ ID NO. 15)
[0281] VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO. 16)
[0282] A SpeI-BamHI fragment encoding an furin cleavage
sequence-insulin fusion protein was generated by RT-PCR
amplification with oligos VR222 and VR221.
[0283] VR222: cACTAGTGCTAGAAACCGTCAGAAGAGATTTGTGAACCAACACCTGTGCGGC
(SEQ ID NO. 17)
[0284] VR221: cGGATCCgcaggctgcgtCTAGTTGCAGTAG (SEQ ID NO. 18)
[0285] The wild type insulin gene and FCS-insulin fusion were
mutagenized to i) alter amino acid B10 to Asp, ii) create a FCS at
the B-C junction, and iii) create a FCS at the C-A junction, using
oligos VR223, VR224, VR225, respectively.
[0286] VR223: CCTGTGCGGCTCAgACCTGGTGGAAGC (SEQ ID NO. 19)
[0287] VR224: CTTCTACACACCCAgGACCaagCGGGAGGCAGAGG (SEQ ID NO.
20)
[0288] VR225: CCCTGGAGGGGTCCCgGCAGAAGCGTGGC (SEQ ID NO. 21)
[0289] Mutation of pC4-hIn produced pC4-hIn-m3. The mutated
FCS-insulin fusions were used to replace the FCS-hGH portion of the
pC4S1-F(36M)-FCS-hGH fusions to create pC4S1-F(36M)-FCS-hIn-m3
fusions.
[0290] (d) LNGFR Fusions
[0291] EcoRI-SpeI fragments containing amino acids 1-274 of the
human low affinity nerve growth factor receptor (LNGFR; Clackson et
al., PNAS 95:10437-42, 1998) and SpeI-BamHI fragments containing 3,
4, or 6 copies of F(36M) were cloned into pC4EN to generate
pC4LNGFR-F(36M) fusions.
[0292] (c) Transcription Factor Fusions
[0293] pCGNN-ZFHD1-F(36M) and pCGNN-F(36M)-p65 fusion proteins were
generated as described for wild type FKBP fusions (Amara et al PNAS
94:10618-23, 1997).
[0294] An XbaI-SpeI fragment containing 6 copies of F(36M) was
inserted into the XbaI or SpeI site of
[0295] pCGNN-ZFHD1-p65 to generate pCGNN- F(36M)6-ZFHD1-p65 and
[0296] pCGNN-ZFHD1-p65-F(36M)6.
[0297] pCGNNZFHD1
[0298] An expression vector for directing the expression of ZFHD1
coding sequence in mammalian cells was prepared as follows. Zif268
sequences were amplified from a cDNA clone by PCR using primers
5'Xba/Zif and 3'Zif+G. Oct1 homeodomain sequences were amplified
from a cDNA clone by PCR using primers 5'Not Oct HD and Spe/Bam
3'Oct. The Zif268 PCR fragment was cut with XbaI and NotI. The OctI
PCR fragment was cut with NotI and BamHI. Both fragments were
ligated in a 3-way ligation between the XbaI and BamHI sites of
pCGNN (Attar and Gilman, 1992) to make pCGNNZFHD1 in which the cDNA
insert is under the transcriptional control of human CMV promoter
and enhancer sequences and is linked to the nuclear localization
sequence from SV40 T antigen. The plasmid pCGNN also contains a
gene for ampicillin resistance which can serve as a selectable
marker.
[0299] pCGNNZFHD1-p65
[0300] An expression vector for directing the expression in
mammalian cells of a chimeric transcription factor containing the
composite DNA-binding domain, ZFHD1, and a transcription activation
domain from p65 (human) was prepared as follows. The sequence
encoding the C-terminal region of p65 containing the activation
domain (amino acid residues 450-550) was amplified from pCGN-p65
using primers p65 5' Xba and p65 3' Spe/Bam. The PCR fragment was
digested with XbaI and BamHI and ligated between the the SpeI and
BamH1 sites of pCGNN ZFHD1 to form pCGNN ZFHD-p65AD.
[0301] The P65 transcription activation sequence contains the
following linear sequence:
2 (SEQ ID NO. 22) CTGGGGGCCTTGCTTGGCAACAGCACAGACCCAGCTGTGTT-
CACAGACCT GGCATCCGTCGACAACTCCGAGTTTCAGCAGCTGCTGAACCAGGGCA- TAC
CTGTGGCCCCCCACACAACTGAGCCCATGCTGATGGAGTACCCTGAGGCT
ATAACTCGCCTAGTGACAGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGC
TCCACTGGGGGCCCCGGGGCTCCCCAATGGCCTCCTTTCAGGAGATGAAG
ACTTCTCCTCCATTGCGGACATGGACTTCTCAGCCCTGCTGAGTCAGATC AGCTCC
Example 2
[0302] Identification and Synthesis of a Ligand for the Conditional
Retention Domain F36M FKBP.
[0303] AP21998 and AP22542 are ligands of FKBP that have particular
utility for CAD applications, because they bind with high affinity
to F36M-FKBP but poorly to the wild-type protein, and are thus
anticipated to lead to minimal interactions with the endogenous
proteins during in vivo applications. The design and assay of such
"bumped" ligands that target a hole created by truncating FKBP
residue Phe36 have been described (Clackson et al., Proc. Natl.
Acad. Sci. USA 95:10437-10442, 1998).
[0304] AP 21998 was prepared via DCC/DMAP-mediated coupling of the
previously described acid AP 1867 (compound 5S in Clackson et al.,
Proc. Natl. Acad. Sci. USA 95:10437-10442, 1998) with commercially
available N,N-dimethyl-1,3-propanediamine (Scheme 1). AP 22542 was
also synthesized by a DCC/DMAP-mediated coupling of acid AP 17362
with alcohol 3 (Scheme 2). Carbinol 3 itself was prepared via a
three step sequence as outlined in Scheme 2. The Claisen-Schmidt
condensation of 3,4-dimethoxybenzaldehyd- e and 3-acetylpyridine
provided unsaturated ketone 1 as a crystalline solid in 68% yield.
Transfer hydrogenation of 1 utilizing ammonium formate as a
hydrogen source provided the propanone adduct 2 as a crystalline
solid in 50% isolated yield. Finally, the enantioselective
reduction of the aryl ketone moiety of 2 to the desired
R-configured carbinol 3 was achieved in 86% by reduction of 2 with
(+)-b-chlorodiisopinocamphenylborane (DIP-Chloride.TM.)
(Chandrasekharan et al. J. Org. Chem. 50:5446, 1985). The synthesis
of the acid component, AP 17362, was prepared as described in
Scheme 3. The commercially available 3,4,5-trimethoxyphenylacetic
acid was converted to the racemic 2-arylbutane derivative 4 in 83%
yield by alkylation with iodoethane of the NaHMDS-generated dianion
of 3,4,5-trimethoxyphenylacetic acid in THF at 0 oC. Resolution of
the acid by repetitive crystallization of the (-)-cinchonidine salt
afforded optically enhanced 4S in 24% yield (48% theoretical) and
of 91% ee. This resolved acid was then coupled with
methyl-L-pipecolate hydrochloride by use of
2-chloro-1-methylpyridinium iodide (Mukaiyama's Reagent). The
resulting coupled product was not isolated, but subjected to
hydrolysis to afford the desired crystalline acid, AP 17362, in 42%
overall yield and >99% de. X-ray structural analysis confirmed
the absolute stereochemistry of the resolved 2-arylbutane center as
the S configuration. 4
[0305] AP 21998
[0306] A solution of AP 1867 (5.0 g, 7.21 mmol) in CH2Cl2 (5.0 mL)
at 0.degree. C. was treated with DCC (178 mg, 0.79 mmol) followed
30 min later by N,N-dimethyl-1,3-propanediamine (880 mg, 8.65 mmol)
and DMAP (5 mg). The reaction 5 mixture was allowed to warm to room
temperature and stir for 5 h, after which time the reaction mixture
was diluted with EtOAc (50 mL), filtered, and the filtrate
extracted with a 5% aqueous citric acid solution (3.times.20 ml).
The acid extract was then made basic by the addition of solid
NaHCO3 and extracted with EtOAc (3.times.50 mL). The organic
extract was dried over Na2SO4, filtered, and evaporated to afford a
crude material which was flash chromatographed on silica gel (5%
then 15% MeOH/CH2Cl2) to afford product (2.2 g, 39%) as a colorless
foam:
[0307] IR (neat) 2940, 1735, 1650, 1510, 1460, 1240, 1130
cm.sup.-1; .sup.1H NMR (CDCl3, 300 MHz) 7.78 (br t, J=5.1 Hz, 1 H),
7.19 (t, J=8.6 Hz, 1 H), 6.92-6.65 (m, 6 H), 6.42 (s, 2 H), 5.63
(dd, J=8.0, 5.5 Hz, 1 H), 5.45 (d, J=4.1 Hz, 1 H), 4.49 (s, 2 H),
3.86-3.70 (m, 16 H), 3.60 (t, J=7.0 Hz, 1 H), 3.47-3.41 (m, 2 H),
2.82 (td, J=13.2, 2.4 Hz, 1 H), 2.62-2.29 (m, 12 H), 2.16-1.23 (m,
10 H), 0.90 (t, J=7.3 Hz, 3 H); .sup.13C NMR (CDCl3, 75 MHz) 172.7,
170.6, 168.5, 157.5, 153.2, 148.9, 147.4, 142.3, 136.7, 135.3,
133.4, 129.8, 120.2, 119.6, 113.9, 112.8, 111.8, 111.4, 105.1,
75.7, 67.3, 60.8, 56.3, 56.0, 52.1, 50.7, 44.3, 43.5, 38.3, 37.4,
31.3, 28.3, 26.8, 25.5, 25.4, 20.9, 12.5; LRMS (ES+): (M+H).sup.+
778; HRMS (FAB): (M+H).sup.+ calcd: 778.4278, meas: 778.4299. 5
[0308] (E)-3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propenone (1): A
solution of 3,4-dimethoxybenzaldehyde (53.7 g, 323 mmol) and
3-acetylpyridine (39.1 g, 323 mmol) in EtOH (400 mL) was treated
with piperdine (4.75 mL, 48 mmol) and heated at reflux for 4 days.
The reaction was then evaporated to a slurry and treated with water
(400 mL). The resulting solids were filtered, air dried, and
recrystallized from EtOAc/hexane to afford product (59.2 g, 68%) as
a yellow colored solid: mp 111-112.5.degree. C.; TLC (EtOAc)
Rf=0.30; 1H NMR (CDCl3, 300 MHz) 9.23 (d, J=1.8 Hz, 1 H), 8.79 (dd,
J=4.8, 1.7 Hz, 1 H), 8.28 (dt, J=7.9, 1.9 Hz, 1 H 7.79 (d, J=15.6
Hz, 1 H), 7.46-7.42 (m, 1 H), 7.35 (d, J=15.6 Hz, 1 H), 7.24 (dd,
J=8.3, 1.9 Hz, 1 H), 7.68 (d, J=1.9 Hz, 1 H), 6.91 (d, J=8.3 Hz, 1
H), 3.95 (s, 3 H), 3.93 (s, 3 H); 13C NMR (CDCl3, 75 MHz) 189.0,
152.9, 151.9, 149.7, 149.4, 146.1, 135.8, 133.8, 127.5, 123.6,
119.4, 111.2, 110.2, 56.0; LRMS (ES+) (M+H)+ 270; Anal. Calcd for
C16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found: C, 71.13; H, 5.70; N,
4.95.
[0309] 3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1-one (2): A
solution of olefin 1 (20.0 g, 74.2 mmol), wet 10% Pd/C (2.0 g), and
ammonium formate (14.0 g, 222 mmol) in MeOH (400 mL) was heated at
reflux for 30 min and filtered, while hot, through a pad of Celite.
The filtrate was allowed to slowly cool and the resulting solids
were filtered and air dried to afford product (10.0 g, 50%) as a
colorless solid: mp 91.5-92.5.degree. C.; TLC (EtOAc) Rf=0.55; 1H
NMR (CDCl3, 300 MHz) 9.16 (d, J=2.0 Hz, 1 H), 8.76 (dd, J=4.8, 1.7
Hz, 1 H), 8.21 (dt, J=8.0, 1.9 Hz, 1 H), 7.40 (dd, J=7.9, 4.8 Hz, 1
H), 6.83-6.77 (m, 3 H), 3.87 (s, 3 H), 3.85 (s, 3 H), 3.30 (d,
J=7.3 Hz, 2 H), 3.03 (d, J=7.7 Hz, 2 H); 13C NMR (CDCl3, 75 MHz)
198.2, 153.5, 149.6, 149.0, 147.6, 135.3, 133.4, 132.1, 123.6,
120.2, 111.9, 111.5, 56.0 (2), 40.9, 29.5; Anal. Calcd for
C16H17NO3: C, 70.83; H, 6.32; N, 5.16. Found: C, 70.63; H, 6.42; N,
5.05.
[0310] (R)-3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1-ol (3):
A solution of (+)-DIP-Chloride.TM. (7.09 g, 22.1 mmol) in THF (10
mL) at -25.degree. C. was treated with ketone 2 (2.0 g, 7.37 mmol).
The resulting mixture was allowed to stand in at -20.degree. C. for
2 h then placed in a -10.degree. C. freezer for 48 h, after which
time the mixture was concentrated and treated with diethyl ether
(50 mL) followed by diethanolamine (4.24 mL, 44.2 mmol). The
viscous mixture was allowed to stir at room temperature for 6 h
after which time it was filtered through a pad of Celite with the
aid of diethyl ether. The filtrate was concentrated and the crude
material flash chromatographed (EtOAc then 10% MeOH/EtOAc) to
afford product. The product was redissolved in diethyl ether (50
mL) and again treated once again with diethanolamine (2.12 mL, 22.1
mmol) as described above to afford product (1.74 g, 86%) as a clear
colorless oil (96% ee by Chiralpak AD HPLC, 15% EtOH/hexane,
retention time 6.1 min for the S-enantiomer and 19.4 min for the
desired R-enantiomer): TLC (EtOAc) Rf=0.25; IR (neat) 3210, 2935,
1590, 1515, 1465, 1420, 1260, 1155, 1070, 1030, 1030 cm-1; 1H NMR
(CDCl3, 300 MHz) 8.50 (d, J=1.7 Hz, 1 H), 8.44 6
[0311] (dd, J=4.7, 1.5 Hz, 1 H), 7.71 (dt, J=7.8, 1.7 Hz, 1 H),
7.28-7.24 (m, 1 H), 6.80-6.70 (m, 1 H), 4.72 (dd, J=7.9, 5.2 Hz, 1
H), 3.85 (s, 6 H), 3.21 (br s, 1 H), 2.77-2.9 (m, 2 H), 2.18-1.96
(m, 2 H); 13C NMR (CDCl3, 75 MHz) 149.0, 148.6, 147.7, 147.4,
140.3, 134.0, 133.8, 123.6, 120.2, 111.8, 111.4, 71.3, 56.0, 55.8,
40.7, 31.5; LRMS (ES+) (M+H)+ 274; HRMS (ES+): (M+H)+ calcd:
274.1462, meas: 274.1443.
[0312]
1-[2(S)-(3,4,5-trimethoxyphenyl)-butyryl]-piperdine-2(S)-carboxylic
acid, 3-(3,4-Dimethoxyphenyl)-1-pyridin-3-yl-propan-1(R)-ol ester
(AP22542): A solution of alcohol 3 (600 mg, 2.20 mmol), acid
AP17362 (882 mg, 2.42 mmol), and DMAP (2.41 mg, 1.98 mmol) in
CH2Cl2 (2.5 mL) at -10.degree. C., was treated with DCC (498 mg,
2.42 mmol). The mixture was allowed to warm to -5.degree. C. over a
1 h period and then placed in a 5.degree. C. refrigerator for an
additional 16 h. The reaction mixture was then diluted with EtOAc
(3 mL), filtered, evaporated, and the crude material flash
chromatographed (75% then 100% EtOAc/hexane) to afford product
(1.15 g, 85%) as a colorless foam: TLC (EtOAc) Rf=0.40; IR (neat)
2940, 1740, 1645, 1590, 1515, 1455, 1420, 1240, 1130, 1030 cm-1; 1H
NMR (CDCl3, 300 MHz) 8.50 (dd, J=4.6, 1.5 Hz, 1 H), 8.42 (d, J=1.7
Hz, 1 H), 7.27 (d, J=8.6 Hz, 1 H), 7.19 (dd, J=7.7, 4.7 Hz, 1 H),
6.78 (d, J=7.7 Hz, 1 H), 6.66-6.64 (m, 2 H), 6.46 (s, 2 H), 5.69
(dd, J=7.7, 6.0 Hz, 1 H), 5.47 (d, J=4.3 Hz, 1 H), 3.86-3.73 (m, 16
H), 3.59 (t, J=7.1 Hz, 1 H), 2.72 (td, J=13.2, 2.6 Hz, 1 H),
2.60-2.38 (m, 2 H), 2.30 (d, J=12.4 Hz, 1 H), 2.16-2.02 (m, 2 H),
1.99-1.90 (m, 1 H), 1.79-1.57 (m, 4 H), 1.46-1.37 (m, 1 H),
1.32-1.19 (m, 1 H), 0.90 (t, J=7.3 Hz, 3 H); 13C NMR (CDCl3, 75
MHz) 172.6, 170.5, 153.3, 149.5, 149.0, 148.3, 147.5, 136.9, 135.6,
135.3, 133.8, 1323.0, 123.6, 120.2, 111.7, 111.5, 105.1, 73.6,
60.9, 56.1, 56.0, 52.0, 50.7, 43.5, 37.9, 31.1, 28.3, 26.7, 25.3,
20.9, 12.5; LRMS (ES+) (M+H)+ 621; HRMS (FAB): (M+H)+ calcd:
621.3176, meas: 621.3178.
[0313] Scheme 3
[0314] (R/S)-2-(3,4,5-Trimethoxyphenyl)butyric acid: A solution of
of 3,4,5-trimethoxyphenylacetic acid (40.0 g, 176.8 mmol) in THF
(125 mL) at 0.degree. C. was treated dropwise with a 2N THF
solution of sodium bis(trimethylsilyl)amide (181 mL, 362 mmol,
Lancaster) over a 1 h period keeping the internal reaction
temperature below 8.degree. C. After 15 min, iodoethane (14.9 mL,
185.7 mmol) was added slowly over a 30 min period keeping the
internal reaction temperature below 6-8.degree. C. and the solution
allowed to warm to room temperature. After 2 h, the mixture was
poured onto EtOAc (700 mL) and acidified by slow addition of a 2.0
N HCl solution (325 mL). The organic component was further washed
with a saturated sodium bisulfite solution (50 mL) followed by
brine (2.times.50 mL), then dried over anhydrous Na2SO4, and
concentrated to a waxy residue (43.8 g). The crude product was
recystallized from hot EtOAc/hexane (30 mL/30 mL) to afford product
(37.1 g, 83%): mp 103-104.degree. C.; TLC (AcOH/EtOAc/hexane,
2:49:49) Rf=0.50.
[0315] (S)-2-(3,4,5-Trimethoxyphenyl)butyric acid (4S): A solution
of 4 (3.09 g, 12.15 mmol) in CH3CN (130 mL) was treated with
(-)-cinchonidine (3.58 g, 12.15 mmol) and the mixture heated to
reflux. The homogeneous solution was allowed to slowly cool to room
temperature with concomitant formation of salts. After a period of
1 h at room temperature, the solution was cooled to 0.degree. C.
for 30 minutes and the solution then filtered to afford 4.05 g of a
chalky colorless solid. This recrystalliztion procedure was then
carried out an addition four times utilizing -20 mL CH3CN/g of
salt. The diastereomeric salt isolated from the fifth
crystallization (1.64 g) was suspended in EtOAc (100 mL) and
treated with a 10% aqueous HCl solution (10 mL). The organic phase
was then washed with water (2.times.15 mL) followed by brine 10
mL), dried over anhydrous MgSO4, and concentrated to afford product
(0.75 g, 24%) as a colorless solid (91% ee by Chiralcel OD HPLC,
1:5:94 formic acid/i-PrOH/hexane, retention time 19.6 min for the
R-enantiomer, and 22.1 min for the desired S-enantiomer): mp
84-85.degree. C. (99.1% ee material); [a]22D +54.8 (c=1.07, MeOH,
30 min, 99.1% ee material); UV (MeOH) lmax 270 (e 895), 232 (e
7,440), 207 (e 40,994) nm; 1H NMR (DMSO-d6, 300 MHz) 6.34 (s, 2 H),
3.52 (s, 6 H), 3.40 (s, 3 H), 3.11 (t, J=7.6 Hz, 1 H) 1.76-164 (m,
1 H), 1.46-1.36 (m, 1 H), 0.60 (t, J=7.3 Hz, 3 H); 1H NMR (CD3OD,
300 MHz) 6.78 (s, 2 H), 4.00 (s, 6 H), 3.90 (s, 3 H), 3.55 (t,
J=7.7 Hz, 1 H) 2.24-2.12 (m, 1 H), 1.97-1.83 (m, 1 H), 1.07 (t,
J=7.3 Hz, 3 H); 13C NMR (DMSO-d6, 75 MHz) 175.1, 153.1, 136.9,
135.8, 105.4, 60.3, 56.2, 53.1, 26.7, 12.4; 13C NMR (CD3OD, 75 MHz)
178.1, 154.9, 138.7, 137.4, 106.8, 61.5, 57.0, 55.3, 28.3, 12.9;
HRMS (FAB): (M-H)- calcd: 253.1076, meas: 253.1063. Anal. Calcd for
C13H1805: C, 61.41; H, 7.13. Found: C, 61.47; H, 7.20.
[0316]
[S-(R*,R*)]-1-[1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-2-piperdineca-
rboxylic acid (AP17362): A solution of 5 (0.75 g, 2.95 mmol, 91%
ee) in CH2Cl2 (15 mL) was treated with methyl-L-pipecolate
hydrochloride (0.539 g, 3.00 mmol) followed by
2-chloro-1-methylpyridinium iodide (0.958 g, 3.75 mmol) and
triethylamine (1.25 mL, 8.95 mmol). The reaction mixture was
allowed to stir for 3.5 h, after which time the solution was
diluted with EtOAc (100 mL), washed with water (15 mL), a 5%
aqueous citric acid solution (25 mL), a saturated Na2CO3 solution
(10 mL), water (15 mL), and finally brine (15 mL). The organic
phase was dried over MgSO4 and concentrated to a yellow oil which
was then dissolved in MeOH (14 mL). The methanolic solution was
treated with water (1 mL) followed by lithium hydroxide monohydrate
(0.620 g, 14.78 mmol). After 4 h, the mixture was diluted with
EtOAc (100 mL), washed with a saturated NaHCO3 solution (3.times.40
mL) followed by water (20 mL). The aqueous portions were combined
and acidified to pH .about.3 by careful addition of a 10% aqueous
HCl solution. The resulting suspension was extracted with EtOAc
(2.times.75 mL) which was then washed with water (2.times.25 mL),
brine (20 mL), dried over MgSO4, and concentrated to a solid which
was dissolved in a refluxing EtOAc (75 mL) solution and allowed to
slowly cool to room temperature. The resulting crystalline material
was filtered and air dried to afford product (0.508 g, 42%) as a
colorless solid: (+99% de by Chiralpak AD HPLC with guard column,
0.2:5:95 formic acid/i-PrOH/hexane, retention time 40.0 min for the
SR-diastereomer, 43.0 min for the desired SS-diastereomer, 46.5 min
for the RR-diastereomer, and 67.5 min for the RS-diastereomer); mp
173.5-174.degree. C.; [a]22D +10.9 (c=1.01, DMSO, 30 min); UV
(MeOH) lmax 270 (e 990), 232 (e 11,161), 207 (e 49,079) nm; 1H NMR
(DMSO-d6, 300 MHz) 6.55 (s, 2 H), 5.13 (d, J=4.4 Hz, 1 H),
3.85-3.64 (m, 11 H), 2.77-2.70 (m, 1 H), 2.12 (d, J=13.4 Hz, 1 H),
1.99-1.85 (m, 1 H), 1.65-1.55 (m, 4 H), 1.38-1.18 (m, 2 H), 0.84
(t, J=7.2 Hz, 3 H); 1H NMR (CD3OD, 300 MHz) 6.74 (s, 2 H), 5.43 (d,
J=4.0 Hz, 1 H), 4.13-3.83 (m, 11 H), 3.03 (td, J=13.5, 3.0 Hz, 1
H), 2.44 (d, J=13.8 Hz, 1 H), 2.24-2.14 (m, 1 H), 1.90-1.40 (m, 6
H) 1.09 (t, J=7.3 Hz, 3 H); 13C NMR (DMSO-d6, 75 MHz) 172.9, 172.2,
153.0, 136.2, 105.4, 60.2, 56.2, 56.0, 51.8, 49.4, 43.1, 28.5,
26.8, 25.3, 21.0, 12.8; 13C NMR (CD3OD, 75 MHz) 175.4, 174.5,
154.9, 137.5, 106.8, 61.5, 57.1, 53.9, 52.1, 45.2, 29.9, 28.2,
26.8, 22.3, 13.2; HRMS (FAB): (M-H)- calcd: 364.1760, meas:
364.1774. Anal. Calcd for C19H27O6: C, 62.45; H, 7.45; N, 3.83.
Found: C, 62.32; H, 7.61; N, 3.88.
Example 3
[0317] The Conditional Retention Domain F(36M) FKBP; Studies with
hGH
[0318] To test whether F(36M) could function as a conditional
retention domain to enable regulated secretion of a fused
heterologous protein, a fusion protein of the design shown in FIG.
2B was constructed. This fusion protein contains a signal sequence
from the human growth hormone (hGH) gene, 4 copies of the F(36M)
domain, a furin cleavage sequence from human stromelysin 3 and
coding sequence from the mature hGH protein. The resulting fusion
protein, in essence, simply contains F(36M) domains and a furin
cleavage signal inserted at the cleavage site between the signal
sequence and the mature hGH peptide sequence. Since the furin
recognition sequence is N-terminal to the cleavage site it can be
situated so that appropriate cleavage will generate the same hGH
amino acid sequence as that generated by natural cleavage of its
own signal sequence.
[0319] A vector driving the expression of this fusion protein,
under control of the strong constitutive enhancer from CMV, was
transiently transfected into HT1080 cells, a human fibrosarcoma
cell line. Following overnight incubation of cells in the absence
of ligand, the medium was washed away and fresh medium added, with
or without ligand. Two hours later, the amount of hGH secreted into
the medium was determined by radioimmunoassay. As shown in FIG. 3A,
in the absence of ligand, the amount of hGH secreted was low. In
contrast, in the presence of ligand, the amount of hGH secreted was
several hundred-fold greater. This demonstrates that F(36M) can act
as a conditional retention domain when fused to a heterologous
protein.
[0320] Next, cell lines were generated by stably transfecting the
F(36M)-hGH expression vector into HT1080 cells. For comparison, the
native hGH gene driven by the same CMV enhancer was also stably
transfected into cells. To allow an initial assessment of any
potential toxic effects of the retained fusion protein, the
selectable marker was expressed from the same transcript as the wt
hGH or F(36M)-hGH fusion proteins through the use of an internal
ribosome entry signal. Equivalent numbers of clones were obtained,
suggesting the there was no toxic effect of the fusion protein.
[0321] Pools of clones stably transfected with the F(36M)-hGH
fusion protein (HT88 pool) were analyzed as described for the
transiently transfected cells. As shown in FIG. 3B, once again, hGH
secretion, which is very low in the absence of ligand, is induced
several hundred fold by incubation with ligand for two hours. To
determine the constitutive rate of hGH secretion, the amount of hGH
secreted in the presence of ligand was measured from cells that had
already been exposed to ligand for 15 hours. As shown in lane 3,
the constitutive rate of secretion from the HT88 cell line was very
similar to the rate of secretion from the HT89 cell line which had
been stably transfected with the wild type hGH protein (lane 4).
Thus, in the presence of ligand the F(36M) domains have no
detectable "retention" activity. Furthermore, this shows that in
the absence of ligand, the fusion protein accumulates to levels
approximately 10-fold higher than that seen when secretion is not
blocked. This steady state level of stored F(36M)-hGH fusion
protein can persist for months in the cell line with no apparent
toxic effect.
Example 4
[0322] Localization/cleavage of Fusion Protein
[0323] To analyze the localization of the fusion protein, EGFP
coding sequence was incorporated in the fusion protein as shown in
FIG. 2C. In cells stably transfected with this fusion protein, in
the absence of ligand, the fusion protein was visible as large
green spots concentrated at multiple points in the perinuclear
space. Co-localization experiments demonstrated that the fusion
protein is aggregated and retained within the ER, as predicted (J.
Rothman, data not shown). Upon addition of ligand, the aggregates
disperse over the next 15 to 60 minutes. This disaggregation
coincides with the appearance of hGH protein in the supernatant of
the cells. As described for the HT88 cell line, ligand induces a
several hundred fold increase in hGH (data not shown).
[0324] To analyze further the state of the fusion protein, cell
lysates and supernatants were prepared from the HT88 cells that had
been incubated in the presence or absence of ligand for 2 hours.
These samples were then immunoblotted with anti-hGH and anti-FKBP
antibodies. As shown in FIG. 4, in the absence of ligand an
approximately 75 kDa-sized band, which corresponds to the expected
size of the F(36M)-hGH fusion protein, is detected in the lysate
(lane 1) but not the supernatant (lane 3) of unstimulated cells
with both the anti-hGH and anti-FKBP antibodies. In cells that had
been stimulated with ligand for 2 hours, very little fusion protein
is detected in the cell lysate, but instead, cleaved proteins are
detected in the supernatant. The anti-hGH blot shows the presence
of a 22 kDa sized protein (lane 6) that co-migrates with purified
recombinant hGH (lane 7). The anti-FKBP blot shows the presence of
a 53 kDa protein that is around the expected size of the remainder
of the fusion protein (F(36M)-FCS). Together these results indicate
that the F(36M)-hGH fusion protein is indeed retained within the ER
in the absence of ligand, released upon interaction with ligand and
subsequently cleaved at the appropriate position, resulting in the
secretion of the F(36M)-FCS portion of the fusion protein and an
intact hGH protein.
Example 5
[0325] Dose Response and Kinetics of hGH Secretion
[0326] The amount of hGH secreted from HT88 cells in response to
ligand is dose-dependent (FIG. 5A). Peak level of secretion occurs
at approximately 2-3 uM AP21998 with half-maximal secretion
occurring at 600 nM.
[0327] To determine the kinetics of secretion, cells were
stimulated with ligand and an aliquot of medium collects at various
time points to measure the accumulation of hGH in the supernatant.
Following addition of saturating levels of ligand, low levels of
hGH are detected in the supernatant within minutes with the peak
rate of secretion occurring between 20-30 minutes (FIG. 5B). This
corresponds to the amount of time it takes for a newly synthesized
protein to be secreted. After the bolus release of stored fusion
protein, the rate of secretion rapidly decreases.
[0328] To further examine the kinetics of secretion in response to
ligand, cells were incubated overnight in the presence or absence
of ligand and then medium collected at 1 hour intervals. The cells
were washed extensively between time points and the medium replaced
with medium containing or lacking ligand as indicated. FIG. 6A
(group A) shows the constitutive rate of secretion from the cells.
In group B, a large bolus release is observed since the cells had
not been exposed to ligand previously. Once ligand is washed away,
however, the rate of hGH secretion quickly decreases, returning to
the low basal rate within 2 hours. Group C shows that if the cells
are exposed to ligand following the large bolus release, within 2
hours the rate of secretion matches that of the constitutively
producing cells (group A).
[0329] Since the constitutive rate of hGH production is only about
75 ng/million cells/hr while 1250 ng/million cells is released in
the first hour after the stores are emptied, it should take some
time for the stores to be refilled. As shown in FIG. 6B, when the
stored hGH is released by incubation with maximal concentration of
ligand, it takes between 8-24 hours for the stores to be refilled
so that the magnitude second bolus release matches that of the
first (or exceeds it since the cell number has increased in the
time). Therefore, in order to achieve consistent, rapid, pulsatile
secretion the stores must not be emptied completely. As shown in
FIG. 6c, if sub-maximal concentrations of ligand are added (e.g.
250 or 500 nM), an equivalent amount of hGH can be secreted 4 hours
later.
[0330] The degree of aggregation increases as the number of F(36M)
domains increases. To test whether the degree of retention could be
manipulated, constructs containing 2, 3 or 6 F(36M) domains were
fused to hGH, stable cell lines generated and hGH secreted in the
presence and absence of ligand assayed. As shown in FIG. 7, the
basal secretion in the absence of ligand increases as the number of
F(36M) domains decreases. This likely reflects a reduction in the
size of aggregates which permits monomeric fusion proteins to
escape retention. An increase in the "leakiness" of fusion protein
secretion is also reflected as a decrease in the amount of stored
fusion protein and, hence, the amount of protein released in bolus
upon addition of ligand. It may be possible to exploit this to
provide transient high level induced secretion against a back drop
of relatively high constitutive basal secretion. Such a situation
may be particularly desirable in the case of insulin production for
the treatment of type 1 diabetes.
Example 6
[0331] Regulated Insulin Secretion
[0332] To test whether the conditional retention domain, F(36M),
could also be used to enable regulated secretion of insulin, a
fusion protein of the design shown in FIG. 3D was constructed. This
fusion protein is analogous to the F(36M)4-hGH fusion protein
described in example X, except the mature hGH coding sequence has
been replaced by coding sequence from the mature human insulin
gene. Normally, in islet cells, proinsulin is processed into the
mature, active, A and B chain complex by endopeptidases that are
expressed exclusively in neuroendocrine cells. Therefore, to allow
insulin to be processed properly in non-endocrine cells mutations
were introduced at the B-C and C-A junctions that would allow
processing by the ubiquitous protease furin (Groskreutz et al., J.
Biol. Chem. 269:6241-6245, 1994). In addition, a third mutation, in
which amino acid 10 of the B chain (histidine) was mutated to
aspartic acid, was introduced to increase the stability of the
protein (Groskreutz et al., J. Biol. Chem. 269:6241-6245 1994).
[0333] A vector driving expression of this F(36M)-insulin fusion
protein (F(36M)4-hIn-m3) was transiently transfected into HT1080
cells. For comparison, vectors driving the expression of insulin
protein alone, with (hIn-m3) or without (hIn-wt) the three
mutations were also transfected. Following overnight incubation of
cells in the absence of ligand, the medium was washed away and
fresh medium added, without or with increasing concentrations of
the monomeric ligand, AP21998. Three hours later, the amount of
insulin secreted into the medium was determined by ELISA using an
assay that recognizes an epitope within the C-peptide (ALPCO). As
shown in FIG. 8, fusion of F(36M) domains to insulin results in a
suppression of its secretion in the absence of monomeric ligand.
Furthermore, secretion is induced in the presence of monomer in a
dose-dependent manner.
Example 7
[0334] Regulated Expression of a Membrane Tethered Protein
[0335] To determine whether the CRD, F(36M), could also be used to
regulate surface expression of a membrane-tethered protein, 3, 4,
or 6 copies of F(36M) were fused to the extracellular and
transmembrane portions of the low-affinity nerve growth factor
receptor (LNGFR; FIG. 3E). In these fusion proteins the F(36M)
domains should be localized to the cytoplasm and tethered to the
plasma membrane, in contrast to the hGH and insulin fusions
described in examples 3 and 6, in which the F(36M) domains were
expressed as part of a soluble protein that localized initially to
the lumen of the ER. Surface expression was assessed by FACS
analysis using anti-LNGFR antibodies (Chromaprobe, Mountain View,
Calif.). As shown in FIG. 9, upon transfection into HT1080 cells
two peaks, corresponding to low and high levels, of LNGFR surface
expression are detected in the absence of monomer with each fusion
protein. The relatively high level of surface expression in the
absence of monomer suggests that the retention activity of the
F(36M) domains is not as strong when the fusion protein is tethered
to the membrane, compared to when it is in solution. This may
reflect the presence of physical constraints that prevent formation
of high order oligomers. However, these results show that the
retention activity of the F(36M) domains clearly increases as the
number of F(36M) domains increase. Furthermore, in the presence of
monomeric ligand, surface expression increases significantly in all
cases. Thus F(36M) domains can also be used to conditionally induce
surface expression of a membrane-tethered protein.
Example 8
[0336] Construction and Testing of a Construct for Conditional
Secretion of hGH Using Rat Retinol Binding Protein as a CRD
[0337] Rat retinol binding protein (rRBP) is conditionally retained
in the ER of a variety of cell types unless retinol is added
(Melhus et al., J Biol Chem 1992 vol 26712036-12041), and so is a
suitable candidate for use as a CRD. We assembled a construct to
test whether rRBP could be used to obtain conditional secretion of
the target protein human growth hormone (hGH) in response to
retinoid ligands. The general structure of the construct is shown
below: 7
[0338] The construct comprises the rRBP cDNA, including the
authentic signal sequence (SS), followed by sequence encoding the
furin cleavage site (FCS) derived from stromelysin E (the amino
acid sequence SARNRQKR (SEQ ID NO. 1) and then the mature 191 amino
acid cDNA coding sequence of hGH (lacking the signal sequence)
followed by an in-frame stop codon. The stromelysin E cleavage site
was chosen because it is of human origin (and therefore expected to
be minimally inuntmogenic in future human therapeutic
applications), and because it is known to be recognised by furin in
the context of fusion to proteins where the P1' residue--the
residue following the cleavage site--is Phe, as in hGH (for a
review see Denault and Leduc, FEBS Lett 1996 vol 379, 113-116). All
junctions between the various sequence motifs and domains are
direct and include no additional sequence, with the exception of an
additional threonine codon between rRBP and FCS to accommodate the
SpeI site. The expression cassette was cloned into the expression
vector pC4EN, placing expression under the control of the strong
hCMV immediate early promoter and enhancer.
[0339] A DNA fragment encompassing the rRBP cDNA was obtained by
RT-PCR from rat liver poly A+RNA (obtained from Clontech, catalog #
6710-1) using the Clontech first strand kit with random primers,
followed by PCR under conventional amplification conditions using
primers RBP-5' (263) and RBP-3' (264). The PCR product was purified
and digested with EcoRI and SpeI. A second DNA fragment encoding
the FCS and mature hGH coding sequence was obtained by PCR
amplification from the hGH cDNA expression vector Z12IHB. The PCR
primers used were FCS-hGH-5' (265) and hGH-3' (266); primer
FCS-hGH-5' (265) includes additional sequence that encodes the FCS.
The PCR product was purified and digested with SpeI and BamHi. The
two DNA fragments were then cloned into EcoRI-BamHI-opened pC4EN in
a three-way ligation to produce the final expression vector
pC4EN-rRBP-hGH. Positive clones were completely sequenced to check
that no errors were incorporated during cloning.
[0340] The construct contains restriction sites that can be used to
add additional modules to the expressed fusion protein. Thus the
stromelysin E FCS can be replaced with alternative cleavage sites
by excising the existing SpeI-AflII fragment and cloning in an
appropriate SpeI-AflII compatible oligonucleotide pair. An epitope
tag can be appended to the rRBP, upstream of the FCS, to allow
immunochemical tracking of the rRBP module inside cells.
Alternative target proteins can be cloned as SpeI-XmaI fragments
(the use of the 3' BamHI site is precluded by the existence of
another BamHI site in the rRBP coding sequence). Alternative CRDs
can be cloned in place of rRBP as EcoRI-SpeI fragments.
[0341] Particularly important additional constructs are those that
incorporate multiple reiterated copies of rRBP. These are obtained
by reamplifying pC4EN-rRBP-hGH using primers 5'-RBP-Xba and
3'-RBP-Spe, generating a fragment containing the mature rRBP
sequence (no signal sequence) flanked by SpeI-compatible 5' XbaI
and 3' SpeI sites. The PCR product is purified, digested with XbaI
and SpeI, and cloned into SpeI-opened pC4EN-rRBP-hGH to generate
pC4EN-(rRBP.times.2)-hGH. An analogous procedure can be used to
prepare constructs encoding higher order concatenates of rRBP.
[0342] PCR Primers:
[0343] RBP-5' (263) 5'
CGTACgaattcCAGAAGCGCGTATGGAGTGGGTGTGGGCGCTCGTGCTG (SEQ ID NO.
23)
[0344] RBP-3' (264) 5'GCATGactagtCAAACTGTTTCTTGAGGGTCTGCTTTGACAG
(SEQ ID NO. 24)
[0345] FCS-hGH-5' (265) (SEQ ID NO. 25)
[0346]
5'GCAACactagtGCTAGAAACCGTCAGAAGAGATTCCCAACCATTCCCTTAAGCAGGCCTTTTGAC-
AACGC (SEQ ID NO. 26)
[0347] hGH-3' (266) 5'GCTCAggatccCGGGCTAGAAGCCACAGCTGCCCTCCACAGAGCG
(SEQ ID NO. 27)
[0348] 5'-RBP-Xba 5'TCAGCtctagaGAGCGCGACTGCAGGGTGAGC (SEQ ID NO.
28)
[0349] 3'-RBP-Spe 5'GAAGCactagtCAAACTGTTTCTTGAGGGTCTG (SEQ ID NO.
29)
[0350] The sequence of the expression cassette is as follows (key
restriction sites underlined):
3 EcoRI rRBP signal sequence--> (SEQ ID NO. 30) 1
gaattccagaagcgcgt ATG GAG TGG GTG TGG GCG CTC GTG CTG CTG GCG GCT
CTG GGA GGC 62 (SEQ ID NO. 31) 1 M E W V W A L V L L A A L G G 15
rRBP mature protein sequence--> 63 GGC AGC GCC GAG CGC GAC TGC
AGG GTG AGC AGC TTC AGA GTC AAG GAG AAC TTC GAC AAG 122 16 G S A E
R D C R V S S F R V K E N F D K 35 BamHI 123 GCT CGT TTC TCT GGG
CTC TGG TAT GCC ATC GCC AAA AAG GAT CCC GAG GGT CTC TTT TTG 182 36
A R F S G L W Y A I A K K D P E G L F L 55 183 CAA GAC AAC ATC ATC
GCT GAG TTT TCT GTC GAC GAG AAG GGT CAT ATG AGC GCT ACA GCC 242 56
Q D N I I A E F S V D E K G H M S A T A 75 243 AAG GGA CGA GTC CGT
CTT CTG AGC AAC TGG GAA GTG TGT GCA GAC ATG GTG GGC ACT TTC 302 76
K G R V R L L S N W E V C A D M V G T F 95 303 ACA GAT ACA GAA GAT
CCT GCC AAG TTC AAG ATG AAG TAC TGG GGT GTA GCC TCC TTT CTC 362 96
T D T E D P A K F K M K Y W G V A S F L 115 363 CAG CGA GGA AAC GAT
GAC CAC TGG ATC ATC GAT ACG GAC TAC GAC ACC TTC GCT CTG CAG 422 116
Q R G N D D H W I I D T D Y D T F A L Q 135 423 TAT TCC TGC CGC CTG
CAG AAT CTG GAT GGC ACC TGT GCA GAC AGC TAC TCC TTT GTG TTT 482 136
Y S C R L Q N L D G T C A D S Y S F V F 155 483 TCT CGT GAC CCC AAT
GGC CTG ACC CCG GAG ACA CGG AGG CTG GTG AGG CAG CGA CAG GAC 542 156
S R D P N G L T P E T R R L V R Q R Q E 175 543 GAG CTG TGC CTA GAG
AGG CAG TAC AGA TGG ATC GAG CAC AAT GGT TAC TGT CAA AGC AGA 602 176
E L C L E R Q Y R W I E H N G Y C Q S R 195 Spel FCS--> mature
hGH--> 603 CCC TCA AGA AAC AGT TTG ACT AGT GCT AGA AAC CGT CAG
AAG AGA TTC CCA ACC ATT CCC 662 196 P S R N S L T S A R N R Q K R F
P T I P 215 AflII 663 TTA AGC AGG CCT TTT GAC AAC GCT ATG CTC CGC
GCC CAT CGT CTG CAC CAG CTG GCC TTT 722 216 L S R P F D N A M L R A
H R L H Q L A F 235 723 GAC ACC TAC CAG GAG TTT GAA GAA GCC TAT ATC
CCA AAG GAA CAG AAG TAT TCA TTC CTG 782 236 D T Y Q E F E E A Y I P
K E Q K Y S P L 255 783 CAG AAC CCC CAG ACC TCC CTC TGT TTC TCA GAG
TCT ATT CCG ACA CCC TCC AAC AGG GAG 842 256 Q N P Q T S L C F S E S
I P T P S N R E 275 843 GAA ACA CAA CAG AAA TCC AAC CTA GAG CTG CTC
CGC ATC TCC CTG CTG CTC ATC CAG TCG 902 276 E T Q Q K S N L E L L R
I S L L L I Q S 295 903 TGG CTG GAG CCC GTG CAG TTC CTC AGG AGT GTC
TTC GCC AAC AGC CTG GTG TAC GGC GCC 962 296 W L E P V Q F L R S V F
A N S L V Y G A 315 963 TCT GAC AGC AAC GTC TAT GAC CTC CTA AAG GAC
CTA GAG GAA GGC ATC CAA ACG CTG ATG 1022 316 S D S N V Y D L L K D
L E E G I Q T L M 335 1023 GGG AGG CTG GAA GAT GGC AGC CCC CGG ACT
GGG CAG ATC TTC AAG CAG ACC TAC AGC AAG 1082 336 G R L E D G S P R
T G Q I F K Q T Y S K 355 1083 TTC GAC ACA AAC TCA CAC AAC GAT GAC
GCA CTA CTC AAG AAC TAC GGG CTG CTC TAC TGC 1142 356 F D T N S H N
D D A L L K N Y G L L Y C 375 1143 TTC AGG AAG GAC ATG GAC AAG GTC
GAG ACA TTC CTG CGC ATC GTG CAG TGC CGC TCT GTG 1202 376 F R K D M
D K V E T F L R K V Q C R S V 395 1203 GAG GGC AGC TGT GGC TTC TAG
cccgggatcctgagaacttcagggtgagtttggggacccttgattgttcttt 1275 396 E G S
C G F * BamHI 402
[0351] The sequence of the encoded protein is as follows:
4 (SEQ ID NO. 31) MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGL-
WYAIAKKDP EGLFLQDNIIAEFSVDEKGHMSATAKGRVRLLSNWEVCADMVGTFTD- TED
PAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLDGTCADS
YSFVFSRDPNGLTPETRRLVRQRQEELCLERQYRWIEHNGYCQSRPSRNS
LTSARNRQKRFPTIPLSRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQ
KYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPV
QFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFK
QTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCG F
[0352] The nucleotide sequence of the expression cassette is as
follows:
5 (SEQ ID NO. 32) ATGGAGTGGGTGTGGGCGCTCGTGCTGCTGGCGGCTCTGGG-
AGGCGGCAGCGCCGAGCGCGACTGCAGGGTGAGCAGCTT
CAGAGTCAAGGAGAACTTCGACAAGGCTCGTTTCTCTGGGCTCTGGTATGCCATCGCCAAAAAGGATCCCGAG-
GGTCTCT TTTTGCAAGACAACATCATCGCTGAGTTTTCTGTCGACGAGAAGGGTCAT-
ATGAGCGCTACAGCCAAGGGACGAGTCCGT CTTCTGAGCAACTGGGAAGTGTGTGCA-
GACATGGTGGGCACTTTCACAGATACAGAAGATCCTGCCAAGTTCAAGATGAA
GTACTGGGGTGTAGCCTCCTTTCTCCAGCGAGGAAACGATGACCACTGGATCATCGATACGGACTACGACACC-
TTCGCTC TGCAGTATTCCTGCCGCCTGCAGAATCTGGATGGCACCTGTGCAGACAGC-
TACTCCTTTGTGTTTTCTCGTGACCCCAAT GGCCTGACCCCGGAGACACGGAGGCTG-
GTGAGGCAGCGACAGGAGGAGCTGTGCCTAGAGAGGCAGTACAGATGGATCGA
GCACAATGGTTACTGTCAAAGCAGACCCTCAAGAAACAGTTTGACTAGTGCTAGAAACCGTCAGAAGAGATTC-
CCAACCA TTCCCTTAAGCAGGCCTTTTGACAACGCTATGCTCCGCGCCCATCGTCTG-
CACCAGCTGGCCTTTGACACCTACCAGGAG TTTGAAGAAGCCTATATCCCAAAGGAA-
CAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTC
TATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTG-
CTCATCC AGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGC-
CTGGTGTACGGCGCCTCTGACAGCAACGTC TATGACCTCCTAAAGGACCTAGAGGAA-
GGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCA
GATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGG-
CTGCTCT ACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTG-
CAGTGCCGCTCTGTGGAGGGCAGCTGTGGC TTCTAG
[0353] To test whether fusion to rRBP can result in
ligand-dependent prevention of secretion of hGH, pC4EN-rRBP-hGH,
pC4EN-(rRBP.times.2)-hGH and their derivatives are transiently
transfected into the human fibrosarcoma cell line HT1080 using
standard methods (eg see Rivera et al., Nature Med 2: 1028-1032
1996; Amara et al., PNAS 94:10618-10623 1997). After overnight
incubation, medium is removed and new medium added, containing
either no drug or retinol at various concentrations. After a
further incubation of 2-24 hours, the amount of hGH secreted into
the medium is determined by radioimmunoassay (Rivera et al., Nature
Med 2: 1028-1032 1996).
[0354] A critical feature for these experiments, as described by
Melhus et al. (J Biol Chem 1992 vol 26712036-12041), is the use of
delipidized serum in the culture medium, since untreated serum
contains significant amounts of retinoids that might lead to
secretion of rRBP in the absence of exogenously added retinol.
Methods for preparing delipidized serum are known (Rothblat et al.,
In Vitro 1976 vol 12, 554-557).
[0355] Increased secretion of hGH upon addition of increasing
concentrations of retinol would indicate that rRBP is acting as a
CRD to retain hGH in secretory compartments until addition of
retinol. Experiments to identify the best configuration of the
system include engineering multimers of rRBP to attempt to enhance
the retention effect, and testing of a variety of different retinol
analogs for activity. Further experiments to confirm the
subcellular location of rRBP fusions include immunocytochemical
subcellular localization of components of the constructs before and
after addition of retinoids using, for example, anti-hGH or
anti-rRBP antibodies.
Example 9
[0356] Physiological Effects of Regulated Insulin Secretion in
vivo
[0357] To test whether this system could be used to regulate
secretion of insulin in vivo and effect changes in serum glucose
levels, 2.times.10e7 HT101-10p cells were implanted intramuscularly
into male nu/nu mice. HT101-10p cells were generated by stably
transfecting HT1080 cells with a vector that drives expression of
the F(36M)4- hIn-m3 fusion protein. Mice were made hyperglycemic by
treatment 2 days earlier with 300 mg/kg streptozotocin (STZ). As
shown in FIG. 11a, STZ treatment elevates serum glucose levels to
-350 mg/dl from -100 mg/dl seen in non-STZ treated mice.
Approximately 1 hr after cells are implanted, animal received
vehicle or the indicated dose of intravenous AP22542 (an analog of
AP21998). Two hours later, serum samples were collected and assayed
for insulin (Ultrasensitive human insulin-specific RIA, Linco) and
glucose (Sigma) concentrations. As shown in FIG. 11a, treatment of
hyperglycemic mice with vehicle or a low dose of AP22542 (1 mg/kg)
fails to increase serum insulin levels above the lower limit of
detection and there is no change in serum glucose. In contrast, in
animals treated with 10 mg/kg AP22542, serum insulin levels
increase to -200 pM and serum glucose levels decline to -75
mg/dl.
[0358] To examine the kinetics of this ligand-induced reduction in
serum glucose, STZ-treated mice implanted with 2.times.10e7
HT101-10p cells were administered a single dose of 30 mg/kg AP22542
intravenously. Serum glucose levels were measured at various times
between 5 minutes and 10 hours later. As shown in FIG. 11b, at 5
and 15 minutes after administration of AP22542, serum glucose
levels are indistinguishable from animals treated with vehicle.
However, within 30 minutes there is a significant reduction in
serum glucose and by two hours serum glucose levels have declined
to 50 mg/dl from initial levels of nearly 500 mg/dl. This effect is
transient as serum glucose levels rise to 350 mg/dl within 5 hours
and return to baseline between 6 and 10 hours later. Since insulin
secretion is dependent on the presence of the drug, administration
of lower doses of AP22542 or of a ligand with a shorter half life
should result in an even more transient production of secreted
protein and resulting physiological effect. Conversely,
administration of higher doses of AP22542 or of a ligand with a
longer half life should result in a more prolonged production of
secreted protein.
Sequence CWU 1
1
38 1 7 PRT Homo sapiens 1 Met Ser Met Arg Val Arg Arg 1 5 2 7 PRT
Homo sapiens 2 Lys Pro Ala Lys Ser Ala Arg 1 5 3 6 PRT Homo sapiens
3 Lys Ser Val Lys Lys Arg 1 5 4 7 PRT Homo sapiens 4 Ala Arg Asn
Arg Gln Lys Arg 1 5 5 7 PRT Homo sapiens 5 Arg Pro Ser Arg Lys Arg
Arg 1 5 6 7 PRT Homo sapiens 6 Thr Glu Lys Arg Lys Lys Arg 1 5 7 40
DNA Artificial Sequence Description of Artificial SequencePCR
primer 7 tcccgcacct cttcggccag cgaattccag aagcgcgtat 40 8 32 DNA
Artificial Sequence Description of Artificial SequencePCR primer 8
gactcactat aggacgcgtt cgagctcgcc cc 32 9 31 DNA Artificial Sequence
Description of Artificial SequencePCR primer 9 catcattttg
gcaaaggatt cactcctcag g 31 10 27 DNA Artificial Sequence
Description of Artificial SequencePCR primer 10 gatggaaaga
aaatggattc ctcccgg 27 11 24 DNA Artificial Sequence Description of
Artificial SequencePCR primer 11 tctagagtga gcaagggcga ggag 24 12
37 DNA Artificial Sequence Description of Artificial SequencePCR
primer 12 ggatccttat taactagtct tgtacagctc gtccatg 37 13 25 DNA
Artificial Sequence Description of Artificial SequencePCR primer 13
aagcttacca ctcagggtcc tgtgg 25 14 19 DNA Artificial Sequence
Description of Artificial SequencePCR primer 14 gaattcgtgg
caacttcca 19 15 29 DNA Artificial Sequence Description of
Artificial SequencePCR primer 15 cacaggaccc tgaattctaa gcttgtggc 29
16 30 DNA Artificial Sequence Description of Artificial SequencePCR
primer 16 ataagggaat ggttctagag gcactgccct 30 17 31 DNA Artificial
Sequence Description of Artificial SequencePCR primer 17 atgccacccg
ggactagtga agccacagct g 31 18 48 DNA Artificial Sequence
Description of Artificial SequencePCR primer 18 actagtgcta
gaaaccgtca gaagagattc ccaaccattc ccttaagc 48 19 31 DNA Artificial
Sequence Description of Artificial SequencePCR primer 19 ggatcccggg
ctagaagcca cagctgccct c 31 20 32 DNA Artificial Sequence
Description of Artificial SequencePCR primer 20 cgaattcttc
tgccatggcc ctgtggatgc gc 32 21 31 DNA Artificial Sequence
Description of Artificial SequencePCR primer 21 cggatccgca
ggctgcgtct agttgcagta g 31 22 52 DNA Artificial Sequence
Description of Artificial SequencePCR primer 22 cactagtgct
agaaaccgtc agaagagatt tgtgaaccaa cacctgtgcg gc 52 23 31 DNA
Artificial Sequence Description of Artificial SequencePCR primer 23
cggatccgca ggctgcgtct agttgcagta g 31 24 27 DNA Artificial Sequence
Description of Artificial SequencePCR primer 24 cctgtgcggc
tcagacctgg tggaagc 27 25 35 DNA Artificial Sequence Description of
Artificial SequencePCR primer 25 cttctacaca cccaggacca agcgggaggc
agagg 35 26 29 DNA Artificial Sequence Description of Artificial
SequencePCR primer 26 ccctggaggg gtcccggcag aagcgtggc 29 27 341 DNA
Homo sapiens 27 ctgggggcct tgcttggcaa cagcacagac ccagctgtgt
tcacagacct ggcatccgtc 60 gacaactccg agtttcagca gctgctgaac
cagggcatac ctgtggcccc ccacacaact 120 gagcccatgc tgatggagta
ccctgaggct ataactcgcc tagtgacagg ggcccagagg 180 ccccccgacc
cagctcctgc tccactgggg gccccggggc tccccaatgg cctcctttca 240
ggagatgaag acttctcctc cattgcggac atggacttct cagccctgct gagtcagatc
300 agctcctaag ggggtgacgc ctgccctccc cagagcactg g 341 28 8 PRT Homo
sapiens 28 Ser Ala Arg Asn Arg Gln Lys Arg 1 5 29 49 DNA Artificial
Sequence Description of Artificial SequencePCR primer 29 cgtacgaatt
ccagaagcgc gtatggagtg ggtgtgggcg ctcgtgctg 49 30 42 DNA Artificial
Sequence Description of Artificial SequencePCR primer 30 gcatgactag
tcaaactgtt tcttgagggt ctgctttgac ag 42 31 70 DNA Artificial
Sequence Description of Artificial SequencePCR primer 31 gcaacactag
tgctagaaac cgtcagaaga gattcccaac cattccctta agcaggcctt 60
ttgacaacgc 70 32 45 DNA Artificial Sequence Description of
Artificial SequencePCR primer 32 gctcaggatc ccgggctaga agccacagct
gccctccaca gagcg 45 33 32 DNA Artificial Sequence Description of
Artificial SequencePCR primer 33 tcagctctag agagcgcgac tgcagggtga
gc 32 34 33 DNA Artificial Sequence Description of Artificial
SequencePCR primer 34 gaagcactag tcaaactgtt tcttgagggt ctg 33 35
1275 DNA Homo sapiens 35 gaattccaga agcgcgtatg gagtgggtgt
gggcgctcgt gctgctggcg gctctgggag 60 gcggcagcgc cgagcgcgac
tgcagggtga gcagcttcag agtcaaggag aacttcgaca 120 aggctcgttt
ctctgggctc tggtatgcca tcgccaaaaa ggatcccgag ggtctctttt 180
tgcaagacaa catcatcgct gagttttctg tcgacgagaa gggtcatatg agcgctacag
240 ccaagggacg agtccgtctt ctgagcaact gggaagtgtg tgcagacatg
gtgggcactt 300 tcacagatac agaagatcct gccaagttca agatgaagta
ctggggtgta gcctcctttc 360 tccagcgagg aaacgatgac cactggatca
tcgatacgga ctacgacacc ttcgctctgc 420 agtattcctg ccgcctgcag
aatctggatg gcacctgtgc agacagctac tcctttgtgt 480 tttctcgtga
ccccaatggc ctgaccccgg agacacggag gctggtgagg cagcgacagg 540
aggagctgtg cctagagagg cagtacagat ggatcgagca caatggttac tgtcaaagca
600 gaccctcaag aaacagtttg actagtgcta gaaaccgtca gaagagattc
ccaaccattc 660 ccttaagcag gccttttgac aacgctatgc tccgcgccca
tcgtctgcac cagctggcct 720 ttgacaccta ccaggagttt gaagaagcct
atatcccaaa ggaacagaag tattcattcc 780 tgcagaaccc ccagacctcc
ctctgtttct cagagtctat tccgacaccc tccaacaggg 840 aggaaacaca
acagaaatcc aacctagagc tgctccgcat ctccctgctg ctcatccagt 900
cgtggctgga gcccgtgcag ttcctcagga gtgtcttcgc caacagcctg gtgtacggcg
960 cctctgacag caacgtctat gacctcctaa aggacctaga ggaaggcatc
caaacgctga 1020 tggggaggct ggaagatggc agcccccgga ctgggcagat
cttcaagcag acctacagca 1080 agttcgacac aaactcacac aacgatgacg
cactactcaa gaactacggg ctgctctact 1140 gcttcaggaa ggacatggac
aaggtcgaga cattcctgcg catcgtgcag tgccgctctg 1200 tggagggcag
ctgtggcttc tagcccggga tcctgagaac ttcagggtga gtttggggac 1260
ccttgattgt tcttt 1275 36 401 PRT Homo sapiens 36 Met Glu Trp Val
Trp Ala Leu Val Leu Leu Ala Ala Leu Gly Gly Gly 1 5 10 15 Ser Ala
Glu Arg Asp Cys Arg Val Ser Ser Phe Arg Val Lys Glu Asn 20 25 30
Phe Asp Lys Ala Arg Phe Ser Gly Leu Trp Tyr Ala Ile Ala Lys Lys 35
40 45 Asp Pro Glu Gly Leu Phe Leu Gln Asp Asn Ile Ile Ala Glu Phe
Ser 50 55 60 Val Asp Glu Lys Gly His Met Ser Ala Thr Ala Lys Gly
Arg Val Arg 65 70 75 80 Leu Leu Ser Asn Trp Glu Val Cys Ala Asp Met
Val Gly Thr Phe Thr 85 90 95 Asp Thr Glu Asp Pro Ala Lys Phe Lys
Met Lys Tyr Trp Gly Val Ala 100 105 110 Ser Phe Leu Gln Arg Gly Asn
Asp Asp His Trp Ile Ile Asp Thr Asp 115 120 125 Tyr Asp Thr Phe Ala
Leu Gln Tyr Ser Cys Arg Leu Gln Asn Leu Asp 130 135 140 Gly Thr Cys
Ala Asp Ser Tyr Ser Phe Val Phe Ser Arg Asp Pro Asn 145 150 155 160
Gly Leu Thr Pro Glu Thr Arg Arg Leu Val Arg Gln Arg Gln Glu Glu 165
170 175 Leu Cys Leu Glu Arg Gln Tyr Arg Trp Ile Glu His Asn Gly Tyr
Cys 180 185 190 Gln Ser Arg Pro Ser Arg Asn Ser Leu Thr Ser Ala Arg
Asn Arg Gln 195 200 205 Lys Arg Phe Pro Thr Ile Pro Leu Ser Arg Pro
Phe Asp Asn Ala Met 210 215 220 Leu Arg Ala His Arg Leu His Gln Leu
Ala Phe Asp Thr Tyr Gln Glu 225 230 235 240 Phe Glu Glu Ala Tyr Ile
Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln 245 250 255 Asn Pro Gln Thr
Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser 260 265 270 Asn Arg
Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile 275 280 285
Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 290
295 300 Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn
Val 305 310 315 320 Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln
Thr Leu Met Gly 325 330 335 Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly
Gln Ile Phe Lys Gln Thr 340 345 350 Tyr Ser Lys Phe Asp Thr Asn Ser
His Asn Asp Asp Ala Leu Leu Lys 355 360 365 Asn Tyr Gly Leu Leu Tyr
Cys Phe Arg Lys Asp Met Asp Lys Val Glu 370 375 380 Thr Phe Leu Arg
Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly 385 390 395 400 Phe
37 400 PRT Homo sapiens 37 Met Glu Trp Val Trp Ala Leu Val Leu Leu
Ala Ala Leu Gly Gly Gly 1 5 10 15 Ser Ala Glu Arg Asp Cys Arg Val
Ser Ser Phe Arg Val Lys Glu Asn 20 25 30 Phe Asp Lys Ala Arg Phe
Ser Gly Leu Trp Tyr Ala Ile Ala Lys Lys 35 40 45 Asp Pro Glu Gly
Leu Phe Leu Gln Asp Asn Ile Ile Ala Glu Phe Ser 50 55 60 Val Asp
Glu Lys Gly His Met Ser Ala Thr Ala Lys Gly Arg Val Arg 65 70 75 80
Leu Leu Ser Asn Trp Glu Val Cys Ala Asp Met Val Gly Thr Phe Thr 85
90 95 Asp Thr Glu Asp Pro Ala Lys Phe Lys Met Lys Tyr Trp Gly Val
Ala 100 105 110 Ser Phe Leu Gln Arg Gly Asn Asp Asp His Trp Ile Ile
Asp Thr Asp 115 120 125 Tyr Asp Thr Phe Ala Leu Gln Tyr Ser Cys Arg
Leu Gln Asn Leu Asp 130 135 140 Gly Thr Cys Ala Asp Ser Tyr Ser Phe
Val Phe Ser Arg Asp Pro Asn 145 150 155 160 Gly Leu Thr Pro Glu Thr
Arg Arg Leu Val Arg Gln Arg Gln Glu Glu 165 170 175 Leu Cys Leu Glu
Arg Gln Tyr Arg Trp Ile Glu His Asn Gly Tyr Cys 180 185 190 Gln Ser
Arg Pro Ser Arg Asn Ser Leu Thr Ser Ala Arg Asn Arg Gln 195 200 205
Lys Arg Phe Pro Thr Ile Pro Leu Ser Arg Pro Phe Asp Asn Ala Met 210
215 220 Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln
Glu 225 230 235 240 Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr
Ser Phe Leu Gln 245 250 255 Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu
Ser Ile Pro Thr Pro Ser 260 265 270 Asn Arg Glu Glu Thr Gln Gln Lys
Ser Asn Leu Glu Leu Leu Arg Ile 275 280 285 Ser Leu Leu Leu Ile Gln
Ser Trp Leu Glu Pro Val Gln Phe Leu Arg 290 295 300 Ser Val Phe Ala
Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val 305 310 315 320 Tyr
Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly 325 330
335 Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr
340 345 350 Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu
Leu Lys 355 360 365 Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met
Asp Lys Val Glu 370 375 380 Thr Phe Leu Arg Ile Val Gln Cys Arg Ser
Val Glu Gly Ser Cys Gly 385 390 395 400 38 1204 DNA Homo sapiens 38
atggagtggg tgtgggcgct cgtgctgctg gcggctctgg gaggcggcag cgccgagcgc
60 gactgcaggg tgagcagctt cagagtcaag gagaacttcg acaaggctcg
tttctctggg 120 ctctggtatg ccatcgccaa aaaggatccc gagggtctct
ttttgcaaga caacatcatc 180 gctgagtttt ctgtcgacga gaagggtcat
atgagcgcta cagccaaggg acgagtccgt 240 cttctgagca actgggaagt
gtgtgcagac atggtgggca ctttcacaga tacagaagat 300 cctgccaagt
tcaagatgaa gtactggggt gtagcctcct ttctccagcg aggaaacgat 360
gaccactgga tcatcgatac ggactacgac accttcgctc tgcagtattc ctgccgcctg
420 cagaatctgg atggcacctg tgcagacagc tactcctttg tgttttctcg
tgaccccaat 480 ggcctgaccc cggagacacg gaggctggtg aggcagcgac
aggaggagct gtgcctagag 540 aggcagtaca gatggatcga gcacaatggt
tactgtcaaa gcagaccctc aagaaacagt 600 ttgactagtg ctagaaaccg
tcagaagaga ttcccaacca ttcccttaag caggcctttt 660 gacaacgcta
tgctccgcgc ccatcgtctg caccagctgg cctttgacac ctaccaggag 720
tttgaagaag cctatatccc aaaggaacag aagtattcat tcctgcagaa cccccagacc
780 tccctctgtt tctcagagtc tattccgaca ccctccaaca gggaggaaac
acaacagaaa 840 tccaacctag agctgctccg catctccctg ctgctcatcc
agtcgtggct ggagcccgtg 900 cagttcctca ggagtgtctt cgccaacagc
ctggtgtacg gcgcctctga cagcaacgtc 960 tatgacctcc taaaggacct
agaggaaggc atccaaacgc tgatggggag gctggaagat 1020 ggcagccccc
ggactgggca gatcttcaag cagacctaca gcaagttcga cacaaactca 1080
cacaacgatg acgcactact caagaactac gggctgctct actgcttcag gaaggacatg
1140 gacaaggtcg agacattcct gcgcatcgtg cagtgccgct ctgtggaggc
agctgtggtt 1200 ctag 1204
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