Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Interferon Alpha Plasmids And Delivery Systems,
And Methods Of Makina And Using' The Same
Related Applications
This application relates to U.S. patent application
Serial No. 08/949,160, filed October 10, 1997 and
International patent application No. PCT/US97/18779, filed
October 10, 1997, (Lyon & Lyon Docket Nos. 226/285 US and
PCT, respectively), both of which are related to U.S. patent
application Serial No. 60/028,676, filed October 18, 1996,
(Lyon & Lyon Docket No. 222/086 US), all three of which are
entitled "IL-12 GENE EXPRESSION AND DELIVERY SYSTEMS AND
USES" (by Nordstrom et al.).
This application is also related to U.S. patent
application Serial No. 08/798,974, filed February 11, 1997,
(Lyon & Lyon Docket No. 224/084 US) and International patent
application No. PCT/US95/17038, filed December 28, 1995,
(Lyon & Lyon Docket No. 210/190 PCT), both of which are
related to U.S. patent application Serial No. 08/372,213,
filed January 13, 1995, (Lyon & Lyon Docket No. 210/190 US),
all three of which are entitled "FORMULATED NUCLEIC ACID
COMPOSITIONS AND METHODS OF ADMINISTERING THE SAME FOR GENE
THERAPY" (by Mumper Rolland).
Each of the above-mentioned applications are
incorporated herein by reference in their entirety,
including any drawings.
Field Of The Invention
The present invention relates to gene delivery and gene
therapy, and provides novel nucleic acid constructs for
expression of interferon alpha in a mammal, formulations for
delivery that incorporate a nucleic acid construct for
expression, and methods for preparing and using such
constructs and formulations. In particular, this invention
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relates to plasmid constructs for delivery of therapeutic
interferon alpha encoding nucleic acids to cells in order to
modulate tumor activity, methods of using those constructs
(including combination therapy with other agents, such as
cytokines, preferably IL-12), as well as methods for
preparing such constructs.
Background Of The Invention
The following discussion of the background of the
invention is merely provided to aid the reader in
understanding the invention and is not admitted to describe
or constitute prior art to the present invention.
Plasmids are an important element in genetic engi-
neering and gene therapy. Plasmids are usually circular DNA
molecules that can be introduced into bacterial cells by
transformation which replicate autonomously in the cell.
Plasmids typically allow for the amplification of cloned
DNA. Some plasmids are present in 20 to 50 copies during
cell growth, and after the arrest of protein synthesis, as
many as 1000 copies per cell of a plasmid can be generated.
Suzuki et al., Genetic Analysis, p. 404, 1989.
Current non-viral approaches to human gene therapy
require that a potential therapeutic gene be cloned into
plasmids. Large quantities of a bacterial host harboring
the plasmid may be fermented and the plasmid DNA may be
purified for subsequent use. Current human clinical trials
using plasmids utilize this approach. Recombinant DNA
Advisory Committee Data Management Report, December, 1994,
Human Gene Therapy 6:535-548. Studies normally focus on the
therapeutic gene and the elements that control its
expression in the patient when designing and constructing
gene therapy plasmids. Generally, therapeutic genes and
regulatory elements are simply inserted into existing
cloning vectors that are convenient and readily available.
Plasmid design and construction utilizes several key
factors. First, plasmid replication origins determine
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plasmid copy number, which affects production yields.
Plasmids that replicate to higher copy number can increase
plasmid yield from a given volume of culture, but excessive
copy number can be deleterious to the bacteria and lead to
undesirable effects (Fitzwater, et al., Embo J. 7:3289-3297
(1988); Uhlin, et al., Mol. Gen. Genet. 165:167-179 (1979)).
Artificially constructed plasmids are sometimes unstably
maintained, leading to accumulation of plasmid-free cells
and reduced production yields.
To overcome this problem of plasmid-free cells, genes
that code for antibiotic resistance phenotype are included
on the plasmid and antibiotics are added to kill or inhibit
plasmid-free cells. Most general purpose cloning vectors
contain ampicillin resistance ((3-lactamase, or b1a) genes.
Use of ampicillin can be problematic because of the
potential for residual antibiotic in the purified DNA, which
can cause an allergic reaction in a treated patient. In
addition, j3-lactam antibiotics are clinically important for
disease treatment. When plasmids containing antibiotic
resistance genes are used, the potential exists for the
transfer of antibiotic resistance genes to a potential
pathogen.
Other studies have used the neo gene which is derived
from the bacterial transposon TnS. The neo gene encodes
resistance to kanamycin and neomycin (Smith, Vaccine
12:1515-1519 (1994)). This gene has been used in a number
of gene therapy studies, including several human clinical
trials (Recombinant DNA Advisory Committee Data Management
Report, December, 1994, Human Gene Therapy 6:535=548). Due
to the mechanism by which resistance is imparted, residual
antibiotic and transmission of the gene to potential
pathogens may be less of a problem than for ~3-lactams.
In addition to elements that affect the behavior of the
plasmid within the host bacteria, such as E. coli, plasmid
vectors have also been shown to affect transfection and
expression in eukaryotic cells. Certain plasmid sequences
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have been shown to reduce expression of eukaryotic genes in
eukaryotic cells when carried in cis (Peterson, et al., Mol.
Cell. Biol. 7:1563-1567 (1987); Yoder et al., Mol. Cell.
Biol. 3:956-959 (1983): Lusky et al., Nature 293:79-81
(1981); and Leite, et al., Gene 82:351-356 (1989)). Plasmid
sequences also have been shown to fortuitously contain
binding sites for transcriptional control proteins (Ghersa,
et al., Gene 151:331-332 (1994) Tully et al., Biochem.
Biophys. Res. Comm. 144:1-10 (1987); and Kushner, et al.,
Mol. Endocrinol. 8:405-407 (1994)). This can cause inappro-
priate levels of gene expression in treated patients.
Interferon alpha is a gene product that has been
proposed for use, either alone or in combination with other
agents, in different delivery systems for the treatment of
certain diseases, including particular cancers.
International patent publication WO/97/00085, published
January 3, 1997, proposes ex vivo transfection of tumor
cells with interferon alpha and another immomodulatory
molecule, such as IL-12. None of the previously proposed
treatments have proven entirely satisfactory, due in part to
the high cost and technical difficulty involved in ex vivo
approaches. Thus there still remains a need in the art for
improved plasmids encoding interferon alpha as well as
improved treatment protocols and technologies.
Summary
The present invention relates to gene delivery and gene
therapy, and provides novel nucleic acid constructs for
expression of interferon alpha in a mammal, formulations for
delivery that incorporate a nucleic acid construct for
expression, and methods for preparing and using such
constructs and formulations. In particular, this invention
relates to plasmid constructs for delivery of therapeutic
interferon alpha encoding nucleic acids to cells in order to
modulate tumor activity, methods of using those constructs
(including combination therapy with other agents, such as
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cytokines, preferably IL-12), as well as methods for
preparing such constructs. The pharmaceutical acceptable,
cost effective and highly efficient delivery system
presented herein represents an unanticipated improvement
5 over the art.
Thus, in a first aspect, the invention features a
plasmid that contains a CMV promoter and optionally a
synthetic 5' intron transcriptionally linked with an
interferon alpha coding sequence, and a 3'-untranslated
region (UTR) . Preferably the 3' UTR is a 3' growth hormone
UTR.
As used herein, the term "plasmid" refers to a
construct made up of genetic material (i.e., nucleic acids).
It includes genetic elements arranged such that an inserted
coding sequence can be transcribed in eukaryotic cells.
Also, while the plasmid may include a sequence from a viral
nucleic acid, such viral sequence does not cause the
incorporation of the plasmid into a viral particle, and the
plasmid is therefore a non-viral vector. Preferably a
plasmid is a closed circular DNA molecule.
"Cytomegalovirus promoter" refers to one or more
sequences from a cytomegalovirus which are functional in
eukaryotic cells as a transcriptional promoter and an
upstream enhancer sequence. The enhancer sequence allows
transcription to occur at a higher frequency from the
associated promoter.
In this context, "transcriptionally linked" means that
in a system suitable for transcription, transcription will
initiate under the direction of the control sequences) and
proceed through sequences which are transcriptionally linked
with that control sequence(s). Preferably no mutation is
created in the resulting transcript, which would alter the
resulting translation product.
The term "coding region" or "coding sequence" refers to
a nucleic acid sequence which encodes a particular gene
product for which expression is desired, according to the
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normal base pairing and codon usage relationships. Thus,
the coding sequence must be placed in such relationship to
transcriptional control sequences (possibly including
control elements and translational initiation and
termination codons) that a proper length transcript will be
produced and will result in translation in the appropriate
reading frame to produce a functional desired product.
In a preferred embodiment the interferon alpha coding
sequence is for human interferon alpha and preferably is a
synthetic sequence having optimal codon usage, such as the
nucleotide sequence of SEQ ID NO:11 or semi-optimal codon
usage, such as the nucleotide sequence of SEQ ID rd0:12.
A particular example of coding regions suitable for use
in the plasmids of this invention are the natural sequences
coding for human interferon alpha. Thus, in a preferred
embodiment coding region has a nucleotide sequence which is
the same as SEQ ID N0:10, which is the natural nucleotide
sequence encoding human interferon alpha. However, it may
be preferable to have an interferon alpha coding sequence
which is a synthetic coding sequence. In a preferred
embodiment, the interferon alpha coding sequence is a
synthetic sequence utilizing optimal or semi-optimal codon
usage, preferably the sequence shown in SEQ ID N0:11 or SEQ
ID N0:12.
Thus, a "sequence coding for the human interferon
alpha" or "a human interferon alpha coding sequence" is a
nucleic acid sequence which encodes the amino acid sequence
of human interferon alpha, based on the normal base pairing
and translational codon usage relationships. It is
preferable that the coding sequence encode the exact, full
amino acid sequence of natural human interferon, but this is
not essential. The encoded polypeptide may differ from
natural human interferon alpha, so long as the polypeptide
retains interferon alpha activity, preferably the
polypeptide is at least 50~, 75$, 90~, or 97~ as active as
natural human interferon alpha, and more preferably fully as
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active as natural human interferon alpha. Thus, the
polypeptide encoded by the interferon alpha coding sequence
may differ from a natural human interferon alpha polypeptide
by a small amount, preferably less than a 15$, 10~, 5~, or
1$ change. Such a change may be of one of more different
types, such as deletion, addition, or substitution of one or
more amino acids.
The term "transcriptional control sequence" refers to
sequences which control the rate of transcription of a
transcriptionally linked coding region. Thus, the term can
include elements such as promoters, operators, and
enhancers. For a particular transcription unit, the
transcriptional control sequences will include at least a
promoter sequence.
A "growth hormone 3' untranslated region" is a sequence
located downstream (i.e., 3') of the region encoding
material polypeptide and including at least part of the
sequence of the natural 3' UTR/poly(a) signal from a growth
hormone gene, preferably the human growth hormone gene.
This region is generally transcribed but not translated.
For expression in eukaryotic cells it is generally
preferable to include sequence which signals the addition of
a poly-A tail. As with other synthetic genetic elements a
synthetic 3' UTR/poly(A) signal has a sequence which differs
from naturally-occurring UTR elements.
The sequence may be modified, for example by the
deletion of ALU repeat sequences. Deletion of such ALU
repeat sequences acts to reduce the possibility of
homologous recombination between the expression cassette and
genomic material in a transfected cell.
The plasmid preferably includes a promoter, a TATA box,
a Cap site and a first intron and intron/exon boundary in
appropriate relationship for expression of the coding
sequence. The plasmid may also include a 5' mRNA leader
sequence inserted between the promoter and the coding
sequence and/or an intron/5' UTR from a chicken skeletal a-
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actin gene. Also, the plasmid may have a nucleotide
sequence which is the same as the nucleotide sequence of
plasmid pIF0921, as shown in Figure 5.
The plasmid may also include: (a) a first transcription
unit containing a first transcriptional control sequence
transcriptionally linked with a first 5'-untranslated
region, a first intron, a first coding sequence, and a first
3'-untranslated region/poly(A) signal, wherein the first
intron is between the control sequence and the first coding
sequence: and (b) a second transcription unit containing a
second transcriptional control sequence transcriptionally
linked with a second 5'-untranslated region, a second
intron, a second coding sequence, and a second 3'-
untranslated region/poly(A) signal, wherein the second
intron is between the control sequence and the second coding
sequence; wherein the first and second coding sequences
contain a sequence having the sequence of SEQ ID N0:2, 3, 4
or 25 coding for a human IL-12 p40 subunit, and a sequence
having the sequence of SEQ ID N0:6, 7, 8 or 24 coding for a
human IL-12 p35 subunit.
The term "transcription unit" or "expression cassette"
refers to a nucleotide sequence which contains at least one
coding sequence along with sequence elements which direct
the initiation and termination of transcription. A
transcription unit may however include additional sequences,
which may include sequences involved in post-transcriptional
or post-translational processes. In preferred embodiments,
the first transcriptional control sequence or the second
transcriptional control sequence contain one or more
cytomegalovirus promoter sequences. The first and second
transcriptional control sequences can be the same or
different.
A "5' untranslated region" or "5' UTR" refers to a
sequence located 3' to promoter region and 5' of the
downstream coding region. Thus, such a sequence, while
transcribed, is upstream of the translation initiation codon
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and therefore is not translated into a portion of the
polypeptide product.
For the plasmids described herein, one or more of a
promoter, 5' untranslated region (5' UTR), the 3'
UTR/poly(A) signal, and introns may be a synthetic sequence.
In this context the term "synthetic" means that the sequence
is not provided directly by the sequence of a naturally
occurring genetic element of that type but rather is an
artificially created sequence (i.e., created by a person by
molecular biological methods). While one or more portions
of such a synthetic sequence may be the same as portions of
naturally occurring sequences, the full sequence over the
specified genetic element is different from a naturally
occurring genetic element of that type. The use of such
synthetic genetic elements allows the functional
characteristics of that element to be appropriately designed
for the desired function.
Thus, a "synthetic intron" refers to a sequence which
is not a naturally occurring intron sequence but which will
be removed from an RNA transcript during normal post
transcriptional processing. Such introns can be designed to
have a variety of different characteristics, in particular
such introns can be designed to have a desired strength of
splice site.
A "subunit" of a therapeutic molecule is a polypeptide
or RNA molecule which combines with one or more other
molecules to form a complex having the relevant
pharmacologic activity. Examples of such complexes include
homodimers and heterodimers as well as complexes having
greater numbers of subunits. A specific example of a
heterodimer is IL-12, having the p40 and p35 subunits.
The "p40 subunit" is the larger of the two subunits of
the IL-12 heterodimer. Thus, it is capable of association
with p35 subunit to form a molecule having activities
characteristic of IL-12. Human p40 has the amino acid
sequence of SEQ ID N0:1. Those skilled in the art will
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recognize that the molecule may have a number of changes
from that sequence, such as deletions, insertions or changes
of one or a few amino acids, while still retaining IL-12
activity when associated with p35. Such active altered
5 molecules are also regarded as p40.
Conversely, the "p35 subunit" is the smaller of the two
heterodimeric subunits of IL-12. For humans, p35 has the
amino acid sequence of SEQ ID N0:5. As for p40, p35 may
have a low level of alterations from that sequence while
10 still being regarded as p35.
A particular example of coding regions suitable for use
in the plasmids of this invention are the natural sequences
coding for the p40 and p35 subunits of human IL-12. Thus,
in a preferred embodiment the first and second coding
regions are coding regions for those sequences and are
preferably in the order p40 then p35 in the 5' to 3'
direction.
Thus, a "sequence coding for the p40 subunit of human
IL-12" is a nucleic acid sequence which encodes the human
p40 subunit as described above, based on the normal base
pairing and translational codon usage relationships. The
sequence coding for p35 subunit of human IL-12 is similarly
defined.
In a preferred embodiment the sequence coding for the
p40 subunit of human IL-12 is 5' to the sequence coding for
the p35 subunit of human IL-12. Those skilled in the art
will appreciate that the interferon alpha, p35 subunit and
p40 subunit may all be on a single transcription unit, that
all three may be on separate transcription units, or that
any two coding sequences may be on one transcription unit
and the other coding sequence on another transcription unit.
The plasmid may also contain an intron having variable
splicing, a first coding sequence, and a second coding
sequence, wherein the first and second coding sequences
include a sequence having the sequence of SEQ ID N0:2, 3, 4
or 25 coding for a human IL-12 p40 subunit, and a sequence
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having the sequence of SEQ ID N0:6, 7, 8 or 24 coding for a
human IL-12 p35 subunit.
In a preferred embodiment, the plasmid also has: (a) a
transcriptional control sequence transcriptionally linked
with a first coding sequence and a second coding sequence;
(b) a 5'-untranslated region; (c) an intron 5' to the first
coding sequence; (d) an alternative splice site 3' to the
first coding sequence and 5' to the second coding sequence;
and (e) a 3'-untranslated region/poly(A) signal. The
transcriptional control sequence preferably includes a
cytomegalovirus promoter sequence.
In a preferred embodiment, the plasmid also has: (a) a
transcriptional control sequence transcriptionally linked
with a first coding sequence, an IRES sequence, a second
coding sequence, and a 3'-untranslated region/poly(A)
signal, wherein the IRES sequence is between the first
coding sequence and the second coding sequence; and (b) an
intron between the promoter and the first coding sequence;
wherein the first and second coding sequences include a
sequence having the sequence of SEQ ID N0:2, 3, 4 or 25
coding for a human IL-12 p40 subunit, and a sequence having
the sequence of SEQ ID N0:6, 7, 8 or 24 coding for a human
IL-12 p35 subunit. The transcriptional control sequence
preferably includes a cytomegalovirus promoter sequence and
the IRES sequence preferably is from an encephalomyocarditis
virus.
For delivery of coding sequences for gene expression,
it is generally useful to provide a delivery composition or
delivery system which includes one or more other components
in addition to the nucleic acid sequences. Such a
composition can, for example, aid in maintaining the
integrity of the DNA and/or in enhancing cellular uptake of
the DNA and/or by acting as an immunogenic enhancer, such as
by the non-DNA components having an immuno-stimulatory
effect of their own.
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Thus, in another aspect, the invention features a
composition containing a plasmid as described above and a
protective, interactive non-condensing compound (PINC).
The PINC enhances the delivery of the nucleic acid
molecule to mammalian cells in vivo, and preferably the
nucleic acid molecule includes a coding sequence for a gene
product to be expressed in the cell. In many cases, the
relevant gene product is a polypeptide or protein.
Preferably the PINC is used under conditions so that the
PINC does not form a gel, or so that no gel form is present
at the time of administration at about 30-40~C. Thus, in
these compositions, the PINC is present at a concentration
of 30~ (w/v) or less. In certain preferred embodiments, the
PINC concentration is still less, for example, 20$ or less,
10$ or less, 5~ or less, or 1~ or less. Thus, these
compositions differ in compound concentration and functional
effect from uses of these or similar compounds in which the
compounds are used at higher concentrations, for example in
the ethylene glycol mediated transfection of plant
protoplasts, or the formation of gels for drug or nucleic
acid delivery. In general, the PINCs are not in gel form in
the conditions in which they are used as PINCs, though
certain of the compounds may form gels under some
conditions.
In connection with the compounds and compositions of
this invention, the term "protects" or "protective" refers
to an effect of the interaction between such a compound and
a nucleic acid such that the rate of degradation of the
nucleic acid is decreased in a particular environment. Such
degradation may be due to a variety of different factors,
which specifically include the enzymatic action of a
nuclease. The protective action may be provided in
different ways, for example, by exclusion of the nuclease
molecules or by exclusion of water.
Some compounds which protect a nucleic acid and/or
prolong the bioavailability of a nucleic acid may also
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interact or associate with the nucleic acid by
intermolecular forces and/or valence bonds such as: Van der
Waals forces, ion-dipole interactions, ion-induced dipole
interactions, hydrogen bonds, or ionic bonds. These
interactions may serve the following functions: (1)
Stereoselectively protect nucleic acids from nucleases by
shielding; (2) facilitate the cellular uptake of nucleic
acid by "piggyback endocytosis". Piggyback endocytosis is
the cellular uptake of a drug or other molecule complexed to
a carrier that may be taken up by endocytosis. CV Uglea and
C Dumitriu-Medvichi, Medical Applications of Synthetic
0ligomers, In: Polymeric Biomaterials_, Severian Dumitriu
ed., Marcel Dekker, Inc., 1993, incorporated herein by
reference.
To achieve the desired effects set forth it is
desirable, but not necessary, that the compounds which
protect the nucleic acid and/or prolong the bioavailability
of a nucleic acid have amphiphilic properties; that is, have
both hydrophilic and hydrophobic regions. The hydrophilic
region of the compounds may associate with the largely ionic
and hydrophilic regions of the nucleic acid, while the
hydrophobic region of the compounds may act to retard
diffusion of nucleic acid and to protect nucleic acid from
nucleases.
Additionally, the hydrophobic region may specifically
interact with cell membranes, possibly facilitating
endocytosis of the compound and thereby also of nucleic acid
associated with the compound. This process may increase the
pericellular concentration of nucleic acid.
Agents which may have amphiphilic properties and are
generally regarded as being pharmaceutically acceptable are
the following: polyvinylpyrrolidones; polyvinylalcohols;
polyvinylacetates; propylene glycol; polyethylene glycols;
poloxamers (Pluronics); poloxamines (Tetronics); ethylene
vinyl acetates; methylcelluloses, hydroxypropylcelluloses,
hydroxypropylmethylcelluloses; heteropolysaccharides
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(pectins); chitosans: phosphatidylcholines (lecithins);
miglyols; polylactic acid: polyhydroxybutyric acid: xanthan
gum. Also, copolymer systems such as polyethylene glycol-
polylactic acid (PEG-PLA), polyethylene glycol-
polyhydroxybutyric acid (PEG-PHB), polyvinylpyrrolidone
polyvinylalcohol (PVP-PVA), and derivatized copolymers such
as copolymers of N-vinyl purine (or pyrimidine) derivatives
and N-vinylpyrrolidone. However, not all of the above
compounds are protective, interactive, non-condensing
compounds as described below.
In connection with the protective, interactive, non-
condensing compounds for these compositions, the term "non-
condensing" means that an associated nucleic acid is not
condensed or collapsed by the interaction with the PINC at
the concentrations used in the compositions. Thus, the
PINCs differ in type and/or use concentration from such
condensing polymers. Examples of commonly used condensing
polymers include polylysine, and cascade polymers (spherical
polycations).
Also in connection with such compounds and an
associated nucleic acid molecule, the term "enhances the
delivery" means that at least in conditions such that the
amounts of PINC and nucleic acid is optimized, a greater
biological effect is obtained than with the delivery of
nucleic acid in saline. Thus, in cases where the expression
of a gene product encoded by the nucleic acid is desired,
the level of expression obtained with the PINC:nucleic acid
composition is greater than the expression obtained with the
same quantity of nucleic acid in saline for delivery by a
method appropriate for the particular PINC/coding sequence
combination.
In preferred embodiments of the above compositions, the
PINC is polyvinyl pyrrolidone (PVP), polyvinyl alcohol
(PVA), a PVP-PVA co-polymer, N-methyl-2-pyrrolidone (NM2P),
ethylene glycol, or propylene glycol. In compositions in
which a Poloxamer (Pluronics) is used, the nucleic acid is
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preferably not a viral vector, i.e., the nucleic acid is a
non-viral vector.
In other preferred embodiments, the PINC is bound with
a targeting ligand. Such targeting ligands can be of a
5 variety of different types, including but not limited to
galactosyl, residues, fucosal residues, mannosyl residues,
carntitine derivatives, monoclonal antibodies, polyclonal
antibodies, peptide ligands, and DNA-binding proteins. The
targeting ligands may bind with receptors on cells such as
10 antigen-presenting cells, hepatocytes, myocytes, epithelial
cells, endothelial cells, and cancer cells.
In connection with the association of a targeting
ligand and a PINC, the term "bound with" means that the
parts have an interaction with each other such that the
15 physical association is thermodynamically favored,
representing at least a local minimum in the free energy
function for that association. Such interaction may involve
covalent binding, or non-covalent interactions such as
ionic, hydrogen bonding, van der Waals interactions,
hydrophobic interactions, and combinations of such
interactions.
While the targeting ligand may be of various types, in
one embodiment the ligand is an antibody. Both monoclonal
antibodies and polyclonal antibodies may be utilized.
The nucleic acid may also be present in various forms.
Preferably the nucleic acid is not associated with a
compounds(s) which alter the physical form, however, in
other embodiments the nucleic acid is condensed (such as
with a condensing polymer), formulated with cationic lipids,
formulated with peptides, or formulated with cationic
polymers.
In preferred embodiments, the protective, interactive
non-condensing compound is polyvinyl pyrrolidone, and/or the
plasmid is in a solution having between 0.5$ and 50~ PVP,
more preferably about 5~ PVP. The DNA preferably is at
least about 80~ supercoiled, more preferably at least about
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90$ supercoiled, and most preferably at least about 95$
supercoiled.
In another aspect the invention features a composition
containing a protective, interactive non-condensing compound
and a plasmid containing an interferon alpha coding
sequence.
In yet another aspect, the invention provides a
composition containing a plasmid of the invention (or a
plasmid containing an interferon alpha coding sequence) and
a cationic lipid with a neutral co-lipid.
Preferably the cationic lipid is DOTMA and the neutral
co-lipid. is cholesterol (chol). DOTMA is 1,2-di-O-
octadecenyl-3-trimethylammonium propane, which is described
and discussed in Eppstein et al., U.S. Patent 9,897,355,
issued January 30, 1990, which is incorporated herein by
reference. However, other lipids and lipid combinations may
be used in other embodiments. A variety of such lipids are
described in Gao & Huang, 1995, Gene Therapy 2:710-722,
which is hereby incorporated by reference.
As the charge ratio of the cationic lipid and the DNA
is also a significant factor, in preferred embodiments the
DNA and the cationic lipid are present is such amounts that
the negative to positive charge ratio is about 1:3. While
preferable, it is not necessary that the ratio be 1:3.
Thus, preferably the charge ratio for the compositions is
between about 1:1 and 1:10, more preferably between about
1:2 and 1:5.
The term "cationic lipid" refers to a lipid which has a
net positive charge at physiological pH, and preferably
carries no negative charges at such pH. An example of such
a lipid is DOTMA. Similarly, "neutral co-lipid" refers to a
lipid which has is usually uncharged at physiological pH.
An example of such a lipid is cholesterol.
Thus, "negative to positive charge ratio" for the DNA
and cationic lipid refers to the ratio between the net
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negative charges on the DNA compared to the net positive
charges on the cationic lipid.
As the form of the DNA affects the expression
efficiency, the DNA preferably is at least about 80$
supercoiled, more preferably at least about 90~ supercoiled,
and most preferably at least about 95~ supercoiled. The
composition preferably includes an isotonic carbohydrate
solution, such as an isotonic carbohydrate solution that
consists essentially of about 10$ lactose. In preferred
embodiments, the composition the cationic lipid and the
neutral co-lipid are prepared as a liposome having an
extrusion size of about 800 nanometers. Preferably the
liposomes are prepared to have an average diameter of
between about 20 and 800 nm, more preferably between about
50 and 400 nm, still more preferably between about 75 and
200 nm, and most preferably about 100 nm. Microfluidization
is the preferred method of preparation of the liposomes.
In another aspect the invention features a composition
containing: (a) a first component having a plasmid including
an interferon alpha coding sequence and a cationic lipid
with a neutral co-lipid, wherein the cationic lipid is DOTMA
and the neutral co-lipid is cholesterol, wherein the DNA in
the plasmid and the cationic lipid are present in amounts
such that the negative to positive charge ratio is about
1:3~ and (b) a second component including a protective,
interactive non-condensing compound, wherein the first
component is present within the second component.
In another aspect, the invention provides a composition
having a protective, interactive non-condensing compound, a
first plasmid including an interferon alpha coding sequence,
and one or more other plasmids independently having an IL-12
p35 or IL-12 p40 subunit coding sequence.
In another aspect, the invention features a method for
making any of the plasmids described above by inserting a
CMV promoter transcriptionally linked with an interferon
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alpha coding sequence, and a growth hormone 3'-untranslated
region into a plasmid.
The invention also provides methods of making the
compositions described above. The method may involve: (a)
preparing a DNA molecule having a transcriptional unit,
wherein the transcriptional unit contains an interferon
alpha coding sequences (b) preparing a protective,
interactive non-condensing compound: and (c) combining the
protective, interactive non-condensing compound with the DNA
in conditions such that a composition capable of delivering
a therapeutically effective amount of an interferon alpha
coding sequence to a mammal is formed.
Preferably, the DNA molecule is a plasmid, wherein the
plasmid includes a CMV promoter transcriptionally linked
with an interferon alpha coding sequence, and a human growth
hormone 3'-untranslated region/poly(A) signal.
The method may involve the steps of: (a) preparing a
DNA having an interferon alpha coding sequence; (b)
preparing a mixture of a cationic lipid and a neutral co-
lipid, wherein the cationic lipid is DOTMA and the neutral
co-lipid is cholesterol; and (c) combining the mixture with
the DNA in amounts such that the cationic lipid and the DNA
are present in a negative to positive charge ratio of about
1:3.
In another embodiment, the method involves the steps
of: (a) preparing a first component having a plasmid
containing an interferon alpha coding sequence and a
cationic lipid with a neutral co-lipid, wherein the cationic
lipid is DOTMA and the neutral co-lipid is cholesterol,
wherein the DNA in the plasmid and the cationic lipid are
present in amounts such that the negative to positive charge
ratio is about 1:3; (b) preparing a second component having
a protective, interactive non-condensing compound: and (c)
combining the first and second components such that the
resulting composition includes the first component within
the second component.
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In another embodiment, the method involves the steps
of: (a) preparing a protective, interactive non-condensing
compound, (b) preparing a first plasmid having an interferon
alpha coding sequence, (c) preparing one or more other
plasmids independently having an IL-12 p35 or IL-12 p40
subunit coding sequence, and (d) combining the protective,
interactive non-condensing compound, the plasmid having the
interferon alpha coding sequence and the other plasmids.
In another aspect, the invention provides a method for
treatment of a mammalian condition or disease, by
administering to a mammal suffering from the condition or
disease a therapeutically effective amount of a plasmid as
described herein.
A "therapeutically effective amount" of a composition
is an amount which is sufficient to cause at least temporary
relief or improvement in a symptom or indication of a
disease or condition. Thus, the amount is also sufficient
to cause a pharmacological effect. The amount of the
composition need not cause permanent improvement or
improvement of all symptoms or indications. A
therapeutically effective amount of a cancer therapeutic
would be one that reduces overall tumor burden in the case
of metastatic disease (i.e., the number of metasteses or
their size) or one that reduces the mass of the tumor in
localized cancers.
The condition or disease preferably is a cancer, such
as epithelial glandular cancer, including adenoma and
adenocarcinoma: squamous and transitional cancer, including
polyp, papilloma, squamous cell and transitional cell
carcinomas connective tissue cancer, including tissue type
positive, sarcoma and other Coma's); hematopoietic and
lymphoreticular cancer, including lymphoma, leukemia and
Hodgkin's diseases neural tissue cancer, including neuroma,
sarcoma, neurofibroma and blastoma; mixed tissues of origin
cancer, including teratoma and teratocarcinoma. Other
cancerous conditions that are applicable to treatment
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include cancer of any of the following: adrenal gland, anus,
bile duct, bladder, brain tumors: adult, breast, cancer of
an unknown primary site, carcinoids of the gastrointestinal
tract, cervix, childhood cancers, colon and rectum,
5 esophagus, gall bladder, head and neck, islet cell and other
pancreatic carcinomas, Kaposi's sarcoma, kidney, leukemia,
liver, lung: non-small cell, lung: small cell, lymphoma:
AIDS-associated, lymphoma: Hodgkin's disease, Lymphomas:
non-Hodgkin's disease, melanoma, mesothelioma, metastatic
10 cancer, multiple myeloma, ovary, ovarian germ cell tumors,
pancreas, parathyroid, penis, pituitary, prostate, sarcomas
of bone and soft tissue, skin, small intestine, stomach,
testis, thymus, thyroid, trophoblastic disease, uterus:
endometrial carcinoma, uterus: uterine sarcomas, vagina, or
15 vulva. The composition preferably is administered by
injection, although the method may also be performed ex
vi vo .
In another aspect, the invention provides a method for
transfection (i.e., the delivery and expression of a gene to
20 cells) of a cell in situ, by contacting the cell with a
plasmid described herein for sufficient time to transfect
the cell. Transfection of the cell preferably is performed
in vivo and the contacting preferably is performed in the
presence of about 5$ PVP solution.
In another aspect, the invention features a method for
delivery and expression of an interferon alpha gene in a
plurality of cells, by: (a) transfecting the plurality of
cells with a plasmid or composition of the inventions and
(b) incubating the plurality of cells under conditions
allowing expression of a nucleic acid sequence in the
vector, wherein the nucleic acid sequence encodes interferon
alpha.
In preferred embodiments, the interferon alpha is human
interferon alpha and the cells are human cells and/or the
contacting is performed in the presence of an about 5~ PVP
solution.
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In another aspect, the invention features a method for
treating a disease or condition, by transfecting a cell in
situ with a plasmid or composition of the invention. The
disease or condition can be a localized disease or condition
or a systemic disease or condition.
In another aspect, the invention features a cell
transfected with a plasmid or composition of the invention.
In yet another aspect, the invention features a method
for treatment of a mammalian condition or disease, by
administering to a mammal suffering from the condition or
disease a therapeutically effective amount of a composition
described herein.
As the compositions are useful for delivery of a
nucleic acid molecule to cells in vivo, in a related aspect
the invention provides a composition at an in vivo site of
administration. In particular this includes at an in vivo
site in a mammal.
In preferred embodiments the nucleic acid molecule
includes a sequence encoding a gene product. Also in
preferred embodiments, the site of administration is in an
interstitial space or a tissue of an animal, particularly of
a mamma 1.
The invention also provides methods for using the above
compositions. Therefore, in further related aspects,
methods of administering the compositions are provided in
which the composition is introduced into a mammal,
preferably into a tissue or an interstitial space.
Various methods of delivery may be utilized, such as
are known in the art, but in preferred embodiments, the
composition is introduced into the tissue or interstitial
space by injection. The compositions may also be delivered
to a variety of different tissues, but in preferred
embodiments the tissue is muscle or tumor.
In another related aspect, the invention provides
methods for treating a mammalian condition or disease by
administering a therapeutically effective amount of a
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composition as described above. In preferred embodiments,
the disease or condition is a cancer.
The summary of the invention described above is non
limiting and other features and advantages of the invention
will be apparent from the following detailed description of
the preferred embodiments, as well as from the claims.
Brief Description Of The DrawincLs
Figure 1 shows the effects of interferon alpha in two
cancer models.
Figure 2 shows a plasmid map and sequence (SEQ ID
N0:18) for an exemplary IL-12 plasmid of the present
invention.
Figure 3 shows optimal codon usage for highly expressed
human genes.
Figure 4 shows a plasmid map and sequence (SEQ ID
N0:19) for plasmid pIF0836, an exemplary interferon alpha
plasmid of the present invention.
Figure 5 shows a plasmid map and sequence (SEQ ID
N0:20) for pIN096, an exemplary IL-I2 plasmid that can be
used with the present invention.
Figure 6 shows the nucleic acid sequence (SEQ ID N0:21)
of plasmid pIF0921, an exemplary interferon alpha plasmid of
the present invention.
Figures 7A and 7B show a plasmid map and sequence (SEQ
ID N0:22) for plasmid pIF0921.
Figure 8 shows an outline of a strategy that can be
used to synthesize a pIF0921 plasmid.
Figure 9 shows interferon alpha and IL-12 gene medicine
(combination therapy) in Renca model.
Detailed Description Of The Preferred Embodiments
The present invention relates to gene delivery and gene
therapy, and provides novel nucleic acid constructs for
expression of interferon alpha in a mammal, formulations for
delivery that incorporate a nucleic acid construct for
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expression, and methods for preparing and using such
constructs and formulations. In particular, this invention
relates to plasmid constructs for delivery of therapeutic
interferon alpha encoding nucleic acids to cells in order to
modulate tumor activity, methods of using those constructs
(including combination therapy with other agents, such as
cytokines, preferably IZ-12), as well as methods for
preparing such constructs.
I. General
As described, this invention concerns expression
systems for the delivery and expression of interferon alpha
coding sequences in mammalian cells, and formulations and
methods for delivering such expression systems or other
expression systems to a mammal.
Therefore, particular genetic constructs are described
which includes nucleotide sequences coding for interferon
alpha, preferably human interferon alpha. Such a construct
can beneficially be formulated and administered as described
herein, utilizing the expression systems of this invention.
To allow convenient production of such plasmids, it is
generally preferable that the plasmid be capable of
replication in a cell to high copy number. Generally such
production is carried out in prokaryotic cells, particularly
including Esherichia coli (E.coli) cells. Thus, the plasmid
preferably contains a replication origin functional in a
prokaryotic cell, and preferably the replication origin is
one which will direct replication to a high copy number.
It is also possible to utilize synthetic genetic
elements in the plasmid constructs.
- As described below, these elements affect post
transcriptional processing in eukaryotic systems. Thus, the
use of synthetic sequences allows the design of processing
characteristics as desired for the particular application.
Commonly, the elements will be designed to provide rapid and
accurate processing.
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For delivery of DNA encoding a desired expression
product to a mammalian system, it is usually preferable to
utilize a delivery system. Such a system can provide
multiple benefits, notably providing stabilization to
protect the integrity of the DNA, as well as assisting in
cellular uptake.
In addition, the non-DNA components of the formulation
may contribute to an immune system enhancement or
activation. As a result, components of a delivery system
can be selected in conjunction with a particular gene
product to enhance or minimize the immuno-stimulatory
effect.
The plasmids may also include elements for expression
of IL-12 or one or more subunits thereof. Similarly, the
treatment may involve administration of an interferon alpha
coding sequence and one or more IL-I2 coding sequences
whether on a single plasmid or on separate plasmids. Such
plasmids may be incorporated into compositions for delivery
with a protective, interactive non-condensing compound, a
cationic lipid and neutral co-lipid, or both.
While these are specific effective examples, other
components may be utilized in formulations containing the
interferon alpha expression vectors of the present invention
to provide effective delivery and expression of interferon
alpha or with other coding sequences for which manipulation
of the activation of immune system components is desirable.
The selection of delivery system components and
preparation methods in conjunction with the selection of
coding sequences provides the ability to balance the
specific effects of the encoded gene products and the immune
system effects of the overall delivery system composition.
This capacity to control the biological effects of delivery
system formulation administration represents an aspect of
the invention in addition to the interferon alpha encoding
constructs and specific formulations of delivery systems.
Co-selection of the encoded gene product and the delivery
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system components and parameters provides enhanced desired
effects rather than merely providing high level gene
expression. In particular, such enhancement is described
below for the antitumor effects of the exemplary PVP
5 containing compositions.
For systems in which IL-12 is also administered, the
antitumor effect can be greater than merely additive (i.e.,
greater than merely the sum of the antitumor effects of
interferon alpha alone and IL-12 alone). Enhancement of
10 immuno-stimulatory effects is also desirable in other
contexts, for example, for vaccine applications.
In contrast, for certain applications, it is preferable
to select a delivery systems which minimizes the immune
system effects. For example, it is often preferred that the
15 immune system activation be minimized for compositions to be
delivered to the lung in order to minimize lung tissue
swelling.
A useful approach for selecting the delivery system
components and preparation techniques for a particular
20 coding sequence can proceed as follows, but is not limited
to these steps or step order.
1. Select a particular genetic construct which
provides appropriate expression in vitro.
2. Select delivery system components based on desired
25 immunostimulatory effects and/or on in vivo
physiological effect. Such effects can be tested
or verified in in vivo model systems.
3. Select the other delivery system parameters
consistent with the desired immuno-stimulatory
effects and/or consistent with enhancing the
desired in vivo physiological effect. Such
parameters can, for example, include the amount
and ratio of DNA to one or more other composition
components, the relative amounts of non-DNA
composition components, the size of delivery
system formulation particles, the percent
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supercoiled DNA for circular dsDNA vectors, and
the specific method of preparation of delivery
system formulation particles. The particular
parameters relevant for specific types of
formulations will be apparent or readily
determined by testing.
The description below illustrates the selection of
components and parameters in the context of interferon alpha
encoding constructs. However, it should be recognized that
the approach is applicable to constructs containing a
variety of other coding sequences.
II. Plasmid Construct Expression Systems
A. Plasmid Design and Construction
For the methods and constructs of this invention, a
number of different plasmids were constructed which are
useful for delivery and expression of sequences encoding
interferon alpha. Thus, these plasmids contain coding
regions for interferon alpha, along with genetic elements
necessary or useful for expression of those coding regions.
While these embodiments utilized interferon alpha cDNA
clones or partial genomic sequences from a particular
source, those skilled in the art could readily obtain
interferon alpha coding sequences from other sources, or
obtain a coding sequence by identifying a cDNA clone in a
library using a probes) based on the published interferon
alpha coding and/or flanking sequences. This also applies
to. the IL-12 coding sequences utilized in the embodiments
described herein.
Coding sequences for interferon alpha were incorporated
into an expression plasmid that contains eukaryotic and
bacterial genetic elements. Eukaryotic genetic elements
include the CMV immediate early promoter and 5' UTR, and a
human growth hormone 3' UTR/poly(a) signal, which influence
gene expression by controlling the accuracy and efficiency
of RNA processing, mRNA stability, and translation.
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The human growth hormone 3' UTR is from a human growth
hormone gene, and preferably includes a poly(a) signal.
This sequence can be linked immediately following the
natural translation termination codon for a cDNA sequence,
genomic sequence, modified genomic sequence, or synthetic
sequence coding for interferon alpha.
An example of a human growth hormone 3' UTR/poly(a)
signal is shown by the human growth hormone 3' UTR
(nucleotides 1 - 100) and 3' flanking sequence (nucleotides
101 - 191; GenBank accession #J03071, HUMGHCSA) below.
(SEQ ID N0:13)
1 GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGT
Poly (a)signal
51 TGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCA
101 TTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTG
151 GTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGC
The 5' and 3' UTR and flanking regions can be further
and more precisely defined by routine methodology, e.g.,
deletion or mutation analysis or their equivalents., and can
be modified to provide other sequences having appropriate
transcriptional and translational functions.
1. Construction of plasmid: Plasmid Backbone, human
interferon alpha cDNA, Final Construct
A diagrammatic representation of the PCR products and
plasmids involved in creation of an exemplary construct is
shown below in Figure 8.
Plasmid pIF0921 was constructed from commercially
available plasmids, and contains the TN5 gene encoding the
kanamycin resistance gene, the pUC origin of replication,
the CMV enhancer and promoter to base +112, a synthetic
intron called IVS8, the human IFN-a2b gene, and the human
growth hormone 3' UTR. The plasmid construction descendancy
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for pIL0697 is shown in Figure 8. pIL0697 was cut with
BamHI and Xba I and the hIFN-a2b PCR product, which had been
amplified from human genomic DNA with BamHI and Xba I ends,
was cloned into the pIL0697 backbone in place of the IL-2
coding region. The resulting plasmid was pIF0863. pIF0863
was cut with Nco I and intron IVS8 from pCT0828 was cloned
in. The resulting plasmid was pIF0890. pIF0890 was cut
with Nde I and Pac I and an additional region of the CMV 5'
UTR to base +112 was cloned in from plasmid pLC0888.
B. Synthetic Genetic Elements
In some embodiments, some or all of the genetic
elements can be synthetic, derived from synthetic
oligonucleotides, and thus are not obtained directly from
natural genetic sequences. These synthetic elements are
appropriate for use in many different expression vectors.
A synthetic intron is designed with splice sites that
ensure that RNA splicing is accurate and efficient. A
synthetic 3' UTR/poly(A) signal is designed to facilitate
mRNA 3' end formation and mRNA stability. A synthetic 5'
UTR is designed to facilitate the initiation of translation.
The design of exemplary synthetic elements is described in
more detail below.
1. Summary of Synthetic Element Features
Exemplary synthetic 5'UTR, intron, and 3'UTR/poly(A)
signal have the general features shown below:
5' UTR Short.
Lack of secondary structure.
Kozak sequence.
Site for intron insertion.
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Intron 5' splice site sequence matches
consensus.
5' splice site sequence is exactly
complementary to 5' end of U1
snRNA.
Branch point sequence matches
consensus.
Branch point sequence is
complementary to U2 snRNA.
3' splice site matches consensus.
Polypyrimidine tract is 16 bases in
length and contains 7 consecutive
T's. (The tract preferably
contains at least 5 consecutive
T's. )
Contains internal restriction
enzyme sites.
BbsI cleaves at the 5'ss, Earl
cleaves at the 3'ss.
3' UTR/Poly(A) Based on rabbit (3-globin 3'
UTR/poly(A) signal.
Consists of two poly(A) signals in
tandem.
2. Features of the Synthetic 5'UTR (UT6):
The 5' untranslated region (5'UTR) influences the
translational efficiency of messenger RNA, and is therefore
an important determinant of eukaryotic gene expression. The
synthetic 5'UTR sequence (UT6) has been designed to
maximize the translational efficiency of mRNAs encoded by
vectors that express genes of therapeutic interest.
The sequence of the synthetic 5' UTR (UT6) is shown
below. The Kozak sequence is in boldface and the initiation
codon is double underlined. The location of the intron
(between residues 48 and 49) is indicated by the filled
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triangle and the sequences that form the exonic portion of
consensus splice sites are single underlined. The
restriction sites for HindIII and NcoI are overlined. (SEQ
ID N0:14)
5
HindIII O NcoI
AAGCTTACTCAACACAATAACAAACTTACTTACAATCTTAATTAACAGGCCACCATGG
The 5' untranslated region (5' UTR), located between
10 the cap site and initiation codon, is known to influence the
efficiency of mRNA translation. Any features that influence
the accessibility of the 5' cap structure to initiation
factors, the binding and subsequent migration of the 43S
preinitiation complex, or the recognition of the initiation
15 codon, will influence mRNA translatability. An efficient 5'
UTR is expected to be one that is moderate in length, devoid
of secondary structure, devoid of upstream initiation
codons, and has an AUG within an optimal local context
(Kozak, 1994, Biochimie 76:815-821; Jansen et al., 1994). A
20 5' UTR with these characteristics should allow efficient
recognition of the 5' cap structure, followed by rapid and
unimpeded ribosome scanning by the ribosome, thereby
facilitating the translation initiation process.
The sequence of the synthetic 5'UTR was designed to be
25 moderate in length (54 nucleotides (nts)), to be deficient
in G but rich in C and A residues, to lack an upstream ATG,
to place the intended ATG within the context of a optimal
Kozak sequence (CCACCATGG), and to lack potential secondary
structure. The synthetic 5' UTR sequence was also designed
30 to lack AU-rich sequences that target mRNAs to be rapidly
degraded in the cytoplasm.
Experiments have demonstrated that introns increase
gene expression from cDNA vectors, and that introns located
in the 5' UTR are more effective than ones located in the 3'
UTR (Huang and Gorman, 1990, Mol. Cell. Biol. 10:1805-1810;
Evans and Scarpulla, 1989, Gene 84:135-142; Brinster et al.,
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1988, Proc. Natl. Acad. Sci. USA 85:836-840; Palmiter et
al., 1991, Proc. Natl. Acad. Sci. USA 88:478-482: Choi et
al., 1991, Mol. Cell. Biol. 11:3070-3074). Accordingly, the
synthetic 5' UTR sequence was designed to accommodate an
intron with consensus splice site sequences. The intron
may, for example, be located between residues 48 and 49 (See
intron sequence structure below). The CAG at position 46-48
is the exonic portion of a consensus 5' splice site. The G
at position 49 is the exonic portion of a consensus 3'
splice site. .
To facilitate cloning manipulations, the synthetic 5'
UTR sequence was designed to begin with a HindIII site and
terminate with a NcoI site.
3. Features of the Synthetic Intron
RNA splicing is required for the expression of most
eukaryotic genes. For optimal gene expression, RNA splicing
must be highly efficient and accurate. A synthetic intron,
termed OPTIVS8B, was designed to be maximally efficient and
accurate.
The structure of the exemplary synthetic intron,
OPTIVS8 is shown below. Sequences for the 5' splice site
(5'ss), branch point (bp), and 3' splice site (3'ss) are
double underlined. The recognition sequences for the
restriction enzymes BbsI and Earl are overlined. The
cleavage site for BbsI corresponds to the 5'ss, and the
cleavage site for Earl corresponds to the 3'ss.
5'ss by 3'ss
I BbsI I Earl I
5'CAG GTAAGTGTCTTC---(77)---TACTAACGGTTCTTTTTTTCTCTTCACAG G 3'
(SEQ ID N0.15) (SEQ ID N0.16)
The 5' splice site (5'ss) sequence matches the
established consensus sequence, MAG 0 GTRAGT, where M = C or
A, and R = G or A. Since the mechanism of splicing involves
an interaction between the 5'ss of the pre-mRNA and U1
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snRNA, the 5'ss sequence of OPTIVSBB was chosen to be
exactly complementary to the 5' end of U1 snRNA.
5'ss 5' CAGGUAAGU 3'
IIIIIIIII
U1 RNA 3' GUCCAUUCA 5'
In mammals, the consensus sequence for branch points
(YNYTRAY, where Y - C or T, R = A or G, N - any base, and
the underlined A residue is the actual branch point) is very
ambiguous. Since the mechanism of splicing involves an
interaction between the branch point (bp) of the pre-mRNA
and U2 snRNA, the branch point sequence of OPTIVSBB was
chosen to maximize this interaction. (Note that the branch
point itself is bulged out). The chosen sequence also
matches the branch point sequence that is known to be
obligatory for pre-mRNA splicing in yeast. The branch point
is typically located 18-38 nts upstream of the 3' splice
site. In OPTIVSBB, the branch point is located 24 nts
upstream from the 3' splice site.
BP 5' UACUAAC 3'
11111 I
U2 RNA 3' AUGAU G 5'
The sequence of the 3' splice site (3'ss) matches the
established consensus sequence, Y11NYAG ~~ G, where Y - C or
T, and N = any base. In 3' splice sites, the polypyrimidine
tract (Y11) is the major determinant of splice site strength.
For optimal splice site function in OPTIVSBB, the length of
the polypyrimidine tract was extended to 16 bases, and its
sequence was adjusted to contain 7 consecutive T residues.
This feature was included because Roscigno et al. (1993)
demonstrated that optimal splicing requires the presence of
at least S consecutive T residues in the polypyrimidine
tract.
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Splicing in vitro is generally optimal when introns are
>80 nts in length (Wieringa, et al., 1984; Ulfendahl et al.,
1985, Nucl. Acids Res. 13:6299-6315). Although many introns
may be thousands of bases in length, most naturally
occurring introns are 90-200 nt in length (Hawkins, 1988,
Nucl. Acids Res. 16:9893-9908). The length of the
synthetic intron (118 nts) falls within this latter range.
OPTIVSBB was designed with three internal restriction
enzyme sites, BbsI, NheI, and Earl. These restriction sites
facilitate the screening and identification of genes that
contain the synthetic intron sequence. In addition, the
BbsI and Earl sites were placed so that their cleavage sites
exactly correspond to the 5'ss (BbsI) or 3'ss (Earl). The
sequence of the polypyrimidine tract was specifically
designed to accommodate the Earl restriction site.
Inclusion of the BbsI and Earl sites at these locations is
useful because they permit the intron to be precisely
deleted from a gene. They also permit the generation of an
"intron cassette" that can be inserted at other locations
within a gene.
The 77 bases between the BbsI site and the branch point
sequence are random in sequence, except for the inclusion of
the NheI restriction site.
4. Features of the Synthetic 3' UTR/poly(A)
Signal:
The 3' ends of eukaryotic mRNAs are formed by the
process of polyadenylation. This process involves site
specific site RNA cleavage, followed by addition of a
poly(A) tail. RNAs that lack a poly(A) tail are highly
unstable. Thus, the efficiency of cleavage/polyadenylation
is a major determinant of mRNA levels, and thereby, of gene
expression levels. 2XPA1 is a synthetic sequence, containing
two efficient poly(A) signals, that is designed to be
maximally effective in polyadenylation.
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A poly(A) signal is required for the formation of the
3' end of most eukaryotic mRNA. The signal directs two RNA
processing reactions: site-specific endonucleolytic cleavage
of the RNA transcript, and stepwise addition of adenylates
(approximately 250) to the newly generated 3' end to form
the poly(A) tail. A poly(A) signal has three parts:
hexanucleotide, cleavage site, and downstream element. The
hexanucleotide is typically AAUAAA and cleavage sites are
most frequently 3' to the dinucleotide CA (Sheets et al.,
1987). Downstream elements are required for optimal poly(A)
signal function and are located downstream of the cleavage
site. The sequence requirement for downstream elements is
not yet fully established, but is generally viewed as UG- or
U-rich sequences (Wickens, 1990; Proudfoot, 1991, Cell
64:671-674; Wahle, 1992, Bioessays 14:113-118: Chen and
Nordstrom, 1992, Nucl. Acids Res. 20:2565-2572).
Naturally occurring poly(A) signals are highly variable
in their effectiveness (Peterson, 1992). The effectiveness
of a particular poly(A) signal is mostly determined by the
quality of the downstream element. (Wahle, 1992). In
expression vectors designed to express genes of therapeutic
interest, it is important to have a poly(A) signal that is
as efficient as possible.
Poly(A) efficiency is important for gene expression,
because transcripts that fail to be cleaved and
polyadenylated are rapidly degraded in the nuclear
compartment. In fact, the efficiency of polyadenylation in
living cells is difficult to measure, since
nonpolyadenylated RNAs are so unstable. In addition to
being required for mRNA stability, poly(A) tails contribute
to the translatability of mRNA, and may influence other RNA
processing reactions such as splicing or RNA transport
((Jackson and Standart,1990, Cell 62:15-24: Wahle, 1992).
Some eukaryotic genes have more than one poly(A) site,
implying that if the cleavage/polyadenylation reaction fails
to occur at the first site, it will occur at one of the
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later sites. In COS cell transfection experiments, a gene
with two strong poly(A) sites yielded approximately two-fold
more mRNA than one with a single strong poly(A) site
(Bordonaro, 1995). These data suggest that a significant
5 fraction of transcripts remain unprocessed even with a
single "efficient" poly(A) signal. Thus, it may be
preferable to include more than one poly(A) site.
The sequence of the exemplary synthetic poly(A) signal
is shown below. The sequence is named 2XPA. The
10 hexanucleotide sequences and downstream element sequences
are double underlined, and the two poly(A) sites are labeled
as pA#1 and pA#2. Convenient restriction sites are
overlined. The entire 2XPA unit may be transferred in
cloning experiments as a XbaI-KpnI fragment. Deletion of
15 the internal BspHI fragment results in the formation of a
1XPA unit. (SEQ ID N0. 17)
XbaI BspHI
TCTAGAGCATTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACG
20 pA#1
Hex I Downstream element
TCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACT
BspHI
25 CGGTACTAGAGCATTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCT
pA#2
Hex I Downstream element
GACGTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCT
30 KpnI
CACTCGGTACC
The sequence of the synthetic poly(A) site shown above
is based on the sequence of the rabbit 0-globin poly(A)
35 signal, a signal that has been characterized in the
literature as strong (Gil and Proudfoot, 1987, Cell 49:399-
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406; Gil. and Proudfoot, 1984, Nature 312:473-474). One of
its key features is the structure of its downstream element,
which contains both UG- and U-rich domains.
A double-stranded DNA sequence corresponding to the
1XPA sequence was constructed from synthetic
oligonucleotides. Two copies of the 1XPA sequence were then
joined to form the 2XPA sequence. The sequences were joined
in such as way as to have a unique XbaI site at the 5' end
of the first poly(A) signal containing fragment, and a
unique KpnI site at the 3' end of the second poly (A) signal
containing fragment.
C. Interferon Alpha and IL-12 Codinct Seguences
The nucleotide sequence of a natural human interferon
alpha coding sequences is known, and is provided below,
along with a synthetic sequence which also codes for human
interferon alpha. The same applies with respect to the
IL-12 coding sequences.
In some cases, instead of the natural sequence coding
for interferon alpha, it is advantageous to utilize
synthetic sequences which encode interferon alpha. Such
synthetic sequences have alternate codon usage from the
natural sequence, and thus have dramatically different
nucleotide sequences from the .natural sequence. In
particular, synthetic sequences can be used which have codon
usage at least partially optimized for expression in a
human. The natural sequences do not have such optimal codon
usage. Preferably, substantially all the codons are
optimized.
Optimal codon usage in humans is indicated by codon
usage frequencies for highly expressed human genes, as shown
in Fig. 3. The codon usage chart is from the program
"Human High. cod" from the Wisconsin Sequence Analysis
Package, Version 8.1, Genetics Computer Group, Madison, WI.
The codons which are most frequently used in highly
expressed human genes are presumptively the optimal codons
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for expression in human host cells, and thus form the basis
for constructing a synthetic coding sequence. An example of
a synthetic interferon alpha coding sequence is shown as the
bottom sequence in the table below.
However, rather than a sequence having fully optimized
codon usage, it may be desirable to utilize an interferon
alpha encoding sequence which has optimized codon usage
except in areas where the same amino acid is too close
together or abundant to make uniform codon usage optimal.
In addition, other synthetic sequences can be used
which have substantial portions of the codon usage
optimized, for example, with at least 50$, 70~, 80g or 90$
optimized codons as compared to a natural coding sequence.
Other particular synthetic sequences for interferon alpha
can be selected by reference to the codon usage chart in
Fig. 3. A sequence is selected by choosing a codon for each
of the amino acids of the polypeptide sequences. DNA
molecules corresponding to each of the polypeptides can then
by constructed by routine chemical synthesis methods. For
example, shorter oligonucleotides can be synthesized, and
then ligated in the appropriate relationships to construct
the full-length coding sequences.
The following sequences are provided in the sequence
listing herein: interferon alpha amino acid sequence, SEQ
ID N0:9~ interferon alpha wild type nucleic acid sequence,
SEQ ID NO:10; interferon alpha synthetic nucleic acid
sequence with optimized codon usage, SEQ ID N0:11;
interferon alpha nucleic acid sequence with additional/semi-
optimized codon usage, SEQ ID N0:12; IL-12 p40 subunit amino
acid sequence, SEQ ID N0:1; IL-12 p40 wild type nucleic acid
sequence, SEQ ID N0:2; IL-12 p40 synthetic nucleic acid
sequence with all codons optimized, SEQ ID N0:3: IL-12 p40
subunit nucleic acid sequence with all codons optimized
except when same nucleic acids were too close/abundant, SEQ
ID N0:4; IL-12 p35 amino acid sequence, SEQ ID N0:5; IL-12
p35 wild type nucleic acid sequence, SEQ ID N0:6; IL-12 p35
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synthetic nucleic acid sequence with all codons optimized,
SEQ ID N0:7; IL-12 p35 subunit nucleic acid sequence with
all codons optimized except when same nucleic acids were too
close/abundant, SEQ ID N0:8. Those skilled in the art will
realize that various nucleic acid sequences with optimized
codon usage can be constructed, for example based on the
various combinations shown below, wherein optimal usage for
each codon is shown below the IL-12 p35 and p40 subunit wild
type sequences and the interferon alpha wild type sequence.
Sequences Encoding Human IL-12 p35
First line = natural sequence (SEQ ID NO. 6)
Second line = all codons optimized (SEQ ID N0. 7)
Third line - all codons optimized except when same
nucleic acids were too close/abundant (changes between
second and third lines bolded) (SEQ ID N0. 8)
ATG TGT CCA GCG CGC AGC CTC CTC CTT GTG GCT ACC CTG GTC CTC CTG GAC CAC CTC
ACT
ATG TGC CCC GCC CGC AGC CTG CTG CTG GTG GCC ACC CTG GTG CTG CTG GAC CAC CTG
AGC
ATG TGC CCC GCC CGC AGC CTG CTG CTC GTG GCC ACC CTG GTG CTC CTG GAC CAC CTC
AGC
TTG GCC AGA AAC CTC CCC GTG GCC ACT CCA GAC CCA GGA ATG TTC CCA TGC CTT CAC
CAC
CTG GCC CGC AAC CTG CCC GTG GCC ACC CCC GAC CCC GGC ATG TTC CCC TGC CTG CAC
CAC
CTG GCC CGC AAC CTC CCC GTG GCC ACC CCA GAC CCC GGC ATG TTC CCA TGC CTG CAC
CAC
2 5 TCC CAA AAC CTG CTG AGG GCC GTC AGC AAC ATG CTC CAG AAG GCC AGA CAA ACT
CTA GAA
AGC CAG AAC CTG CTG GCG GCC GTG AGC AAC ATG CTG CAG AAG GCC GCG CAG ACC CTG
GAG
AGC CAG AAC CTG CTG GCG GCC GTG AGC AAC ATG CTG CAG AAG GCC GCG CAG ACC CTG
GAG
TTT TAC CCT TGC ACT TCT GAA GAG ATT GAT CAT GAA GAT ATC ACA AAA GAT AAA ACC
AGC
3 O TTC TAC CCC TGC ACC AGC GAG GAG ATC GAC CAC GAG GAC ATC ACC AAG GAC AAG
ACC AGC
TTC TAC CCC TGC ACC AGC GAG GAG ATC GAC CAC GAG GAC ATC ACC AAG GAC AAG ACC
AGC
ACA GTG GAG GCC TGT TTA CCA TTG GAA TTA ACC AAG AAT GAG AGT TGC CTA AAT TCC
AGA
ACC GTG GAG GCC TGC CTG CCC CTG GAG CTG ACC AAG AAC GAG AGC TGC CTG AAC AGC
CGC
3 5 ACC GTG GAG GCC TGC CTG CCC CTC GAG TTA ACC AAG AAC GAG AGC TGC CTC AAC
AGC CGC
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GAG ACC TCT TTC ATA ACT AAT GGG AGT TGC CTG GCC TCC AGA AAG ACC TCT TTT ATG
ATG
GAG ACC AGC TTC ATC ACC AAC GGC AGC TGC CTG GCC AGC CGC AAG ACC AGC TTC ATG
ATG
GAG ACC TCC TTC ATC ACC AAC GGC ACT TGC CTG GCC TCC CGC AAG ACC AGC TTC ATG
ATG
GCC CTG TGC CTT AGT AGT ATT TAT GAA GAC TTG AAG ATG TAC CAG GTG GAG TTC AAG
ACC
GCC CTG TGC CTG AGC AGC ATC TAC GAG GAC CTG AAG ATG TAC CAG GTG GAG TTC AAG
ACC
GCC CTG TGC CTG AGC TCC ATC TAC GAG GAC CTG AAG ATG TAC CAG GTG GAG TTC AAG
ACC
Z O ATG AAT GCA AAG CTT CTG ATG GAT CCT AAG AGG CAG ATC TTT CTA GAT CAA AAC
ATG CTG
ATG AAC GCC AAG CTG CTG ATG GAC CCC AAG CTC CAG ATC TTC CTG GAC CAG AAC ATG
CTG
ATG AAC GCC AAG CTC CTG ATG GAC CCC AAG CTC CAG ATC TTC CTG GAC CAG AAC ATG
CTG
GCA GTT ATT GAT GAG CTG ATG CAG GCC CTG AAT TTC AAC AGT GAG ACT GTG CCA CAA
AAA
Z 5 GCC GTG ATC GAC GAG CTG ATG CAG GCC CTG AAC TTC AAC AGC GAG ACC GTG CCC
CAG AAG
GCC GTG ATC GAC GAG CTG ATG CAG GCC CTG AAC TTC AAC AGC GAG ACC GTG CCC CAG
AAG
TCC TCC CTT GAA GAA CCG GAT TTT TAT AAA ACT AAA ATC AAG CTC TGC ATA CTT CTT
CAT
AGC AGC CTG GAG GAG CCC GAC TTC TAC AAG ACC AAG ATC AAG CTG TGC ATC CTG CTG
CAC
2 O AGC AGC CTG GAG GAG CCC GAC TTC TAC AAG ACC AAG ATC AAG CTG TGC ATC CTG
CTG CAC
GCT TTC AGA ATT CGG GCA GTG ACT ATT GAC AGA GTG ACG AGC TAT CTG AAT GCT TCC
TAA
GCC TTC CGC ATC CGC GCC GTG ACC ATC GAC CGC GTG ACC AGC TAC CTG AAC GCC ACC
TGA
GCC TTC CGC ATC CGG GCC GTG ACC ATC GAC CGC GTG ACC AGC TAC CTG AAC GCC ACG
TGA
25 Additional Optimized Seguences Coding For IL-12 35 Subunit
(Second Line = SEQ ID N0:24)
20
Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu Asp His Leu
Ser
ATG TGY CCN GCN MGN WSN YTN YTN YTN GTN GCN ACN YTN GTN YTN YTN GAY CAY YTN
WSN
3 0 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
ATG TGT CCT GCT CGT TCT TTA TTA TTA GTT GCT ACT TTA GTT TTA TTA GAT CAT TTA
TCT
TGC CCC GCC CGC TCC TTG TTG TTG GTC GCC ACC TTG GTC TTG TTG GAC CAC TTG TCC
CCA GCA CGA TCA CTT CTT CTT GTA GCA ACA CTT GTA CTT CTT CTT TCA
CCG GCG CGG TCG CTC CTC CTC GTG GCG ACG CTC GTG CTC CTC CTC TCG
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AGA AGT CTA CTA CTA CTA CTA CTA CTA AGT
AGG AGC CTG CTG CTG CTG CTG CTG CTG AGC
30 40
5 Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His
His
YTN GCN MGN AAY YTN CCN GTN GCN ACN CCN GAY CCN GGN ATG TTY CCN TGY YTN CAY
CAY
TTA GCT CGT AAT TTA CCT GTT GCT ACT CCT GAT CCT GGT ATG TTT CCT TGT TTA CAT
CAT
TTG GCC CGC AAC TTG CCC GTC GCC ACC CCC GAC CCC GGC TTC CCC TGC TTG CAC CAC
Z O CTT GCA CGA CTT CCA GTA GCA ACA CCA CCA GGA CCA CTT
CTC GCG CGG CTC CCG GTG GCG ACG CCG CCG GGG CCG CTC
CTA AGA CTA CTA
CTG AGG CTG CTG
1 S 50 60
Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu
Glu
WSN CAR AAY YTN YTN MGN GCN GTN WSN AAY ATG YTN CAR AAR GCN MGN CAR ACN YTN
GAR
TCT CAA AAT TTA TTA CGT GCT GTT TCT AAT ATG TTA CAA AAA GCT CGT CAA ACT TTA
GAA
2 O TCC TTG
CAG AAC CAG
TTG TTG AAG
CGC GCC GCC
GTC TCC CGC
AAC CAG
ACC
TTG
GAG
TCA CTT CTT CGA GCA GTA TCA CTT GCA CGA ACA CTT
TCG CTC CTC CGG GCG GTG TCG CTC GCG CGG ACG CTC
AGT CTA CTA AGA AGT CTA AGA CTA
AGC CTG CTG AGG AGC CTG AGG CTG
25
so
Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
Ser
TTY TAY CCN TGY ACN WSN GAR GAR ATH GAY CAY GAR GAY ATH ACN AAR GAY AAR ACN
WSN
3 O TTT TAT CCT TGT ACT TCT GAA GAA ATT GAT CAT GAA GAT ATT ACT AAA GAT AAA
ACT TCT
TTC TAC CCC TGC ACC TCC GAG GAG ATC GAC CAC GAG GAC ATC ACC AAG GAC AAG ACC
TCC
CCA ACA TCA ATA ATA ACA ACA TCA
CCG ACG TCG ACG ACG TCG
AGT AGT
3 S AGC AGC
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90 100
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser
Arg
ACN GTN GAR GCN TGY YTN CCN YTN GAR YTN ACN AAR AAY GAR WSN TGY YTN AAY WSN
MGN
ACT GTT GAA GCT TGT TTA CCT TTA AAT TCT
TTA GAA ACT CGT
AAA
AAT
GAA
TCT
TGT
TTA
ACC GTC GAG GCC TGC TTG CCC TTG AAG AAC GAG TCC AAC TCC
TTG GAG ACC TGC TTG CGC
ACA GTA GCA CTT CCA CTT CTT TCA CTT TCA CGA
ACA
ACG GTG GCG CTC CCG CTC CTC TCG CTC TCG CGG
ACG
CTA CTA CTA AGT CTA AGT AGA
1 O CTG CTG CTG AGC CTG AGC AGG
110 120
Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met
Met
GAR ACN WSN TTY ATH ACN AAY GGN WSN TGY YTN GCN WSN MGN AAR ACN WSN TTY ATG
ATG
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
GAA ACT TCT TTT ATT ACT AAT GGT TCT TGT TTA GCT TCT CGT AAA ACT TCT TTT ATG
ATG
GAG ACC TCC TTC ATC ACC AAC TGC GCC CGC ACC TCC TTC
GGC TCC TTG TCC AAG
ACA TCA ATA ACA GGA TCA CTT GCA CGA ACA TCA
TCA
ACG TCG ACG GGG TCG CTC GCG CGG ACG TCG
TCG
2 O AGT AGT CTA AGT AGA AGT
AGC AGC CTG AGC AGG AGC
130 140
Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys
Thr
2 5 GCN YTN TGY YTN WSN WSN ATH TAY GAR GAY YTN AAR ATG TAY CAR GTN GAR TTY
AAR ACN
GCT TTA TGT TTA TCT TCT ATT TAT GAA GAT TTA AAA ATG TAT CAA GTT GAA TTT AAA
ACT
GCC TTG TGC TTG TCC TCC ATC TAC GAG GAC TTG AAG TAC CAG GTC GAG TTC AAG ACC
GCA CTT CTT TCA TCA ATA CTT GTA ACA
3 O GCG CTC CTC TCG TCG CTC GTG ACG
CTA CTA AGT AGT CTA
CTG CTG AGC AGC CTG
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150 160
Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
Leu
ATG AAY GCN AAR YTN YTN ATG GAY CCN AAR MGN CAR ATH TTY YTN GAY CAR AAY ATG
YTN
S ATG AAT GCT AAA TTA TTA ATG GAT CCT AAA CGT CAA ATT TTT TTA GAT CAA AAT ATG
TTA
AAC GCC AAG TTG TTG GAC CCC AAG CGC CAG ATC TTC TTG GAC CAG AAC TTG
GCA CTT CTT CCA CGA ATA CTT CTT
GCG CTC CTC CCG CGG CTC CTC
CTA CTA AGA CTA CTA
Z O CTG CTG AGG CTG CTG
170 180
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln
Lys
GCN GTN ATH GAY GAR YTN ATG CAR GCN YTN AAY TTY AAY WSN GAR ACN GTN CCN CAR
AAR
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
GCT GTT ATT GAT GAA TTA ATG CAA GCT TTA AAT TTT AAT TCT GAA ACT GTT CCT CAA
AAA
GCC GTC ATC GAC GAG TTG CAG GCC TTG AAC TTC AAC TCC GAG ACC GTC CCC CAG AAG
GCA GTA ATA CTT GCA CTT TCA ACA GTA CCA
GCG GTG CTC GCG CTC TCG ACG GTG CCG
2 O CTA CTA AGT
CTG CTG AGC
190 200
Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu
His
2 5 WSN WSN YTN GAR GAR CCN GAY TTY TAY AAR ACN AAR ATH AAR YTN TGY ATH YTN
YTN CAY
TCT TCT TTA GAA GAA CCT GAT TTT TAT AAA ACT AAA ATT AAA TTA TGT ATT TTA TTA
CAT
TCC TCC TTG GAG GAG CCC GAC TTC TAC AAG ACC AAG ATC AAG TTG TGC ATC TTG TTG
CAC
TCA TCA CTT CCA ACA ATA CTT ATA CTT CTT
3 O TCG TCG CTC CCG ACG CTC CTC CTC
AGT AGT CTA CTA CTA CTA
AGC AGC CTG CTG CTG CTG
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210 220
Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser
***
GCN TTY MGN ATH MGN GCN GTN ACN ATH GAY MGN GTN ACN WSN TAY YTN AAY GCN WSN
TRR
GCT TTT CGT ATT CGT GCT GTT ACT ATT GAT CGT GTT ACT TCT TAT TTA AAT GCT TCT
TAA
GCC TTC CGC ATC CGC GCC GTC ACC ATC GAC CGC GTC ACC TCC TAC TTG AAC GCC TCC
TAG
GCA CGA ATA CGA GCA GTA ACA ATA CGA GTA ACA TCA CTT GCA TCA TGA
GCG CGG CGG GCG GTG ACG CGG GTG ACG TCG CTC GCG TCG
AGA AGA AGA AGT CTA AGT
1 O AGG AGG AGG AGC CTG AGC
Sequences Encoding Human IZ-12 p40
First line = natural sequence (SEQ ID N0. 2)
Second line = all codons optimized (SEQ ID N0. 3)
Third line - all codons optimized except when same
nucleic acids were too close/abundant (changes between
second and third lines bolded) (SEQ ID N0. 4)
ATG TGT CAC CAG CAG TTG GTC ATC TCT TGG TTT TCC CTG GTT TTT CTG GCA TCT CCC
CTC
ATG TGC CAC CAG CAG CTG GTG ATC AGC TGG TTC AGC CTG GTG TTC CTG GCC AGC CCC
CTG
ATG TGC CAC CAG CAG CTG GTG ATC AGC TGG TTC TCC CTG GTG TTT CTG GCC AGC CCC
CTC
GTG GCC ATA TGG GAA CTG AAG AAA GAT GTT TAT GTC GTA GAA TTG GAT TGG TAT CCG
GAT
GTG GCC ATC TGG GAG CTG AAG AAG GAC GTG TAC GTG GTG GAG CTG GAC TGG TAC CCC
GAC
GTG GCC ATC TGG GAG CTG AAG AAA GAC GTG TAC GTG GTC GAG CTG GAC TGG TAC CCC
GAC
2 5 GCC CCT GGA GAA ATG GTG GTC CTC ACC TGT GAC ACC CCT GAA GAA GAT GGT ATC
ACC TGG
GCC CCC GGC GAG ATG GTG GTG CTG ACC TGC GAC ACC CCC GAG GAG GAC GGC ATC ACC
TGG
GCC CCC GGC GAG ATG GTG GTC CTG ACC TGC GAC ACC CCC GAG GAA GAC GGC ATC ACC
TGG
ACC TTG GAC CAG AGC AGT GAG GTC TTA GGC TCT GGC AAA ACC CTG ACC ATC CAA GTC
AAA
3 O ACC CTG GAC CAG AGC AGC GAG GTG CTG GGC AGC GGC AAG ACC CTG ACC ATC CAG
GTG AAG
ACC CTG GAC CAG AGC AGT GAG GTG CTG GGC TCC GGC AAG ACC CTG ACC ATC CAG GTG
AAG
GAG TTT GGA GAT GCT GGC CAG TAC ACC TGT CAC AAA GGA GGC GAG GTT CTA AGC CAT
TCG
GAG TTC GGC GAC GCC GGC CAG TAC ACC TGC CAC AAG GGC GGC GAG GTG CTG AGC CAC
AGC
3 5 GAG TTC GGC GAC GCC GGC CAG TAC ACC TGC CAC AAG GGA GGC GAG GTG CTG AGC
CAC TCC
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CTC CTG CTG CTT CAC AAA AAG GAA GAT GGA ATT TGG TCC ACT GAT ATT TTA AAG GAC
CAG
CTG CTG CTG CTG CAC AAG AAG GAG GAC GGC ATC TGG AGC ACC GAC ATC CTG AAG GAC
CAG
CTC CTG CTG CTC CAC AAA AAG GAG GAC GGC ATC TGG AGC ACC GAC ATC CTG AAG GAC
CAG
AAA GAA CCC AAA AAT AAG ACC TTT CTA AGA TGC GAG GCC AAG AAT TAT TCT GGA CGT
TTC
AAG GAG CCC AAG AAC AAG ACC TTC CTG CGC TGC GAG GCC AAG AAC TAC AGC GGC CGC
TTC
AAG GAG CCC AAG AAC AAG ACC TTC CTG CGC TGC GAG GCC AAG AAC TAC AGC GGC CGC
TTC
Z O ACC TGC TGG TGG CTG ACG ACA ATC AGT ACT GAT TTG ACA TTC AGT GTC AAA AGC
AGC AGA
ACC TGC TGG TGG CTG ACC ACC ATC AGC ACC GAC CTG ACC TTC AGC GTG AAG AGC AGC
AGG
ACC TGC TGG TGG CTG ACC ACG ATC AGC ACC GAC CTG ACC TTC AGT GTG AAG AGC AGC
AGG
GGC TCT TCT GAC CCC CAA GGG GTG ACG TGC GGA GCT GCT ACA CTC TCT GCA GAG AGA
GTC
Z 5 GGC AGC AGC GAC CCC CAG GGC GTG ACC TGC GGC GCC GCC ACC CTG AGC GCC GAG
CGC GTG
GGC TCC AGC GAC CCC CAG GGC GTG ACC TGC GGC GCT GCC ACC CTG AGC GCC GAG CGC
GTG
AGA GGG GAC AAC AAG GAG TAT GAG TAC TCA GTG GAG TGC CAG GAG GAC AGT GCC TGC
CCA
CGC GGC GAC AAC AAG GAG TAC GAG TAC AGC GTG GAG TGC CAG GAG GAC AGC GCC TGC
CCC
2 O CGC GGC GAC AAC AAG GAG TAC GAG TAC AGC GTG GAG TGC CAG GAA GAC TCC GCC
TGC CCC
GCT GCT GAG GAG AGT CTG CCC ATT GAG GTC ATG GTG GAT GCC GTT CAC AAG CTC AAG
TAT
GCC GCC GAG GAG AGC CTG CCC ATC GAG GTG ATG GTG GAC GCC GTC CAC AAG CTG AAG
TAC
GCC GCT GAG GAG AGC CTG CCC ATC GAG GTG ATG GTG GAC GCC GTT CAC AAG CTG AAG
TAC
GAA AAC TAC ACC AGC AGC TTC TTC ATC AGG GAC ATC ATC AAA CCT GAC CCA CCC AAG
AAC
GAG AAC TAC ACC AGC AGC TTC TTC ATC CGC GAC ATC ATC AAG CCC GAC CCC CCC AAG
AAC
GAG AAC TAC ACC AGC AGC TTC TTC ATC CGC GAC ATC ATC AAG CCT GAC CCA CCC AAG
AAC
3 O TTG CAG CTG AAG CCA TTA AAG AAT TCT CGG CAG GTG GAG GTC AGC TGG GAG TAC
CCT GAC
CTG CAG CTG AAG CCC CTG AAG AAC AGC CGC CAG GTG GAG GTG AGC TGG GAG TAC CCC
GAC
CTC CAG CTG AAG CCC CTC AAG AAC TCC CGC CAG GTG GAG GTG AGC TGG GAG TAC CCC
GAC
ACC TGG AGT ACT CCA CAT TCC TAC TTC TCC CTG ACA TTC TGC GTT CAG GTC CAG GGC
AAG
ACC TGG AGC ACC CCC CAC AGC TAC TTC AGC CTG ACC TTC TGC GTG CAG GTG CAG GGC
AAG
3 5 ACC TGG AGC ACG CCC CAC TCC TAC TTC TCC CTG ACC TTC TGC GTG CAG GTC CAG
GGC AAG
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AGC AAG AGA GAA AAG AAA GAT AGA GTC TTC ACG GAC AAG ACC TCA GCC ACG GTC ATC
TGC
AGC AAG CGC GAG AAG AAG GAC CGC GTG TTC ACC GAC AAG ACC AGC GCC ACC GTG ATC
TGC
AGC AAG CGC GAG AAG AAA GAC CGG GTG TTC ACC GAC AAG ACC AGC GCC ACC GTC ATC
TGC
5 CGC AAA AAT GCC AGC ATT AGC GTG CGG GCC CAG GAC CGC TAC TAT AGC TCA TCT TGG
AGC
CGC AAG AAC GCC AGC ATC AGC GTG CGC GCC CAG GAC CGC TAC TAC AGC AGC AGC TGG
AGC
CGC AAG AAC GCC AGC ATC AGC GTG CGC GCC CAG GAC CGC TAC TAT AGC TCC TCT TGG
AGC
GAA TGG GCA TCT GTG CCC TGC AGT TAG
Z O GAG TGG GCC AGC GTG CCC TGC AGC TAG
GAG TGG GCC AGC GTG CCC TGC TCC TAG
Additional Optimized Sequences Coding For IL-12 p40 Subunit
(Second Line = SEQ ID N0:25)
to zo
1 5 Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu Ala Ser
Pro Leu
ATG TGY CAY CAR CAR YTN GTN ATH WSN TGG TTY WSN YTN GTN TTY YTN GCN WSN CCN
YTN
ATG TGT CAT CAA CAA TTA GTT ATT TCT TGG TTT TCT TTA GTT TTT TTA GCT TCT CCT
TTA
TGC CAC CAG CAG TTG GTC ATC TCC TTC TCC TTG GTC TTC TTG GCC TCC CCC TTG
2 O CTT GTA ATA TCA TCA CTT GTA CTT GCA TCA CCA CTT
CTC GTG TCG TCG CTC GTG CTC GCG TCG CCG CTC
CTA AGT AGT CTA CTA AGT CTA
CTG AGC AGC CTG CTG AGC CTG
25 3o Qo
Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro
Asp
GTN GCN ATH TGG GAR YTN AAR AAR GAY GTN TAY GTN GTN GAR YTN GAY TGG TAY CCN
GAY
GTT GCT ATT TGG GAA TTA AAA AAA GAT GTT TAT GTT GTT GAA TTA GAT TGG TAT CCT
GAT
3 O GTC GCC ATC GAG TTG AAG AAG GAC GTC TAC GTC GTC GAG TTG GAC TAC CCC GAC
GTA GCA ATA CTT GTA GTA GTA CTT CCA
GTG GCG CTC GTG GTG GTG CTC CCG
CTA CTA
CTG CTG
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50 60
Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr
Trp
GCN CCN GGN GAR ATG GTN GTN YTN ACN TGY GAY ACN CCN GAR GAR GAY GGN ATH ACN
TGG
S GCT CCT GGT GAA ATG GTT GTT TTA ACT TGT GAT ACT CCT GAA GAA GAT GGT ATT ACT
TGG
GCC CCC GGC GAG GTC GTC TTG ACC TGC GAC ACC CCC GAG GAG GAC GGC ATC ACC
GCA CCA GGA GTA GTA CTT ACA ACA CCA GGA ATA ACA
GCG CCG GGG GTG GTG CTC ACG ACG CCG GGG ACG
CTA
Z O CTG
Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val
Lys
ACN YTN GAY CAR WSN WSN GAR GTN YTN GGN WSN GGN AAR ACN YTN ACN ATH CAR GTN
AAR
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
ACT TTA GAT CAA TCT GAA GTT TTA GGT
TCT TCT GGT AAA ACT
TTA ACT ATT CAA
GTT AAA
ACC TTG GAC CAG TCC GAG GTC TTG GGC AAG ACC TTG ACC ATC CAG
TCC TCC GGC GTC AAG
ACA CTT TCA TCA GTA CTT GGA TCA ACA CTT ACA ATA GTA
GGA
ACG CTC TCG TCG GTG CTC GGG TCG ACG CTC ACG GTG
GGG
2 O CTA AGT AGT CTA AGT CTA
CTG AGC AGC CTG AGC CTG
100
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His
Ser
Z S GAR TTY GGN GAY GCN GGN CAR TAY ACN TGY CAY AAR GGN GGN GAR GTN YTN WSN
CAY WSN
GAA TTT GGT GAT GCT GGT CAA TAT ACT TGT CAT AAA GGT GGT GAA GTT TTA TCT CAT
TCT
GAG TTC GGC GAC GCC GGC CAG TAC ACC TGC CAC AAG GGC GGC GAG GTC TTG TCC CAC
TCC
GGA GCA GGA ACA GGA GGA GTA CTT TCA TCA
3 O GGG GCG GGG ACG GGG GGG GTG CTC TCG TCG
CTA AGT AGT
CTG AGC AGC
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ll0 120
Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp
Gln
YTN YTN YTN YTN CAY AAR AAR GAR GAY GGN ATH TGG WSN ACN GAY ATH YTN AAR GAY
CAR
S TTA TTA TTA TTA CAT AAA AAA GAA GAT GGT ATT TGG TCT ACT GAT ATT TTA AAA GAT
CAA
TTG TTG TTGTTG CAC AAG AAG GAG GAC TCC ACC ATC TTG AAG
GGC ATC GAC GAC CAG
CTT CTT CTTCTT GGA ATA TCA ACA ATA CTT
CTC CTC CTCCTC GGG TCG ACG CTC
CTA CTA CTACTA AGT CTA
Z O CTG CTGCTG AGC CTG
CTG
130 190
Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg
Phe
AAR GAR CCN AAR AAY AAR ACN TTY YTN MGN TGY GAR GCN AAR AAY TAY WSN GGN MGN
TTY
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
AAA GAA CCT AAA AAT AAA ACT TTT TTA CGT TGT GAA GCT AAA AAT TAT TCT GGT CGT
TTT
AAG GAG CCC AAG AAC AAG ACC TTC TTG CGC TGC GAG GCC AAG AAC TAC TCC GGC CGC
TTC
CCA ACA CTT CGA GCA TCA GGA CGA
CCG ACG CTC CGG GCG TCG GGG CGG
2 O CTA AGA AGT AGA
CTG AGG AGC AGG
150 160
Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser
Arg
2 S ACN TGY TGG TGG YTN ACN ACN ATH WSN ACN GAY YTN ACN TTY WSN GTN AAR WSN
WSN MGN
ACT TGT TGG TGG TTA ACT ACT ATT TCT ACT GAT TTA ACT TTT TCT GTT AAA TCT TCT
CGT
ACC TGC TTG ACC ACC ATC TCC ACC GAC TTG ACC TTC TCC GTC AAG TCC TCC CGC
ACA CTT ACA ACA ATA TCA ACA CTT ACA TCA GTA TCA TCA CGA
3 O ACG CTC ACG ACG TCG ACG CTC ACG TCG GTG TCG TCG CGG
CTA AGT CTA AGT AGT AGT AGA
CTG AGC CTG AGC AGC AGC AGG
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l~o lso
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg
Val
GGN WSN WSN GAY CCN CAR GGN GTN ACN TGY GGN GCN GCN ACN YTN WSN GCN GAR MGN
GTN
S GGT TCT TCT GAT CCT CAA GGT GTT ACT TGT GGT GCT GCT ACT TTA TCT GCT GAA CGT
GTT
GGC TCC TCC GAC CCC CAG GGC GTC ACC TGC GGC GCC GCC ACC TTG TCC GCC GAG CGC
GTC
GGA TCA TCA CCA GGA GTA ACA GGA GCA GCA ACA CTT TCA GCA CGA GTA
GGG TCG TCG CCG GGG GTG ACG GGG GCG GCG ACG CTC TCG GCG CGG GTG
AGT AGT CTA AGT AGA
Z O AGC AGC CTG AGC AGG
190 200
Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys
Pro
MGN GGN GAY AAY AAR GAR TAY GAR TAY WSN GTN GAR TGY CAR GAR GAY WSN GCN TGY
CCN
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
CGT GGT GAT AAT AAA GAA TAT GAA TAT TCT GTT GAA TGT CAA GAA GAT TCT GCT TGT
CCT
CGC GGC GAC AAC AAG GAG TAC GAG TAC TCC GTC GAG TGC CAG GAG GAC TCC GCC TGC
CCC
CGA GGA TCA GTA TCA GCA CCA
CGG GGG TCG GTG TCG GCG CCG
Z O AGA AGT AGT
AGC AGC
210 220
Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys
Tyr
2 r'J GCN GCN GAR GAR WSN YTN CCN ATH GAR GTN ATG GTN GAY GCN GTN CAY AAR YTN
AAR TAY
GCT GCT GAA GAA TCT TTA CCT ATT GAA GTT ATG GTT GAT GCT GTT CAT AAA TTA AAA
TAT
GCC GCC GAG GAG TCC TTG CCC ATC GAG GTC GTC GAC GCC GTC CAC AAG TTG AAG TAC
GCA GCA TCA CTT CCA ATA GTA GTA GCA GTA CTT
3 O GCG GCG TCG CTC CCG GTG GTG GCG GTG CTC
AGT CTA CTA
AGC CTG CTG
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230 240
Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys
Asn
GAR AAY TAY ACN WSN WSN TTY TTY ATH MGN GAY ATH ATH AAR CCN GAY CCN CCN AAR
AAY
GAA AAT TAT ACT TCT TCT TTT TTT ATT CGT GAT ATT ATT AAA CCT GAT CCT CCT AAA
AAT
GAG AAC TAC ACC TCC TCC TTC TTC ATC CGC GAC ATC ATC AAG CCC GAC CCC CCC AAG
AAC
ACA TCA TCA ATA CGA ATA ATA CCA CCA CCA
ACG TCG TCG CGG CCG CCG CCG
AGT AGT AGA
Z O AGC AGC AGG
250 260
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro
Asp
YTN CAR YTN AAR CCN YTN AAR AAY WSN MGN CAR GTN GAR GTN WSN TGG GAR TAY CCN
GAY
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
TTA CAA TTA AAA CCT TTA AAA AAT TCT CGT CAA GTT GAA GTT TCT TGG GAA TAT CCT
GAT
TTG CAG TTG AAG CCC TTG AAG AAC TCC CGC CAG GTC GAG GTC TCC GAG TAC CCC GAC
CTT CTT CCA CTT TCA CGA GTA GTA TCA CCA
CTC CTC CCG CTC TCG CGG GTG GTG TCG CCG
2 O CTA CTA CTA AGT AGA AGT
CTG CTG CTG AGC AGG AGC
2~0 280
Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln Gly
Lys
2 S ACN TGG WSN ACN CCN CAY WSN TAY TTY WSN YTN ACN TTY TGY GTN CAR GTN CAR
GGN AAR
ACT TGG TCT ACT CCT CAT TCT TAT TTT TCT TTA ACT TTT TGT GTT CAA GTT CAA GGT
AAA
ACC TCC ACC CCC CAC TCC TAC TTC TCC TTG ACC TTC TGC GTC CAG GTC CAG GGC AAG
ACA TCA ACA CCA TCA TCA CTT ACA GTA GTA GGA
3 O ACG TCG ACG CCG TCG TCG CTC ACG GTG GTG GGG
AGT AGT AGT CTA
AGC AGC AGC CTG
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Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile
Cys
WSN AAR MGN GAR AAR AAR GAY MGN GTN TTY ACN GAY AAR ACN WSN GCN ACN GTN ATH
TGY
5 TCT CGT GTT TTT
AAA ACT GAT AAA
CGT ACT TCT GCT
GAA ACT GTT ATT
AAA TGT
AAA
GAT
TCC AAG CGC GAG AAG CGC GTC TTC AAG ACC TCC GCC ACC GTC
AAG GAC ACC GAC ATC TGC
TCA CGA CGA GTA ACA ACA TCA GCA ACA GTA ATA
TCG CGG CGG GTG ACG ACG TCG GCG ACG GTG
AGT AGA AGA AGT
Z O AGG AGG AGC
AGC
310 320
Arg Lys Asn Ala 5er Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp
Ser
MGN AAR AAY GCN WSN ATH WSN GTN MGN GCN CAR GAY MGN TAY TAY WSN WSN WSN TGG
WSN
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ _ ___ ___ ___ ___ ___ ___ ___
___
CGT AAA AAT GCT TCT ATT TCT GTT CGT GCT CAA GAT CGT TAT TAT TCT TCT TCT TGG
TCT
CGC AAG TCC
AAC GCC
TCC ATC
TCC GTC
CGC GCC
CAG GAC
CGC TAC
TAC TCC
TCC TCC
CGA GCA TCA ATA TCA GTA CGA CGA TCA TCA TCA TCA
GCA
CGG GCG TCG TCG GTG CGG GCG CGG TCG TCG TCG TCG
2 O AGA AGT AGT AGA AGA AGT AGT AGT AGT
AGG AGC AGC AGG AGG AGC AGC AGC AGC
Glu Trp Ala Ser Val Pro Cys Ser ***
2 5 GAR TGG GCN WSN GTN CCN TGY WSN TRR
GAA TGG GCT TCT GTT CCT TGT TCT TAA
GAG GCC TCC GTC CCC TGC TCC TAG
GCA TCA GTA CCA TCA TGA
3 O GCG TCG GTG CCG TCG
AGT AGT
AGC AGC
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Wild Type Sequence Coding For Interferon Alpha
9 18 27 36 45 54
5' ATG GCC TTG ACC TTT GCT TTA CTG GTG GCC CTC CTG GTG CTC AGC TGC AAG TCA
M A L T F A L L V A L L V L S C K S
63 72 81 90 99 108
AGC TGC TCT GTG GGC TGT GAT CTG CCT CAA ACC CAC AGC CTG GGT AGC AGG AGG
ZO S C S V G C D I. P Q T H S L G S R R
117 126 135 144 153 162
ACC TTG ATG CTC CTG GCA CAG ATG AGG AGA ATC TCT CTT TTC TCC TGC TTG AAG
1 5 T L M L L A Q M R R I S L F S C L K
171 180 189 198 207 216
GAC AGA CAT GAC TTT GGA TTT CCC CAG GAG GAG TTT GGC AAC CAG TTC CAA AAG
2 O D R H D F G F P Q E E F G N Q F Q K
225 239 243 252 261 270
GCT GAA ACC ATC CCT GTC CTC CAT GAG ATG ATC CAG CAG ATC TTC AAT CTC TTC
Z 5 A E T I P V L H E M I Q Q I F N L F
279 288 297 306 315 324
AGC ACA AAG GAC TCA TCT GCT GCT TGG GAT GAG ACC CTC CTA GAC AAA TTC TAC
3 O S T K D S S A A W D E T L L D K F Y
333 392 351 360 369 378
ACT GAA CTC TAC CAG CAG CTG AAT GAC CTG GAA GCC TGT GTG ATA CAG GGG GTG
35 T E L Y- Q Q L N D L E A C V I Q G V
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387 396 405 414 923 432
GGG GTG ACA GAG ACT CCC CTG ATG AAG GAG GAC TCC ATT CTG GCT GTG AGG AAA
G V T E T P L M K E D S I L A V R K
441 950 459 468 477 486
TAC TTC CAA AGA ATC ACT CTC TAT CTG AAA GAG AAG AAA TAC AGC CCT TGT GCC
Y F Q R I T L Y L K E K K Y S P C A
495 504 513 522 531 540
TGG GAG GTT GTC AGA GCA GAA ATC ATG AGA TCT TTT TCT TTG TCA ACA AAC TTG
W E V V R A E I M R S F S L S T N L
549 558 567
CAA GAA AGT TTA AGA AGT AAG GAA TGA 3'
Q E S L R S K E
Interferon Alpha Coding Sequence with All Codons Optimized
(SEQ ID NO:11)
ATG GCC CTG ACC TTC GCC CTG CTG GTG GCC CTG CTG GTG CTG AGC TGC AAG AGC AGC
TGC
2 5 TCC GTG GGG TGC GAC CTG CCC CAG ACC CAC AGC CTG GGG AGC CGG CGG ACC CTG
ATG CTG
CTG GCC CAG ATG CGG CGG ATC AGC CTG TTC AGC TGC CTG AAG GAC CGG CAC GAC TTC
GGG
TTC CCC CAG GAG GAG TTC GGG AAC CAG TTC CAG AAG GCC GAG ACC ATC CCC GTG CTG
CAC
GAG ATG ATC CAG CAG ATC TTC AAC CTG TTC AGC ACC AAG GAC AGC AGC GCC GCC TGG
GAC
GAG ACC CTG CTG GAC AAG TTC TAC ACC GAG CTG TAC CAG CAG CTG AAC GAC CTG GAG
GCC
3 5 TGC GTG ATC CAG GGG GTG GGG GTG ACC GAG ACC CCC CTG ATG AAG GAG GAC AGC
ATC CTG
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GCC GTG CGG AAG TAC TTC CAG CGG ATC ACC CTG TAC CTG AAG GAG AAG AAG TAC TCC
CCC
TGC GCC TGG GAG GTG GTG CGG GCC GAG ATC ATG CGG AGC TTC AGC CTG AGC ACC AAC
CTG
CAG GAG AGC CTG CGG AGC AAG GAG TGA
Additional/Semi Optimized Sequence Coding For Interferon
Alpha (Second Line = SEQ ID N0:12)
l0 20
MET ALA LEU THR PHE ALA LEU LEU VAL ALA LEU LEU VAL LEU SER CYS LYS SER SER
CYS
ATG GCN YTN ACN TTY GCN YTN YTN GTN GCN YTN YTN GTN YTN WSN TGY AAR WSN WSN
TGY
ATG GCT TTA ACT TTT GCT TTA TTA GTT GCT TTA TTA GTT TTA TCT TGT AAA TCT TCT
TGT
1 5 GCC TTG ACC TTC GCC TTG TTG GTC GCC TTG TTG GTC TTG TCC TGC AAG TCC TCC
TGC
GCA CTT ACA GCA CTT CTT GTA GCA CTT CTT GTA CTT TCA TCA TCA
GCG CTC ACG GCG CTC CTC GTG GCG CTC CTC GTG CTC TCG TCG TCG
CTA CTA CTA CTA CTA CTA AGT AGT AGT
CTG CTG CTG CTG CTG CTG AGC AGC AGC
34 qp
SER VAL GLY CYS ASP LEU PRO GLN THR HIS SER LEU GLY SER ARG ARG THR LEU MET
LEU
WSN GTN GGN TGY GAY YTN CCN CAR ACN CAY WSN YTN GGN WSN MGN MGN ACN YTN ATG
YTN
2 5 TCT GTT GGT TGT GAT TTA CCT CAA ACT CAT TCT TTA GGT TCT CGT CGT ACT TTA
ATG TTA
TCC GTC GGC TGC CAC TCC GGC TCC CGC CGC ACC TTG
GAC TTG CCC TTG TTG
CAG ACC
TCA GTA GGA CTT CCA ACA TCA CTT GGA TCA CGA CGA ACA CTT
CTT
TCG GTG GGG CTC CCG ACG TCG CTC GGG TCG CGG CGG ACG CTC
CTC
AGT CTA AGT CTA AGT AGA AGA CTA CTA
3 O AGC CTG AGC CTG AGC AGG AGG CTG CTG
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SO 60
LEU ALA GLN MET ARG ARG ILE SER LEU PHE SER CYS LEU LYS ASP ARG HIS ASP PHE
GLY
YTN GCN CAR ATG MGN MGN ATH WSN YTN TTY WSN TGY YTN AAR GAY MGN CAY GAY TTY
GGN
TTA GCT CAA ATG CGT CGT ATT TCT TTA TTT TCT TGT TTA AAA GAT CGT CAT GAT TTT
GGT
TTG GCC CAG CGC CGC ATC TCC AAG GAC CGC CAC GAC
TTG TTC TCC TGC TTC GGC
TTG
CTT GCA CGA CGA ATA TCA TCA CTT CGA GGA
CTT
CTC GCG CGG CGG TCG CTC TCG CTC CGG GGG
CTA AGA AGA AGT CTA AGT CTA AGA
Z O CTG AGG AGG AGC CTG AGC CTG AGG
70 80
PHE PRO GLN GLU GLU PHE GLY ASN GLN PHE GLN LYS ALA GLU THR ILE PRO VAL LEU
HIS
TTY CCN CAR GAR GAR TTY GGN AAY CAR TTY CAR AAR GCN GAR ACN ATH CCN GTN YTN
CAY
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
TTT CCT CAA GAA GAA TTT GGT AAT CAA TTT CAA AAA GCT GAA ACT ATT CCT GTT TTA
CAT
TTC CCC CAG GAG GAG TTC GGC AAC CAG TTC CAG AAG GCC GAG ACC ATC CCC GTC TTG
CAC
CCA GGA GCA ACA ATA CCA GTA CTT
CCG GGG GCG ACG CCG GTG CTC
2 O CTA
CTG
90 100
GLU MET ILE GLN GLN ILE PHE ASN LEU PHE SER THR LYS ASP SER SER ALA ALA TRP
ASP
2 5 GAR ATG ATH CAR CAR ATH TTY AAY YTN TTY WSN ACN AAR GAY WSN WSN GCN GCN
TGG GAY
GAA ATG ATT CAA CAA ATT TTT AAT TTA TTT TCT ACT AAA GAT TCT TCT GCT GCT TGG
GAT
GAG ATC CAG CAG ATC TTC AAC TTG TTC TCC ACC AAG GAC TCC TCC GCC GCC GAC
ATA ATA CTT TCA ACA TCA TCA GCA GCA
3 O CTC TCG ACG TCG TCG GCG GCG
CTA AGT AGT AGT
CTG AGC AGC AGC
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GLU THR LEU LEU ASP LYS PHE TYR THR GLU LEU TYR GLN GLN LEU ASN ASP LEU GLU
ALA
GAR ACN YTN YTN GAY AAR TTY TAY ACN GAR YTN TAY CAR CAR YTN AAY GAY YTN GAR
GCN
5 GAA ACT TTA TTA GAT AAA TTT TAT ACT GAA TTA TAT CAA CAA TTA AAT GAT TTA GAA
GCT
GAG ACC TTG TTG GAC AAG TTC TAC ACC GAG TTG TAC CAG CAG TTG AAC GAC TTG GAG
GCC
ACA CTT CTT ACA CTT CTT CTT GCA
ACG CTC CTC ACG CTC CTC CTC GCG
CTA CTA CTA CTA CTA
Z O CTG CTG CTG CTG CTG
130 140
CYS VAL ILE GLN GLY VAL GLY VAL THR GLU THR PRO LEU MET LYS GLU ASP SER ILE
LEU
TGY GTN ATH CAR GGN GTN GGN GTN ACN GAR ACN CCN YTN ATG AAR GAR GAY WSN ATH
YTN
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
___ ___
TGT GTT ATT CAA GGT GTT GGT GTT ACT GAA ACT CCT TTA ATG AAA GAA GAT TCT ATT
TTA
TGC GTC ATC CAG GGC GTC GGC GTC ACC GAG ACC CCC TTG AAG GAG GAC TCC ATC TTG
GTA ATA GGA GTA GGA GTA ACA ACA CCA CTT TCA ATA CTT
GTG GGG GTG GGG GTG ACG ACG CCG CTC TCG CTC
2 O CTA AGT CTA
CTG AGC CTG
150 160
ALA VAL ARG LYS TYR PHE GLN ARG ILE THR LEU TYR LEU LYS GLU LYS LYS TYR SER
PRO
2 5 GCN GTN MGN AAR TAY TTY CAR MGN ATH ACN YTN TAY YTN AAR GAR AAR AAR TAY
WSN CCN
GCT GTT CGT AAA TAT TTT CAA CGT ATT ACT TTA TAT TTA AAA GAA AAA AAA TAT TCT
CCT
GCC GTC CGC AAG TAC TTC CAG CGC ATC ACC TTG TAC TTG AAG GAG AAG AAG TAC TCC
CCC
GCA GTA CGA CGA ATA ACA CTT CTT TCA CCA
3 O GCG GTG CGG CGG ACG CTC CTC TCG CCG
AGA AGA CTA CTA AGT
AGG AGG CTG CTG AGC
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m o iao
CYS ALA TRP GLU VAL VAL ARG ALA GLU ILE MET ARG SER PHE SER LEU SER THR ASN
LEU
TGY GCN TGG GAR GTN GTN MGN GCN GAR ATH ATG MGN WSN TTY WSN YTN WSN ACN AAY
YTN
S TGT GCT TGG GAA GTT GTT CGT GCT GAA ATT ATG CGT TCT TTT TCT TTA TCT ACT AAT
TTA
TGC GCC GAG GTC GTC CGC GCC GAG ATC CGC TCC AAC
TTC TCC TTG
TTG TCC
ACC
GGA GTA GTA CGA GCA ATA CGA TCA TCA CTT TCA CTT
ACA
GCG GTG GTG CGG GCG CGG TCG TCG CTC TCG CTC
ACG
AGA AGA AGT AGT CTA AGT CTA
Z O AGG AGG AGC AGC CTG AGC CTG
GLN GLU SER LEU ARG SER LYS GLU ***
CAR GAR WSN YTN MGN WSN AAR GAR TRR
1 5 ___ ___ ___ ___ ___ ___ ___ ___ ___
CAA GAA TCT TTA CGT TCT AAA GAA TAA
CAG GAG TCC TTG CGC TCC AAG GAG TAG
TCA CTT CGA TCA TGA
TCG CTC CGG TCG
Z O AGT CTA AGA AGT
AGC CTG AGG AGC
Delivery and expression of nucleic acids in many
formulations is limited due to degradation of the nucleic
25 acids by components of organisms, such as nucleases. Thus,
protection of the nucleic acids when delivered in vivo can
greatly enhance the resulting expression, thereby enhancing
a desired pharmacological or therapeutic effect. It was
found that certain types of compounds which interact with a
30 nucleic acid (e.g., DNA) in solution but do not condense the
nucleic acid provide in vivo protection to the nucleic acid,
and correspondingly enhance the expression of an encoded
gene product.
We have described the use of delivery systems designed
35 to interact with plasmids and protect plasmids from rapid
extracellular nuclease degradation [Mumper, R.J., et al.,
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1996, Pharm. Res. 13:701-709; Mumper, R.J., et al., 1997.
Submitted to Gene Therapy]. A characteristic of the PINC
systems is that they are non-condensing systems that allow
the plasmid to maintain flexibility and diffuse freely
throughout the muscle while being protected from nuclease
degradation. While the PINC systems are primarily discussed
below, it will be understood that cationic lipid based
systems and systems utilizing both PINCS and cationic lipids
are also within the scope of the present invention.
A common structural component of the PINC systems is
that they are amphiphilic molecules, having both a
hydrophilic and a hydrophobic portion. The hydrophilic
portion of the PINC is meant to interact with plasmids by
hydrogen bonding (via hydrogen bond acceptor or donor
groups), Van der Waals interactions, or/and by ionic
interactions. For example, PVP and N-methyl-2-pyrrolidone
(NM2P) are hydrogen bond acceptors while PVA and PG are
hydrogen bond donors.
All four molecules have been reported to form complexes
with various (poly)anionic molecules [Buhler V., BASF
Aktiengescellschaft Feinchemie, Ludwigshafen, pp 39-42;
Galaev Y, et al., J. Chrom. A. 684:45-54 (1994); Tarantino
R, et al. J. Pharm. Sci. 83:1213-1216 (1994); Zia, H., et
al., Pharm. Res. 8:502-504 (1991);]. The hydrophobic
portion of the PINC systems is designed to result in a
coating on the plasmid rendering its surface more
hydrophobic. Kabanov et al. have described previously the
use of cationic polyvinyl derivatives for plasmid
condensation designed to increase plasmid hydrophobicity,
protect plasmid from nuclease degradation, and increase its
affinity for biological membranes [Kabanov, A.V., and
Kabanov, V.A., 1995, Bioconj. Chem. 6:7-20: Kabanov, A.V.,
et al., 1991, Biopolymers 31:1437-1443: Yaroslavov, A.A., et
al., 1996, FEBS Letters 384:177-180].
Substantial protective effect is observed; up to at
least a one log enhancement of gene expression in rat muscle
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over plasmid formulated in saline has been demonstrated with
these exemplary PINC systems. We have also found that the
expression of reporter genes in muscle using plasmids
complexed with the PINC systems was more reproducible than
when the plasmid was formulated in saline. For example, the
coefficient of variation for reporter gene expression in
muscle using plasmid formulated in saline was 96 _+ 35~k (n =
20 studies; 8-12 muscles/study) whereas with coefficient of
variation with plasmids complexed with PINC systems was 40 _+
19~ (n - 30 studies; 8-12 muscles/study). The high
coefficient of variation for reporter gene expression with
plasmid formulated in saline has been described previously
[Davis, H.L., et al., 1993, Hum. Gene Ther. 4:151-9]. In
addition, in contrast with the results for DNA: saline, there
was no significant difference in gene expression in muscle
when plasmid with different topologies were complexed with
polyvinyl pyrrolidone (PVP). This suggests that PVP is able
to protect all forms of the plasmid from rapid nuclease
degradation.
1. Summary of interactions between a PINC
polymer (PVP) and lasmid
We have demonstrated using molecular modeling that an
exemplary PINC polymer, PVP, forms hydrogen bonds with the
base pairs of a plasmid within its major groove and results
in a hydrophobic surface on the plasmid due to the vinyl
backbone of PVP. These interactions are supported by the
modulation of plasmid zeta potential by PVP as well as by
the inhibition of ethidium bromide intercalation into
complexed plasmid. We have correlated apparent binding
between PVP and plasmid to pH and salt concentration and
have demonstrated the effect of these parameters on D-gal
expression after intramuscular injection of plasmid/PVP
complexes [Mumper, R.J., et al., 1997. Submitted to Gene
Therapy]. A summary of the physico-chemical properties of
plasmid/PVP complexes is listed in Table I below.
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Table I: Summary of the Physico-Chemical Properties of
Plasmid/PVP Complexes
Method Result
Molecular modeling Hydrogen bonding and
Fourier-transformed hydrophobic plasmid surface
Infra-red Hydrogen observed bonding demonstrated
DNase I challenge Decreased rate of plasmid
degradation in the presence
of PVP
Microtitration Positive heats of reaction
Calorimetry indicative of an endothermic
process
Potentiometric titration One unit pH drop when plasmid
and PVP are complexed
Dynamic Dialysis Rate of diffusion of PVP
reduced in the presence of
plasmid
Zeta potential Surface charge of plasmid
modulation decreased by PVP
Ethidium bromide Ethidium bromide
Intercalation intercalation reduced by
plasmid/PVP complexation
Osmotic pressure Hyper-osmotic formulation
(i.e., 340 mOsm/kg H20)
Luminescence Plasmid/PVP binding decreased
Spectroscopy in salt and/or at pH 7
2. Histology of expression in muscle
Immunohistochemistry for ~3-gal using a slide scanning
technology has revealed the uniform distribution of (3-gal
expression sites across the whole cross-sections of rat
tibialis muscles. Very localized areas were stained positive
far ~i-gal when CMV-(3-gal plasmid was formulated in saline.
~i-gal positive cells were observed exclusively around the
needle tract when plasmid was injected in saline. This is
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in agreement with previously published results [Wolff, J.A.,
et al., 1990, Science 247:1465-68; Davis, H.L., et al.,
1993, Hum. Gene Ther. 4:151-9; Davis, H.L., et al., 1993,
Hum. Gene Ther. 4:733-40].
5 In comparison, immunoreactivity for (3-gal was observed
in a wide area of muscle tissue after intramuscular
injection of CMV-~i-gal plasmid/PVP complex (1:17 w/w) in 150
mM NaCl. It appeared that the majority of positive muscle
fibers were located at the edge of muscle bundles. Thus,
10 staining for ~i-gal in rat muscle demonstrated that, using a
plasmid/PVP complex, the number of muscle fibers stained
positive for (3-gal was approximately 8-fold greater than
found using a saline formulation. Positively stained
muscle fibers were also observed over a much larger area in
15 the muscle tissue using the plasmid/PVP complex providing
evidence that the injected plasmid was widely dispersed
after intramuscular injection.
We conclude that the enhanced plasmid distribution and
expression in rat skeletal muscle was a result of both
20 protection from extracellular nuclease degradation due to
complexation and hyper-osmotic effects of the plasmid/PVP
complex. However, Dowty and Wolff et al. have demonstrated
that osmolarity, up to twice physiologic osmolarity, did not
significantly effect gene expression in muscle [Dowty, M.E.,
25 and Wolff, J.A. In: J.A. Wolff (Ed.), 1994, Gene
Therapeutics: Methods and Applications of Direct Gene
Transfer. Birkhauser, Boston, pp. 82-98]. This suggests
that the enhanced expression of plasmid due to PVP
complexation is most likely due to nuclease protection and
30 less to osmotic effects. Further, the surface modification
of plasmids by PVP (e.g., increased hydrophobicity and
decreased negative surface charge) may also facilitate the
uptake of plasmids by muscle cells.
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3. Structure-activity relationship of PINC
polymers
We have found a linear relationship between the
structure of a series of co-polymers of vinyl pyrrolidone
and vinyl acetate and the levels of gene expression in rat
muscle. We have found that the substitution of some vinyl
pyrrolidone monomers with vinyl acetate monomers in PVP
resulted in a co-polymer with reduced ability to form
hydrogen bonds with plasmids. The reduced interaction
subsequently led to decreased levels of gene expression in
rat muscle after intramuscular injection. The expression of
(3-gal decreased linearly (R - 0.97) as the extent of vinyl
pyrrolidone monomer (VPM) content in the co-polymers
decreased.
These data demonstrate that pH and viscosity are not
the most important parameters effecting delivery of plasmid
to muscle cells since these values were equivalent for all
complexes. These data suggest that enhanced binding of the
PINC polymers to plasmid results in increased protection and
bioavailability of plasmid in muscle.
4. Additional PINC systems
The structure-activity relationship described above can
be used to design novel co-polymers that will also have
enhanced interaction with plasmids. It is expected that
there is "an interactive window of opportunity" whereby
enhanced binding affinity of the PINC systems will result in
a further enhancement of gene expression after their
intramuscular injection due to more extensive protection of
plasmids from nuclease degradation. It is expected that
there will be an optimal interaction beyond which either
condensation of plasmids will occur or "triplex" type
formation, either of which can result in decreased
bioavailability in muscle and consequently reduced gene
expression.
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As indicated above, the PINC compounds are generally
amphiphilic compounds having both a hydrophobic portion and
a hydrophilic portion. In many cases the hydrophilic
portion is provided by a polar group. It is recognized in
the art that such polar groups can be provided by groups
such as, but not limited to, pyrrolidone, alcohol, acetate,
amine or heterocyclic groups such as those shown on pp. 2-73
and 2-74 of CRC Handbook of Chemistry and Physics (72nd
Edition), David R. Zide, editor, including pyrroles,
pyrazoles, imidazoles, triazoles, dithiols, oxazoles,
(iso)thiazoles, oxadiazoles, oxatriazoles, diaoxazoles,
oxathioles, pyrones, dioxins, pyridines, pyridazines,
pyrimidines, pyrazines, piperazines, (iso)oxazines, indoles,
indazoles, carpazoles, and purines and derivatives of these
groups, hereby incorporated by reference.
Compounds also contain hydrophobic groups which, in the
case of a polymer, are typically contained in the backbone
of the molecule, but which may also be part of a non-
polymeric molecule. Examples of such hydrophobic backbone
groups include, but are not limited to, vinyls, ethyls,
acrylates, acrylamides, esters, celluloses, amides,
hydrides, ethers, carbonates, phosphazenes, sulfones,
propylenes, and derivatives of these groups. The polarity
characteristics of various groups are quite well known to
those skilled in the art as illustrated, for example, by
discussions of polarity in any introductory organic
chemistry textbook.
The ability of such molecules to interact with nucleic
acids is also understood by those skilled in the art, and
can be predicted by the use of computer programs which model
such intermolecular interactions. Alternatively or in
addition to such modeling, effective compounds can readily
be identified using one or more of such tests as 1)
determination of inhibition of the rate of nuclease
digestion, 2) alteration of the zeta potential of the DNA,
which indicates coating of DNA, 3) or inhibition of the
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ability of intercalating agents, such as ethidium bromide to
intercalate with DNA.
5. Targeting Ligands
In addition to the nucleic acid/PINC complexes
described above for delivery and expression of nucleic acid
sequences, in particular embodiments it is also useful to
provide a targeting ligand in order to preferentially obtain
expression in particular tissues, cells, or cellular regions
or compartments.
Such a targeted PINC complex includes a PINC system
(monomeric or polymeric PINC compound) complexed to plasmid
(or other nucleic acid molecule). The PINC system is
covalently or non-covalently attached to (bound to) a
targeting ligand (TL) which binds to receptors having an
affinity for the ligand. Such receptors may be on the
surface or within compartments of a cell. Such targeting
provides enhanced uptake or intracellular trafficking of the
nucleic acid.
The targeting ligand may include, but is not limited
to, galactosyl residues, fucosal residues, mannosyl
residues, carnitine derivatives, monoclonal antibodies,
polyclonal antibodies, peptide ligands, arid DNA-binding
proteins. Examples of cells which may usefully be targeted
include, but are not limited to, antigen-presenting cells,
hepatocytes, myocytes, epithelial cells, endothelial cells,
and cancer cells.
Formation of such a targeted complex is illustrated by
the following example of covalently attached targeting
ligand (TL) to PINC system:
TL-PINC + Plasmid ----------> TL-PINC::::::Plasmid
Formation of such a targeted complex is also
illustrated by the following example of non-covalently
attached targeting ligand (TL) to PINC system
TL::...:PINC + Plasmid --------> TL::...:PINC::...:Plasmid
or alternatively,
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PINC + Plasmid ------------> PINC:.....:Plasmid + TL ---
---------> TL::...:PINC::....:Plasmid
In these examples ........ is non-covalent interaction such
as ionic, hydrogen-bonding, Van der Waals interaction,
hydrophobic interaction, or combinations of such
interactions.
A targeting method for cytotoxic agents is described in
Subramanian et al., International Application No.
PCT/US96/08852, International Publication No. WO 96/39124,
hereby incorporated by reference. This application
describes the use of polymer affinity systems for targeting
cytotoxic materials using a two-step targeting method
involving zip polymers, i.e., pairs of interacting polymers.
An antibody attached to one of the interacting polymers
binds to a cellular target. That polymer then acts as a
target for a second polymer attached to a cytotoxic agent.
As referenced in Subramanian et al., other two-step (or
multi-step) systems for delivery of toxic agents are also
described.
In another aspect, nucleic acid coding sequences can be
delivered and expressed using a two-step targeting approach
involving a non-natural target for a PINC system or PINC-
targeting ligand complex. Thus, for example, a PINC-plasmid
complex can target a binding pair member which is itself
attached to a ligand which binds to a cellular target (e. g.,
a MAB). Binding pairs for certain of the compounds
identified herein as PINC compounds as identified in
Subramanian et al. Alternatively, the PINC can be complexed
to a tareting ligand, such as an antibody. That antibody
can be targeted to a non-natural target which binds to, for
example, a second antibody.
III. Model Systems for Evaluation of Interferon Alpha
Constructs and Formulations
In accord with the concept of using interferon alpha
expressing plasmid constructs and formulations in anti-
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cancer treatment, murine model systems were utilized based
on murine tumor cell lines. The line primarily used was
S.C. VII/SF, which is a cell line derived from murine
squamous cell carcinoma (S.C.).
5 Squamous cell carcinoma of the head and neck begins
with the cells lining the oral and pharyngeal cavities.
Clinical disease progresses via infiltration and spreads
into the underlying tissues and lymphatics. The
undifferentiated, in vivo passage tumor line S.C. VII/SF
10 displays this typical growth pattern. In addition, its
rapid growth rate provides a relatively short test period
for individual experiments. Other murine tumor cell lines
include another SCC line KLN-205, a keratinocyte line I-7,
and a colon adenocarcinoma line MC-38.
15 An optimal model system preferably satisfies the
criteria based on having tumor growth rate in vivo (i.e.,
tumors are ready for treatment in 4-10 days post implant),
invasiveness, and local spread similar to those observed in
clinical disease, and providing accessibility for
20 experimental treatment. As indicated, the SCC VII/SF cell
line was utilized as the primary model system cell line.
This cell line typically grows rapidly, resulting in death
of untreated syngeneic mice 14-17 days after tumor cell
implantation.
25 This cell line can be utilized in a variety of ways to
provide model system suitable for a variety of different
tests. Four such possibilities are described below.
First, SCCVII cells can be utilized in cell culture to
provide an in vitro evaluation of interferon alpha
30 expression construct and formulation characteristics, such
as expression levels and cellular toxicities.
Second, the cells can be implanted subcutaneously in
mice. This system can be utilized in tests in which
accessibility of the implant site is beneficial. As an
35 example, the method was utilized in evaluations of
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expression efficiencies based on the expression of
chloramphenicol acetyltransferase (CAT).
Third, the cells can be implanted transcutaneously into
the fascia of digastric muscle.
Fourth, the cells can be implanted transcutaneously
into digrastric/mylohyoid muscles. The important features
of models 3 and 4 are shown in the table below.
TABLE II: Comparison of submandibular tumor models
Feature Mouse Tumor Model Mouse Tumor Model 4
3
Tumor implant 2-4 x 105 cells 5 x 105
procedure transcutaneously intotranscutaneously into
fascia of digastric digastric/mylohyoid
muscle muscles
Tumor growth and Prominent More variable,
invasiveness submandibular bulge; invasion of
characteristics invasion of digastric/mylohyoid
digastric/mylohyoid muscles and lymphatics
muscles and
lymphatics
Treatment Transcutaneous, Lower jaw skin flap
procedure needle inserted and raised to expose
(primary moved within tumor tumor, needle inserted
to
treatment) produce a 4 quadrant and moved within tumor
distribution of gene to produce a 4
medicine quadrant distribution
of gene medicine
Days treated Day 5, day 10 (both Day 5 (tumor exposed),
(post-implant) transcutaneously) day 8
(transcutaneously)
Measurement External calipering First caliper when
procedure 2-3 x per week until tumor exposed for
death treatment, second
caliper at sacrifice
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Feature Mouse Tumor Model Mouse Tumor Model 4
3
Advantages Non-surgical, closed Surgical, open model
model allows larger allows direct
experiments and more treatment of exposed
frequent treatments; tumor: Local
Sacrifice unnecessaryinflammation from
to caliper (=more surgery may
time points) additionally stimulate
immune response; More
like clinical
situation for protocol
development
Disadvantages Transcutaneous Labor intensive:
treatment is Smaller, fewer
potentially less experiments possible;
accurate and Tumors deeper and more
intensive: less like difficult to treat
expected clinical transcutaneously (for
treatments than secondary treatments):
surgical approaches Fewer treatments and
caliperings possible
The tumor size treated in the mouse models is generally
20-50 mm3. A 50 mm3 mouse tumor is approximately equivalent
to 150 cc3 human tumor having an average diameter of about
6.6 cm. This tumor size is approximately 10-fold larger
than the size proposed to be treated in the phase I clinical
trials. This indicates that the mouse models are strongly
biased towards over estimating the expected tumor burden in
human patients.
IV. Formulations for In Vivo Delivery
i0 A. General
While expression systems such as those described above
provide the potential for expression when delivered to an
appropriate location, it is beneficial to provide the
expression system constructs) in a delivery system which
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can assist both the delivery and the cellular uptake of the
construct. Thus, this invention also provides particular
formulations which include one or more expression system
constructs (e.g., DNA plasmids as described above), and a
protective, interactive non-condensing compound.
An additional significant factor relating to the
plasmid construct is the percentage of plasmids which are in
a supercoiled (SC) form rather than the open circular (OC)
form.
B. Delivery and Expression
A variety of delivery methods can be used with the
constructs and formulations described above, in particular,
delivery by injection to the site of a tumor can be used.
The submandibular tumor models utilized injection into four
quadrants of the tumor being treated.
C. Anti-Cancer Efficacy of Human Interferon Al ha
Formulations
The effects of the administration of the interferon
alpha formulations described above were evaluated using the
S.C. VII mouse tumor models. Plasmid constructs as
described above were incorporated in delivery formulations.
The formulations were delivered by injection.
D. Synergistic Effects of Interferon A1 ha lasmid
and IL-12 Plasmid and Effect of Human Interferon
Alpha Formulation Administration o_n _Production of
Secondary Cytokines
The effects of the expression of the human interferon
alpha plasmids in tumor cells on the progress of the mouse
tumors demonstrates that such interferon alpha expression is
effective against such tumors. However, it was also shown
that IL-12 can act synergistically with the interferon alpha
expression to exercise the antitumor effect (see Figure 9).
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E. Toxicity Evaluation of Exemplary Formulations
The exemplary formulations do not show high cellular
toxicity at the concentrations tested, suggesting that the
formulations do not significantly kill cells by direct toxic
action in vivo. Moreover, the anti-tumor activity induced
by IFNa gene therapy is dependent upon activation of the
immune system, which is demonstrated by depletion studies in
vivo. Removal of a specific T lymphocyte population (CD8+)
abrogates the anti-tumor activity elicited by IFNa gene
therapy.
V. Administration
Administration as used herein refers to the route of
introduction of a plasmid or carrier of DNA into the body.
In addition to the methods of delivery described above, the
expression systems constructs and the delivery system
formulations can be administered by a variety of different
methods.
Administration can be directly to a target tissue or by
targeted delivery to the target tissue after systemic
administration. In particular, the present invention can be
used for treating disease by administration of the
expression system or formulation to the body in order to
establishing controlled expression of any specific nucleic
acid sequence within tissues at certain levels that are
useful for gene therapy.
The preferred means for administration of vector
(plasmid) and use of formulations for delivery are described
above. The preferred embodiments are by direct injection
using needle injection.
The route of administration of any selected vector
construct will depend on the particular use for the expres-
sion vectors. In general, a specific formulation for each
vector construct used will focus on vector uptake with
regard to the particular targeted tissue, followed by
demonstration of efficacy. Uptake studies will include
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uptake assays to evaluate cellular uptake of the vectors and
expression of the DNA of choice. Such assays will also
determine the localization of the target DNA after uptake,
and establishing the requirements for maintenance of steady-
5 state concentrations of expressed protein. Efficacy and
cytotoxicity can then be tested. Toxicity will not only
include cell viability but also cell function.
Muscle cells have the unique ability to take up DNA
from the extracellular space after simple injection of DNA
10 particles as a solution, suspension, or colloid into the
muscle. Expression of DNA by this method can be sustained
for several months.
Delivery of formulated DNA vectors involves
incorporating DNA into macromolecular complexes that undergo
15 endocytosis by the target cell. Such complexes may include
lipids, proteins, carbohydrates, synthetic organic
compounds, or inorganic compounds. Preferably, the complex
includes DNA, a cationic lipid, and a neutral lipid in
particular proportions. The characteristics of the complex
20 formed with the vector (size, charge, surface character-
istics, composition) determines the bioavailability of the
vector within the body. Other elements of the formulation
function as ligand which interact with specific receptors on
the surface or interior of the cell. Other elements of the
25 formulation function to enhance entry into the cell, release
from the endosome, and entry into the nucleus.
Delivery can also be through use of DNA transporters.
DNA transporters refers to molecules which bind to DNA
vectors and are capable of being taken up by epidermal
30 cells. DNA transporters contain a molecular complex capable
of noncovalently binding to DNA and efficiently transporting
the DNA through the cell membrane. It is preferable that
the transporter also transport the DNA through the nuclear
membrane. See, e.g., the following applications all of
35 which (including drawings) are hereby incorporated by
reference herein: (1) Woo et al., U.S. Serial No.
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07/855,389, entitled "A DNA Transporter System and Method of
Use " filed March 20, 1992, now abandoned (2) Woo et al.,
PCT/US93/02725, International Publ. W093/18759, entitled "A
DNA Transporter System and Method of Use", (designating the
U.S. and other countries) filed March 19, 1993; (3)
continuation-in-part application by Woo et al., entitled
"Nucleic Acid Transporter Systems and Methods of Use", filed
December 14, 1993, U.S. Serial No. 08/167,641: (4) Szoka et
al. , U.S. Serial No. 07/913,669, entitled "Self-Assembling
Polynucleotide Delivery System", filed July 14, 1992 and (5)
Szoka et al., PCT/US93/03406, International Publ. W093/19768
entitled "Self-Assembling Polynucleotide Delivery System",
(designating the U.S. and other countries) filed April 5,
1993. A DNA transporter system can consist of particles
containing several elements that are independently and non-
covalently bound to DNA. Each element consists of a ligand
which recognizes specific receptors or other functional
groups such as a protein complexed with a cationic group
that binds to DNA. Examples of cations which may be used
are spermine, spermine derivatives, histone, cationic
peptides and/or polylysine. One element is capable of
binding both to the DNA vector and to a cell surface
receptor on the target cell. Examples of such elements are
organic compounds which interact with the asialoglycoprotein
receptor, the folate receptor, the mannose-6-phosphate
receptor, or the carnitine receptor. A second element is
capable of binding both to the DNA vector and to a receptor
on the nuclear membrane. The nuclear ligand is capable of
recognizing and transporting a transporter system through a
nuclear membrane. An example of such ligand is the nuclear
targeting sequence from SV40 large T antigen or histone. A
third element is capable of binding to both the DNA vector
and to elements which induce episomal lysis. Examples
include inactivated virus particles such as adenovirus,
peptides related to influenza virus hemagglutinin, or the
GALA peptide described in the Szoka patent cited above.
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Transfer of genes directly into a tumor has been very
effective. Experiments show that administration by direct
injection of DNA into tumor cells results in expression of
the gene in the area of injection. Injection of plasmids
containing human interferon alpha results in expression of
the gene for 5 days following a single intra-tumoral
injection. Human IFNa production was highest in tumors
harvested 1 day post-tumor injection and steadily declined
thereafter. The injected DNA appears to persist in an
unintegrated extrachromosomal state. This means of transfer
is a preferred embodiment.
Administration may also involve lipids as described in
preferred embodiments above. The lipids may form liposomes
which are hollow spherical vesicles composed of lipids
arranged in unilamellar, bilamellar, or multilamellar
fashion and an internal aqueous space for entrapping water
soluble compounds, such as DNA, ranging in size from 0.05 to
several microns in diameter. Lipids may be useful without
forming liposomes. Specific examples include the use of
cationic lipids and complexes containing DOPE which interact
with DNA and with the membrane of the target cell to
facilitate entry of DNA into the cell.
Gene delivery can also be performed by transplanting
genetically engineered cells. For example, immature muscle
cells called myoblasts may be used to carry genes into the
muscle fibers. Myoblast genetically engineered to express
recombinant human growth hormone can secrete the growth
hormone into the animal's blood. Secretion of the incorpor-
ated gene can be sustained over periods up to 3 months.
Myoblasts eventually differentiate and fuse to existing
muscle tissue. Because the cell is incorporated into an
existing structure, it is not just tolerated but nurtured.
Myoblasts can easily be obtained by taking muscle tissue
from an individual who needs gene therapy and the
genetically engineered cells can also be easily put back
with out causing damage to the patient's muscle. Similarly,
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keratinocytes may be used to delivery genes to tissues.
Large numbers of keratinocytes can be generated by
cultivation of a small biopsy. The cultures can be prepared
as stratified sheets and when grafted to humans, generate
epidermis which continues to improve in histotypic quality
over many years. The keratinocytes are genetically
engineered while in culture by transfecting the
keratinocytes with the appropriate vector. Although
keratinocytes are separated from the circulation by the
basement membrane dividing the epidermis from the dermis,
human keratinocytes secrete into circulation the protein
produced.
The chosen method of delivery should result in
expression of the gene product encoded within the nucleic
acid cassette at levels which exert an appropriate
biological effect. The rate of expression will depend upon
the disease, the pharmacokinetics of the vector and gene
product, and the route of administration, but should be in
the range 0.001-100 mg/kg of body weight /day, and
preferably 0.01-10 mg/kg of body weight/day. This level is
readily determinable by standard methods. It could be more
or less depending on the optimal dosing. The duration of
treatment will extend through the course of the disease
symptoms, possibly continuously. The number of doses will
depend upon the disease, delivery vehicle, and efficacy data
from clinical trials.
Examples
The present invention will be more fully described in
conjunction with the following specific examples which are
not to be construed in any way as limiting the scope of the
invention. As shown below, mIFN- gene medicine reduces the
growth of tumors in syngeneic murine tumor models. Lipid
formulations of mIFN- gene medicine display anti tumor
activity in both SCC-VII and MC-38 tumor models. PINC and
peptide formulations of mIFN- gene medicine display anti
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tumor effects in the MC-38 tumor model. The anti tumor
effects of mIFN- gene medicine are dose dependent. In
addition, the examples demonstrate that treatment of tumors
with the combination of IFNa and IL-12 gives an
unanticipated more than additive (synergystic) anti-tumor
activity using either a PINC or a lipid formulation.
Example 1
A plasmid expression system encoding murine IFNa4 and
formulated in a polymeric delivery system was used for in
vivo immunotherapeutic activity against an immunogenic
murine renal cell carcinoma, Renca, and a non-immunogenic
mammary adenocarcinoma, TS/A. Mice bearing established
tumors were treated with IFNa/polyvinyl-pyrrolidone (PVP)
expression complexes via direct intra-tumoral injection. Up
to 100 ~ tumor growth inhibition was observed in the treated
mice. By using an optimal dose of 96 and 48 ~g of
formulated IFN-a plasmid for the treatment of Renca and TS/A
respectively, 30$ (Renca) and 10~ (TS/A) of the treated
animals remained tumor-free. Tumor inhibition was dependent
upon activation of the immune system. The anti-tumor
activity elicited by IFN-a gene therapy was abrogated when
mice were selectively depleted of CD8+ T cells. By contrast,
removal of CD4+ resulted in increased tumor rejection
following IFN-a/PVP treatments. Finally, mice that remained
tumor-free following IFN-a gene therapy displayed immune
resistance to a subsequent challenge of tumor. These data
provide evidence that non-viral IFNa gene therapy can be
used to induce an efficient anti-tumor response.
Local presence of cytokines in tumors can activate an
immune response that in some cases leads to induction of
specific long-lasting anti-tumor immunity. By direct intra
tumoral injection of plasmid encoding murine IFNa4 and
formulated in a polymeric delivery system, tumor-bearing
mice develop an immune response, which leads to inhibition
and eradication of the tumor. We have shown by depletion
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studies in vivo that the immune response induced by IFNa is
mainly CD8-mediated and that this treatment results in a
long-term immunity in mice demonstrating complete tumor
regression. Thus, non-viral IFNa gene therapy may be an
5 effective alternative to IFNa protein therapy for human
cancers.
Transduction of tumor cells with cytokine genes has
proven to be a very efficient technique to induce cytokine
mediated anti-tumor immunity. In experimental models, the
10 local presence of IL-2, IL-1, IL-4, IL-6, IL-7, IL-12, IFNs
and . CSFs ( i . a . , GM-CSF) at the site of the tumor can result
in significant tumor growth inhibition (Colombo et al.,
"Local Cytokine Availability Elicits Tumor Rejection and
Systemic Immunity Through Granulocyte-T-Lymphocyte Cross-
15 Talk", Cancer Research, 52, 4853-4857 (1992)). In these
systems, cytokines have limited effect on tumor
proliferation directly but are capable of activating a rapid
and potent anti-tumor immune response, which impedes tumor
progression. Established parental tumors, however, are
20 difficult to eradicate with ex vivo cytokine-transduced
tumor cells because efficacy of vaccination is highly
dependent on the size, growth rate and invasiveness of the
tumor.
To overcome these problems, cytokine-based gene therapy
25 approaches, which can deliver transgenic cytokines locally
and induce an anti-tumor immune response, have been recently
evaluated by a number of investigators (Forni et al.,
"Cytokine-Induced Immunogenicity: From Exogenous Cytokines
to Gene Therapy", Journal of Immunotherapy, 14, 253-257,
30 (1993): Pericle et al., "An Efficient Th2-type Memory
Follows Cd8+ Lymphocyte-driven and Eosinophil-mediated
Rejection of a Spontaneous Mouse Mamary Adenocarcicoma
Engineered to Release I1-4", The Journal of Immunology, 153,
5660-5673. (1994); Pardoll et al., "Gene Modified Tumor
35 Vaccines, In Cytokine-Induced Tumor Immunogenicity", eds.
Academic Press, London, p. 71-86. (1994); and Musiani et
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76
al., "Cytokines, Tumor-cell Death and Immunogenicity: A
Question of Choice", Immunology Today. 1, 32-36 (1997)).
Technological breakthroughs in gene therapy using
adenoviral, retroviral, and liposomal vectors have provided
powerful tools with which to study the biological effects of
specific cytokine mediators as well as to develop novel and
clinically applicable anti-tumor immunotherapies (Pardoll,
"Paracrine Cytokine Adjuvants in Cancer Immunotherapy",
Annu. Rev. Immunol. 13, 399-415 (1995); Bramson et al.,
"Direct Intratumoral Injection of an Adenovirus Expressing
Interleukin-12 Induces Regression and Long-lasting Immunity
That Is Associated with Highly Localized Expression of
Interleukin-12", Hum. Gene Ther., 7, 1995-2002 (1996); Rao
et al., "I1-12 Is an Effective Adjuvant to Recombinant
Vaccinia Virus-based Tumor Vaccines", J. Immunol. 156,
3357-3365. 1996; Rakhmilevich et al., "Gene Gun-mediated
Skin Transfection with Interleukin 12 Gene Results in
Regression of Established Primary and Metastatic Murine
Tumors", Proc. Natl. Acad. Sci. USA. 93, 6291-6296 (1996);
and Rakhmilevich et al, "Cytokine Gene Therapy of Cancer
Using Gene Gun Technology: Superior Antitumor Activity of
Interleukin-12", Hum. Gene Ther. 8, 1303-1311, (1997)).
A gene therapy approach utilizing an interactive
polymeric gene delivery system that increases protein
expression by protecting plasmid DNA (pDNA) from nucleases
and controlling the dispersion and retention of pDNA in
muscle cells is described in Mumper et al., 1996. These
polymeric interactive non-condensing (PINC) systems
routinely result in a greater amount of gene expression from
tissues as compared to delivery of unformulated plasmid in
saline (Mumper et al., 1996). By using a plasmid that
encodes human insulin growth factor-1 (hIGF-1) and
formulated as a PINC complex, production of biologically
active h IGF-1 in vivo following intra-muscular injection
has been shown (Alila et al., "Expression of Biologically
Active Human Insulin-Like Growth Factor-1 Following
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Intramuscular Injection of a Formlated Plasmid in Rats",
Human Gene Therapy, 8, 1785-1795 (1997)). The specific
objective of this study was to determine whether a plasmid
expression system encoding murine IFNa4 and formulated as a
complex with PVP could induce an anti-tumor immune response
following direct injection into subcutaneous murine tumors.
The IFN family consists of three major glycoproteins,
IFNa, IFN(3 and IFNy. Although IFNs were first developed as
antiviral agents, it is now clear that they also control
cell growth and differentiation, and modulate various
aspects of host immunity (Gresser et al., "Antitumor effects
of interferon", Acta Oncol. 28, 347-353 (1989)). Clinical
data concluded that systemic chronic administration of IFNa
could produce regression of vascular tumors, including
Kaposi's sarcoma, pulmonary hemangiomastosis, and
hemangiomas (Singh et al., "Interferons A and B Down-
regulate the Expression of Basic Fibroblast Growth Factor in
Human Carcicomas", Proc. Nati. Acad. Sci. USA. 92, 4562-4566
(1995)). Although IFNa was the first cytokine to be used in
clinical trials that proved to be effective against certain
types of human cancer, only recently has this cytokine been
considered as a candidate for gene therapy (Ogura et al.
1993, Belldegrun et al., "Human Renal Carcinoma Line
Transfected With Interleukin-2 and/or Interferon a Gene(s):
Implications for Live Cancer Vaccines, Journal of the
National Cancer Institute, 85, 207-216 (1993).
Initial studies have shown that the injection of
genetically modified tumor cells producing IFNa into
syngeneic mice induces tumor growth inhibition and elicits a
tumor-specific immune memory (Ferrantini et al., Interferon
Alpha-1-Interferon Gene Transfer into Metastatic Friend
Lukemia Cells Abrogated Tumorigenicity in Immunocompetent
Mice: Antitumor Therapy by Means of Interferon-Producing
Cells: Cancer Res. 53, 1107-4615 (1993); Ferrantini et al.,
"Ifn-al Gene Expression into a Metastatic Murine
Adenocarcicoma (Ts/a) Results in Cd8+ T Cell-Mediated Tumor
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Rejection and Development of Antitumor Immunity: Comparative
Studies with Ifn-y-producing Ts/a Cells" Journal of
Immunology, 153, 4604-4615, (1994); Musiani et al. 1997).
However, the real value of this potential form of vaccine in
inducing the regression of established tumors remains to be
demonstrated.
In this study we present evidence that direct injection
of IFNa plasmid formulated in PVP into subcutaneous murine
tumors results in a host-dependent tumor rejection,
primarily mediated by CD8+ T cells, and elicits a protective
immunity against subsequent tumor re-challenge.
Materials And Methods
Plasmid construction and formulation
A plasmid expression system containing an expression
cassette for mIFN-la4 was constructed as follows. The coding
sequence of the murine IFN-a4 gene (Genebank X01973 M15956 M23830
X01967) was amplified by PCR from mouse genomic DNA. The
amplified mIFN-a4 sequence was then subcloned into a plasmid
backbone, and the sequence fidelity was verified by DNA sequence
analysis (data not shown). The coding sequence for mIFN-a4 was
then subcloned as an XbaI-BamHlfragment into the expression
plasmid pIL0697 to create the mIFN-a4 expression system pIF0836.
Plasmid pVC0612 (empty plasmid, EP) contains expression elements
including the cytomegalovirus immediate early promoter and the 3'
UTR/poly(A) signal from the bovine growth gene in the pVC0289
backbone described by Alila et al. (1997). Plasmid pVC0612 was
used as a control plasmid in all in vivo experiments. Plasmids
for intra-tumoral injection were grown under kanamycin selection
in E. coli host strains DHSa and purified using conventional
alkaline lysis and chromatographic methods. Purified plasmid
utilized for intra-tumoral injections had the following
specifications: endotoxin (< 500 Eu/mg plasmid); protein (< 1~);
and chromosomal DNA (< 20 $). Purified pIF0836 and control
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plasmids~were formulated at a concentration of 3 mg DNA/ml in a
solution of 5 ~ w/v polyvinyl-pyrrolidone (Plasdone C-30, ISP
Technologies, Wayne, NJ), 150 mM NaCl on the day of injection, as
described previously (Mumper et al., 1996).
Western blot analysis and bioassay for mIFNa.
HeLa cells were plated in 6 well plates at 3 x 105 cells
per well, and transfected using 1 a~,g of mouse IFNa4 plasmid
pIF0836C and 3 ~g of Lipofectamine (Life Technologies, Inc.,
Gaithersburg, MD) in serum-free DMEM. Same supernatants
were harvested 24 hours later and immunoprecipitated using
anti-mouse interferon a/(3 polyclonal antibody (BioSource
International, Camarillo, CA) and protein A and G agarose
(Boehringer Mannheim, Indianapolis, IN). Samples were run
on a 12$ Tris-glycine gel and electroblotted to Millipore
PVDF membrane. Anti-mouse interferon a/(3 polyclonal antibody
was used at 1:1000, followed by anti-sheep Ig HRP
(Boehringer Mannheim) at 1:1000. Biotinylated molecular
weight markers were detected using Streptavidin-HRP
(Amersham, Arlington Heights, IL). Detection was performed
using the Amersham ECL kit. Supernatants were also tested
for IFNa biological activity using L929 cells treated with
encephalomyocarditis virus, in parallel with a NIH mouse
IFNa reference reagent (Access Biomedical, San Diego, CA).
Animals
Normal 8-week-old female BALB/c mice were purchased
from Harlan Laboratories, Houston, TX. Mice were maintained
on ad libitum rodent feed and water at 23° C, 40$ humidity,
and a 12-h/12-h light-dark cycle. Animals were acclimated
for at least 4 days before the start of the study.
Tumors
Three established mouse tumor models were used in this
study. TS/A is a tumor cell line established by Dr. P.
Nanni, University of Bologna, Italy, from the first in vivo
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transplant of a moderately differentiated mammary
adenocarcinoma that spontaneously arose in a BALB/c mouse
(Nanni et al., 1983). A number of pre-immunization-
challenge experiments suggested that TS/A does not elicit a
5 long-lasting anti-tumor immunity (Forni et al., 1987). TS/A
was generously provided by Dr. Guido Forni, University of
Turin, Italy. Renca, a spontaneously arising murine renal
cell carcinoma, and CT-26, a colon adenocarcinoma, were
generously provided by Dr. Drew M. Pardoll, John Hopkins
10 Hospital, Baltimore, MD. Tumor cell cultures were
maintained in sterile disposable flasks from Corning
(Corning, NY) at 37° C in a humidified 5~ COZ atmosphere,
using RPMI 1640 supplemented with 10$ FBS, 100 U/ml
penicillin, 100 U/ml streptomycin and 50 ~.g/ml gentamycin;
15 all from Life Technologies.
In vivo evaluation of tumor growth and treatments
BALB/c mice were challenged s.c. in the middle of the
left flank with 30 ~1 of a single-cell suspension contained
the specified number of cells. Seven days later when the
20 tumor size reached approximately 10 mm3, treatments with
IFNa/PVP or EP/PVP started and were repeated at 1-2 day
intervals for 2 weeks (total of 8 treatments: 4/week).
Tumor volume was measured with electronic caliper in the two
perpendicular diameters and in the depth. Measurements of
25 the tumor masses (mm3) were performed twice a week for 40-50
days. All mice bearing tumor masses exceeding 1 cm3 volume
were sacrificed for humane reasons. When depletion of
immunocompetent cells in vivo was required, a group of mice
received i.v 0.5 ml of a-CD4 (GK1.5 hybridoma, 207-TIB,
30 ATCC, Rockville, MD) ascite (1:10), or a-CD8 (2.43
hybridoma, 210-TIB, ATCC) ascite (1:100) or i.p. 100 ~.g
a-GRl (RB6-8C5 hybridoma, Pharmingen, San Diego, CA).
Control mice received i.v. 0.5 ml isotype control IgG
(Pharmingen). Antibody treatments were performed twice:
35 first injection 1 day before starting the gene therapy
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treatment and the second injection (i.p at the same dosage)
7 days later.
CTL assay
A standard 6-hour 51-chromium (SlCr)-release assay was
performed following 5 days of in vitro effector cell
stimulation. Single cell suspensions of splenocytes were
prepared 3 weeks following tumor challenge by mashing the
spleens in RPMI 1640 medium (Life Technologies) and passing
the cells through 70 Eun nylon mesh cell strainers (Falcon,
Becton Dickinson, Lincoln Park, NJ) into 50m1 centrifuge
tubes (Falcon). After centrifugation, red blood cells were
lysed with ACK Lysing Buffer (Biofluids, Inc., Rockville,
MD) and the splenocytes washed twice with RPMI. In vitro
stimulation cultures contained 3 X 106 splenocytes/effectors
per ml with 6 X 105 mitomycin-C-treated Renca/stimulator
cells per ml and 10 Units per ml recombinant murine IL-2
(Genzyme, Cambridge, MA) in RPMI containing 10$ FBS, 22mM
HEPES buffer (Research Organics Inc., Cleveland OH), Penn-
Streptomycin, 5 X 10-5 M 2-(3-mercapto-ethanol (Life
Technologies), OPI media supplement (Sigma, St. Louis, MO),
and essential and non-essential amino acids (Life
Technologies) (for a 5 . 1 responder . stimulator ratio).
Stimulators were prepared by incubating Renca cells at 3 X
10' per ml in RPMI with 30 )tg per ml mitomycin-C (Sigma) at
37° C for 60 minutes, followed by four washes in HBSS with
2.5~ FBS. After 5 days at 37° C, effector cells were
pelleted, resuspended in complete RPMI, counted, and mixed
with SlCr -labeled targets in a 96 well V-bottomed plate
(Costar/Corning, Cambridge, MA). Renca and CT26 targets
were labeled by incubating them at 2 X 106 cells per ml in
complete RPMI with 150 uCi SlCr (Amersham) for 2.5 hours.
Targets were washed 3 times in HBSS with 2.5~ FBS and
resuspended in complete RPMI before addition to the assay.
After mixing effectors and targets (in triplicate wells) and
a brief pelleting, plates were placed at 37° C for 6 hours.
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Approximately 90~ of the supernatants were then collected
from each well with the Skatron Harvesting Press and
Supernatant Collection System (Skatron Instruments, Norway).
SiCr release was detected using a WALLAC 1470 Wizard
automatic gamma counter (WALLAC Inc., Gaithersburg MD).
Specific release was determined with the following equation:
(experimental cpm - spontaneous cpm) / (total cpm-
spontaneous cpm) X 100. Spontaneous release from the
targets was less than 18~, and the standard error of the
triplicate experimental counts was less than 14$.
Statistical analysis
Data for the effects of mIFN-a gene therapy on tumor
growth were analyzed by repeated measures analysis.
Individual treatment means were compared using Duncan's
multiple range test when the main effect was significant.
Data for the effect of mIFN-a gene therapy on tumor
rejection were analyzed by ANOVA. In all cases a p value of
less than 0.05 was considered to be statistically
significant.
Results
Expression of mIFN-a
Murine IFN-a expression plasmid (pIF0836) was
transfected into Cos-1 cells, and the resulting conditioned
media was assayed for mIFN-a by Western blot and by
bioassay. Western blot analysis of conditioned media
indicated that the recombinant mIFN-a expressed from pIF0836
template was present as a single band with an approximate
molecular weight of 23 kDa. This band was not observed in
conditioned media from mock-transfected cells and likely
represents a glycosylated form of mIFN-a. Recombinant mIFN-
a ran with an approximate molecular weight of 18 kDa, which
corresponds to the predicted molecular weight of non-
glycosylated mIFN-a. Assay of conditioned media using an
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anti-viral bioassay for mIFN-a indicated that approximately
175 x 103 IU/ml mIFN-a were present.
Anti-tumor activity of IFN-a gene therapy. The anti
tumor effect of murine IFNa4 plasmid formulated as a complex
with PVP (IFNa/PVP) was tested in a syngeneic murine renal
cell carcinoma (Renca) and a mammary adenocarcinoma (TS/A)
tumor model. BALB/c mice were challenged subcutaneously
with 7 X105 Renca or 1 X105 CT26 cells, and IFNa/PVP
injections were initiated 7 days later when tumors reached
approximately 10 mm3 size. Each group of mice received at
interval of 1-2 days 8 treatments (4 injections/week) of
IFNa/PVP at scalar doses (from 12 to 96 ~g/mouse), EP/PVP
(96 ~,g/mouse) or no treatments for control (ctrl). Tumor
size increased progressively in mice injected with EP/PVP
(Renca, TS/A) or low doses of IFNa/PVP (TS/A), while tumors
in mice injected with each dose of IFNa/PVP (Renca) or high
dose of IFNa/PVP (TS/A) showed marked growth inhibition.
Tumor growth inhibition is associated to systemic immune
response
Treatments of Renca and TS/A tumors with IFNa/PVP at 96
~.g/mouse and 48 ~.g/mouse respectively, induced complete
regression in 6 out of 20 (Renca) and 2 out of 20 (TS/A) of
challenged mice. To test whether the rejection of these
tumors leads to specific immune memory, mice with no tumors
for 40-50 days following IFNa treatments were re-challenged
with a greater number of fresh tumors in the right flank.
All mice that rejected primary tumors displayed protection
against the second tumor challenge whereas mice used as the
control group and injected for the first time with same
number of tumor cells (1 X 106 Renca or 2 X 105 TS/A)
developed tumors.
To evaluate the requirements for the induction of anti-
tumor immune memory, Renca and TS/A were injected into
BALB/c rendered immunosuppressed by treatment with anti-CD4,
anti-CD8 or anti-polymorphonuclear cells (PMN). Depletion
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of CD8+ T cells allowed both Renca and TS/A to grow in all
animals following IFNa/PVP treatments, showing that this
population is crucial for the immune response induced by
IFNa gene therapy. No increase in tumor growth was found in
mice treated with anti-PMN (a-GR1) monoclonal Ab (mAb).
Increase in tumor rejection was observed in mice depleted of
CD4+ T and treated with IFNa/PVP suggesting that depletion
of CD4+ T cells can enhance the anti-tumor effect of IFNa
gene therapy.
Expression of IFN-a within the tumor induces a CTL
response. To assess whether CD8+ tumor specific CTL were
induced in vivo by IFNa/PVP treatments, splenocytes from
Renca tumor-challenged mice were tested for their cytolytic
activity following IFNa gene therapy. Cytotoxic activity
against Renca, and not against CT26 cells used as control
for tumor specificity, was found in 2 of 9 mice that had
received IFNa gene therapy. Moreover, splenocytes from mice
depleted of CD4+ T cells and treated with IFNa/PVP
demonstrated potent CTL activity against Renca cells. By
contrast, little CTL activity against Renca cells was
evident from splenocytes isolated from mice treated with
EP/PVP.
Discussion
The data reported herein demonstrate that direct
administration of IFNa gene formulated in a polymeric
delivery system into subcutaneous renal cell carcinoma and
mammary adenocarcinoma inhibits tumor growth and induces a
long-lasting immunity to secondary tumor challenges. Of
considerable significance is the fact that the anti-tumor
response was observed against both an immunogenic carcinoma
as well a more aggressive and poorly immunogenic
adenocarcinoma, a phenotype which is similar to many
spontaneously arising tumors in man.
A variety of genetic abnormalities arise in human
cancers that contribute to neoplastic transformation and
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malignancy. Despite increasing understanding of the
molecular basis of cancer, many malignancies remain
resistant to established forms of treatment. More recently,
molecular genetic interventions have been designed in an
5 attempt to improve the efficacy of immunotherapy. While
numerous experimental studies have been performed in marine
models with tumor cells transduced with cytokine-gene ex
vivo, a major limitation in the translation of this strategy
to large-scale human tumor vaccine therapy is the labor
10 intensity and variability of establishing each individual
tumor in culture and transducing it with an appropriate
vector (i.e., retrovirus). Our approach to this problem is
to use a non-viral delivery system to modify tumor cells in
vivo with cytokine cDNAs so that the tumor cells can supply
15 the cytokine of interest in a paracrine fashion to the anti-
tumor responder cells present within the tumor.
Using a plasmid containing IFNa4 gene and formulated in
PVP, we have shown that intra-tumoral injections of this
DNA-PINC complex can lead to complete tumor regression in 30
20 ~ of the cases (Renca model) with an overall response rate
of 100 $ tumor growth inhibition. These results are in
agreement with a recent study that described an anti-tumor
activity elicited by genetically modified TS/A cells
producing marine IFN-al (Ferrantini et al., 1994). Although
25 the anti-tumor effect of IFNa using cytokine-gene transduced
tumor cells has been described (Scarpa et al.,
"Extracellular Matrix Remodelling in a Marine Mamary
Adenocarcicoma Transfected with the Interferon-alphal Gene",
Journal of Pathology. 181, 116-123 1997), the real value of
30 IFNa gene therapy in blocking or inhibiting advanced tumors
remains to be explored. The advantage of using a non-viral
IFNa gene delivery system over retrovirus is that tumor
cells could be transduced directly in vivo without the need
of first establishing tumor cells in vitro. Moreover, IFNa
35 has a potent anti-viral activity limiting the use of this
gene in combination with viral vectors.
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Therapeutic levels of gene expression for IGF-I using a
similar interactive PVP-based delivery system have previusly
been described (Alila et al., 1997). Direct intra-tumor
injection of the same PINC delivery system as a complex with
IFNa gene, resulted in dispersion in vivo of plasmid into
target cells inducing an IFNa-specific anti-tumor activity.
Tumors treated with the same plasmid but in the absence of
IFNa coding region and formulated as a complex with PVP, did
not respond to this treatment and grew in a similar rate to
untreated tumors. By using an optimal dose of IFNa/PVP,
tumor-bearing mice were able to reject the tumors mounting a
specific long-lasting tumor immunity. Although, the numbers
of mice rejecting a second tumor challenge was low, this
observation indicates that a considerable portion of the
activity of IFNa in inducing the rejection of established
tumors is not anti-angiogenic or anti-proliferative but
immunostimulatory. Our result demonstrating that IFNa-
induced regression of advanced tumors was prevented by in
vivo administration of anti-CD8 mAb provides direct evidence
for a key role of CD8+ T cells in the anti-tumor effect of
IFNa.
Depletion of CD4+ T cells in tumor-bearing mice treated
with IFNa gene therapy significantly enhanced the
therapeutic effect of IFNa, resulting in tumor regression
and prolonged survival of up to 80$ of treated mice. A CD4-
mediated suppression during tumor progression has been
previously reported and it has also been shown that
depletion of CD4+ T cells in tumor-bearing mice results in
augmentation of anti-tumor therapy with either IL-2 or IL-I2
(Rackmilevich et al., 1994 and Martinotti et al., "Cd4 T
Cells Inhibit in Vivo the Cd8-Mediated Immune Response
Against Murine Colon Carcinoma Cells Transducted with
Interleukin-12 Genes", Eur. J. Immunol, 25, 137-146.
( 1995 ) ) . They have shown that depletion of CD4 +T cells in
tumor-bearing mice in the absence of treatment did not
affect the growth of tumor itself suggesting that removal of
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CD4+ T cells does not deprive the tumor of growth factors
(Rackmilevich et al., 1994). A possible explanation for
this phenomenon is that depletion of CD4+ T cells from tumor-
bearing mice augments the anti-tumor efficacy of IFNa-
activated CD8+ T cells by releasing them from
immunosuppression. The mechanism driving CD4 suppression is
poorly understood, although Th2 type cytokines, directly or
through B cell activation, may inhibit cell-mediated
immunity (Mossman et al., 1989; Powrie et al., Eur-J-
Immunol, 23(11):3043-9 (1993)). CTL can be generated in
both CD4-depleted and non-depleted mice from lymphocytes
obtained from spleens by in vitro re-stimulation with
mitomycin-treated Renca cells and rIL-2. Thus, CD4-mediated
suppression appears to be exerted on CD8 expansion and not
priming. In accord with the in vivo results, stronger CTL
activity was observed from mice depleted of CD4 and treated
with IFNa/PVP suggesting CD4+ T cells inhibit an IFNa-
mediated CD8+ T cell response in vivo. This study suggests
that direct administration of cytokine genes, like IFNa,
into tumors, which have been found to suppress malignancy
growth, provide a new therapeutic option for the treatment
of human cancers.
Example 2: Pharmacology of mIFN - Gene Medicine in Syngeneic
Tumor Models
Gene expression systems encoding either mIFN-2 or mIFN-
4 were tested for anti tumor activity when formulated in
either cationic lipid, peptide, or PINC delivery systems and
injected intratumoraliy into subcutaneous squamous cell
carcinoma (SCC-VII) or adenocarcinoma (MC-38) tumors.
Experimental design and treatment regimen
Experiments to test the anti tumor activity of mIFN-
gene medicine were conducted in either SCC-VII or MC-38
tumor models. Tumor cells (4 x 105) were injected
subcutaneously into the flank region of mice, and treatment
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was initiated when tumor volume reached approximately 50 mm3.
Treatment was begun approximately 6 days (SCC-VII tumors)
and 10 days (MC-38 tumors) after tumor initiation and
repeated at 3 to 5 day intervals.
All formulations of mIFN- gene medicine were
administered in a dose volume of 50 ul. The faster growing
SCC-VII tumors typically received 3 treatments, whereas the
relatively slower growing MC-38 tumors typically received 4
treatments. Experiments were terminated when lactose
vehicle control tumors reached approximately 1000 mm3.
The anti-tumor effects of murine IFN gene medicine
(IFNa/PVP) was tested in syngeneic murine renal cell
carcinoma (Renca) and mammary adenocarcinoma (TS/A) tumor
model. BALB/c mice were challenged subcutaneously with 7
X105 or 1 X105 CT26, and IFNa/PVP injections were initiated 7
days later when tumors reached approximately 10 mm3 size.
Each group of mice received 8 treatments (4 injections for 2
weeks) of IFNa/PVP at scalar doses (from 12 to 96 ~,g/mouse),
empty plasmid/PVP (EP/PVP, 96 ~tg/mouse) or no treatments for
control (ctrl). Tumor size increased progressively in mice
injected with EP/PVP (Renca, TS/A) or low doses of IFNa/PVP
(TS/A), while tumors in mice injected with each dose of
IFNa/PVP (Renca) or high dose of IFNa/PVP (TS/A) showed
marked growth inhibition.
Example 3: mIFN- Gene Medicine Formulated in Cationic lipid
Reduces the Growth of SCC-VII Tumors
Experiments were conducted in the SCC-VII tumor model
as described in the preceding example. mIFN- gene medicine
formulated in cationic lipid, peptide, and PINC delivery
systems was tested. Results show that cationic lipid
formulations significantly reduce the growth of SCC-VII
tumors relative both to lactose vehicle injected tumors and
to tumors injected with control (non coding) plasmid
formulated in cationic lipid. The effect of mIFN- gene
medicine formulated in cationic lipid is dose dependent and
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there is no effect of mIFN- gene medicine when formulated
in PVA. In addition, analysis of tumors from this
experiment using immunohistochemical methods revealed
infiltration of CD8+ lymphocytes in tumors injected with
cationic lipid formulations, but not in tumors injected with
PVA formulations.
mIFN- gene medicine significantly reduces the growth of
SCC-VII tumors as compared to control plasmid or lactose
injected tumors. Differences between control plasmid and
mIFN- plasmid were consistent across formulation. Plasmid
dose was 46 pg/treatment. Growth of tumors injected with
control plasmid was compared to that of tumors injected with
mIFN- gene medicine using repeated measures analysis.
mIFN- reduced SCC-VII tumor growth relative to control
plasmid (p=.035).
Example 4: mIFN- Gene Medicine Reduces the Growth of MC-38
Tumors
Experiments were carried out as described in the
preceding examples. The effects of various prototype
formulations of mIFN- gene medicine on the growth of
subcutaneous MC-38 tumors were compared. mIFN- gene
medicine elicited reduced tumor growth in all formulations
tested (cationic lipid, peptide, and PINC). Subsequent
experiments in the MC-38 tumor model have shown similar
results.
Example 5: Dose Responses
After demonstrating anti tumor effects of mIFN- gene
medicine, the dose response for these effects was
investigated in the MC-38 tumor model. Both cationic lipid
(DOTMA:Chol) and PINC (PVA) delivery systems were evaluated.
Results clearly show that mIFN- gene medicine elicited a
dose dependent reduction in tumor growth. Tumor volume in
this experiment was maximally reduced by approximately 50 ~
with mIFN- /DOTMA:Chol and 60 ~ with mIFN- /PVA after 4
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treatments. Maximal reduction in tumor volume was observed
at a plasmid dose of approximately 50 ug/treatment
(cumulative dose of approximately 200 ug). These
experiments will be conducted primarily in the MC-38 tumor
5 model because it provides a broader treatment window than
does the SCC-VII model.
Example 6: IFN-alpha Formulations
The formulations for the IFN-a are: (1) PVP 4 vial, (2)
PVP three vial, (3) PVP two vial. The details are listed
10 below:
PVP 4 vial
Materials: 25~ PVP (50 kDa) stock solution, plasmid
stock solution, 5 M NaCl stock solution, and water.
Method: Add in order of water, plasmid, 25$ PVP and 5 M
15 NaCl into a vial to make a plasmid in 5~ PVP in saline
formulation. The final concentration of PVP and NaCl are
fixed (5~ and 150 mM) and plasmid concentration could be
varied (but based on the IGF-1 data, 3 mg DNA/ml in 5g PVP
in saline should be the best formulation). The quality of
20 the formulation is characterized by pH, DNA concentration,
osmolality, and gel electrophoresis. The DNA concentration
could be varied from 0.1-5 mg/ml. The pH may be varied from
3-5, osmolality may be 250 - 400 mOsm.
Three vial
25 Material: lyophilized PVP, plasmid stock solution (4
mg/ml), 115 mM Na-Citrate/5~ NaCl stock buffer (pH = 4).
Method: Add in order of plasmid and buffer into the PVP
to make final 3 mg DNA/ml in 5~ PVP in 25 mM citrate/saline
buffer (pH =4) . DNA expression is higher in saline than in
30 the citrate buffer.
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m..." ~~; ~ i
Materials: Co-lyophilized plasmid and PVP, saline. Add
saline into the co-lyophilized DNA and PVP to make final 3
mg/ml DNA in 5$ PVP in saline.
The final formulation is 3mg/mL DNA, 5~ PVP as a single
vial. The formulation is prepared by adding (5~) PVP to
(4mg/mL) DNA and recirculating the two components for a
finite period of time (using static mixer). Then the
formulation is lyophilized and rehydrated with 0.9~ sodium
chloride, to obtain a final composition of 3mg/mL, 5$PVP in
saline.
Example 7: Treatment of Human Tumors
The murine studies are predictive of the response of
Human tumors to therapy using a plasmid construct encoding
the human IFN alpha gene sequence such as that depicted in
SEQ ID NO: 10, 11 or 12. A patient in need of anti-cancer
therapy is injected with up to 3mg of plasmid formulation at
daily intervals. The plasmid formulation may contain INF
alpha plasmid alone or optionally a mixture of IFN-alpha
encoding plasmid and an additional plasmid coding for a
cytokine. The preffered cytokine is IL-12. The treatments
are continued and the patient monitored as is the usual
practice for anti-cancer chemotherapeutic regimes.
One skilled in the art would readily appreciate that
the present invention is well adapted to carry out the
objects and obtain the ends and advantages mentioned, as
well as those inherent therein. The molecular complexes and
the methods, procedures, treatments, molecules, specific
compounds described herein are presently representative of
preferred embodiments are exemplary and are not intended as
limitations on the scope of the invention. Changes therein
and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention are
defined by the scope of the claims.
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It will be readily apparent to one skilled in the art
that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the
scope and spirit of the invention.
All patents and publications mentioned in the speci-
fication are indicative of the levels of those skilled in
the art to which the invention pertains. All patents and
publications are herein incorporated by reference to the
same extent as if each individual publication was specific-
ally and individually indicated to be incorporated by
reference.
The invention illustratively described herein suitably
may be practiced in the absence of any element or elements,
limitation or limitations which is not specifically
disclosed herein. Thus, for example, in each instance
herein any of the terms "comprising", "consisting
essentially of" and "consisting of" may be replaced with
either of the other two terms. The terms and expressions
which have been employed are used as terms of description
and not of limitation, and there is no intention that in the
use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus,
it should be understood that although the present invention
has been specifically disclosed by preferred embodiments and
optional features, modification and variation of the
concepts herein disclosed may be resorted to by those
skilled in the art, and that such modifications and
variations are considered to be within the scope of this
invention as defined by the appended claims.
In addition, where features or aspects of the invention
are described in terms of Markush groups, those skilled in
the art will recognize that the invention is also thereby
described in terms of any individual member or subgroup of
members of the Markush group. For example, if X is
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described as selected from the group consisting of bromine,
chlorine, and iodine, claims for X being bromine and claims
for X being bromine and chlorine are fully described.
Other embodiments are within the following claims.
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1
Sequence Listing Part
<110> NORDSTROM, JEFF; PERICLE, FEDERICA; ROLLAND,
ALLAIN; RALSTON, ROBERT
<120> INTERFERON ALPHA PLASMIDS AND DELIVERY SYSTEMS,
AND METHODS OF MAKING AND USING THE SAME
<150> US 08/949,160 and PCT/US97/18779
<151> October 10, 1997
<160> 25
<210> 1
<211> 328
<212> amino acid
<400> 1
Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu
1 5 10 15
Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
25 30
20 Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
50 55 60
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys
65 70 75 80
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val
85 90 95
Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
100 105 110
Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
115 120 125
Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
130 135 140
Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg
145 150 155 160
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser
165 170 175
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190
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Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
195 200 205
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr
210 215 220
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
225 230 235 240
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp
245 250 255
Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
260 265 270
Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg
275 280 2g5
Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala
290 295 300
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
305 310 315 320
Glu Trp Ala Ser Val Pro Cys Ser
325
<210> 2
<211> 987
<212> nucleic acid
<400> 2
ATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTC60
3 GTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGAT120
5
GCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGG180
ACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAA240
GAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCG300
CTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAG360
4 AAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTC420
O
ACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGA480
GGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTC540
AGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCA600
GCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTAT660
4 GAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAAC720
5
TTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGAC780
ACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGB40
AGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGC900
CGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGC960
5 GAATGGGCATCTGTGCCCTGCAGTTAG 9g7
O
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3
<210> 3
<211> 987
<212> nucleic acid
<400> 3
ATGTGCCACCAGCAGCTGGTGATCAGCTGG TGTTCCTGGCCAGCCCCCTG60
TTCAGCCTGG
GTGGCCATCTGGGAGCTGAAGAAGGACGTGTACGTGGTGGAGCTGGACTGGTACCCCGAC120
GCCCCCGGCGAGATGGTGGTGCTGACCTGCGACACCCCCGAGGAGGACGGCATCACCTGG180
ACCCTGGACCAGAGCAGCGAGGTGCTGGGCAGCGGCAAGACCCTGACCATCCAGGTGAAG240
1 GAGTTCGGCGACGCCGGCCAGTACACCTGCCACAAGGGCGGCGAGGTGCTGAGCCACAGC300
O
CTGCTGCTGCTGCACAAGAAGGAGGACGGCATCTGGAGCACCGACATCCTGAAGGACCAG360
AAGGAGCCCAAGAACAAGACCTTCCTGCGCTGCGAGGCCAAGAACTACAGCGGCCGCTTC420
ACCTGCTGGTGGCTGACCACCATCAGCACCGACCTGACCTTCAGCGTGAAGAGCAGCAGG980
GGCAGCAGCGACCCCCAGGGCGTGACCTGCGGCGCCGCCACCCTGAGCGCCGAGCGCGTG540
1 CGCGGCGACAACAAGGAGTACGAGTACAGCGTGGAGTGCCAGGAGGACAGCGCCTGCCCC600
5
GCCGCCGAGGAGAGCCTGCCCATCGAGGTGATGGTGGACGCCGTCCACAAGCTGAAGTAC660
GAGAACTACACCAGCAGCTTCTTCATCCGCGACATCATCAAGCCCGACCCCCCCAAGAAC720
CTGCAGCTGAAGCCCCTGAAGAACAGCCGCCAGGTGGAGGTGAGCTGGGAGTACCCCGAC780
ACCTGGAGCACCCCCCACAGCTACTTCAGCCTGACCTTCTGCGTGCAGGTGCAGGGCAAG840
2 AGCAAGCGCGAGAAGAAGGACCGCGTGTTCACCGACAAGACCAGCGCCACCGTGATCTGC900
O
CGCAAGAACGCCAGCATCAGCGTGCGCGCCCAGGACCGCTACTACAGCAGCAGCTGGAGC960
GAGTGGGCCAGCGTGCCCTGCAGCTAG 987
<210> 4
25 <211> 987
<212> nucleic acid
<400> 4
ATGTGCCACCAGCAGCTGGTGATCAGCTGGTTCTCCCTGGTGTTTCTGGC 60
CAGCCCCCTC
3 GTGGCCATCTGGGAGCTGAAGAAAGACGTGTACGTGGTCGAGCTGGACTGGTACCCCGAC120
O
GCCCCCGGCGAGATGGTGGTCCTGACCTGCGACACCCCCGAGGAAGACGGCATCACCTGG180
ACCCTGGACCAGAGCAGTGAGGTGCTGGGCTCCGGCAAGACCCTGACCATCCAGGTGAAG240
GAGTTCGGCGACGCCGGCCAGTACACCTGCCACAAGGGAGGCGAGGTGCTGAGCCACTCC300
CTCCTGCTGCTCCACAAAAAGGAGGACGGCATCTGGAGCACCGACATCCTGAAGGACCAG360
3 AAGGAGCCCAAGAACAAGACCTTCCTGCGCTGCGAGGCCAAGAACTACAGCGGCCGCTTC420
5
ACCTGCTGGTGGCTGACCACGATCAGCACCGACCTGACCTTCAGTGTGAAGAGCAGCAGG480
GGCTCCAGCGACCCCCAGGGCGTGACCTGCGGCGCTGCCACCCTGAGCGCCGAGCGCGTG540
CGCGGCGACAACAAGGAGTACGAGTACAGCGTGGAGTGCCAGGAAGACTCCGCCTGCCCC600
GCCGCTGAGGAGAGCCTGCCCATCGAGGTGATGGTGGACGCCGTTCACAAGCTGAAGTAC660
4 GAGAACTACACCAGCAGCTTCTTCATCCGCGACATCATCAAGCCTGACCCACCCAAGAAC720
O
CTCCAGCTGAAGCCCCTCAAGAACTCCCGCCAGGTGGAGGTGAGCTGGGAGTACCCCGAC780
ACCTGGAGCACGCCCCACTCCTACTTCTCCCTGACCTTCTGCGTGCAGGTCCAGGGCAAG840
AGCAAGCGCGAGAAGAAAGACCGGGTGTTCACCGACAAGACCAGCGCCACCGTCATCTGC900
CGCAAGAACGCCAGCATCAGCGTGCGCGCCCAGGACCGCTACTATAGCTCCTCTTGGAGC960
4 GAGTGGGCCAGCGTGCCCTGCTCCTAG 987
5
<210> 5
<211> 219
<212> amino acid
50 <400> 5
Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val Zeu Leu
1 5 10 15
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Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro
20 25 30
Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val
35 40 45
Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
50 55 60
Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser
65 70 75 80
Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
85 90 95
Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala
100 105 110
Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr
115 120 125
Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys
130 135 140
Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu
145 150 155 160
Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr
165 170 175
Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
180 185 190
Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr
195 200 205
Ile Asp Arg Val Thr Ser Tyr Leu Asn Ala Ser
210 215
<210> 6
<211> 660
<212> nucleic acid
<400> 6
4 5 ATGTGTCCAG CGCGCAGCCT CCTCCTTGTG GCTACCCTGG TCCTCCTGGA CCACCTCACT 60
TTGGCCAGAA ACCTCCCCGTGGCCACTCCAGACCCAGGAA 120
TGTTCCCATG
CCTTCACCAC
TCCCAAAACC TGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACA AACTCTAGAA180
TTTTACCCTT GCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGA TAAAACCAGC240
ACAGTGGAGG CCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCT AAATTCCAGA300
S O GAGACCTCTT TCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTC TTTTATGATG360
GCCCTGTGCC TTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGA GTTCAAGACC420
ATGAATGCAA AGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCA AAACATGCTG480
GCAGTTATTG ATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGT GCCACAAAAA540
TCCTCCCTTG AAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCAT ACTTCTTCAT600
5 5 GCTTTCAGAA TTCGGGCAGTGACTATTGACAGAGTGACGAGCTATCTGAA TGCTTCCTAA660
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<210> 7
<211> 660
<212> nucleic acid
<400> 7
ATGTGCCCCG CCCGCAGCCTGCTGCTGGTGGCCACCCTGGTGCTGCTGGACCACCTGAGC60
CTGGCCCGCA ACCTGCCCGTGGCCACCCCCGACCCCGGCATGTTCCCCTGCCTGCACCAC120
AGCCAGAACC TGCTGGCGGCCGTGAGCAACATGCTGCAGAAGGCCGCGCAGACCCTGGAG180
TTCTACCCCT GCACCAGCGAGGAGATCGACCACGAGGACATCACCAAGGACAAGACCAGC240
1 O ACCGTGGAGGCCTGCCTGCCCCTGGAGCTGACCAAGAACGAGAGCTGCCTGAACAGCCGC300
GAGACCAGCT TCATCACCAACGGCAGCTGCCTGGCCAGCCGCAAGACCAGCTTCATGATG360
GCCCTGTGCC TGAGCAGCATCTACGAGGACCTGAAGATGTACCAGGTGGAGTTCAAGACC420
ATGAACGCCA AGCTGCTGATGGACCCCAAGCTCCAGATCTTCCTGGACCAGAACATGCTG480
GCCGTGATCG ACGAGCTGATGCAGGCCCTGAACTTCAACAGCGAGACCGTGCCCCAGAAG540
1 5 AGCAGCCTGGAGGAGCCCGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCAC600
GCCTTCCGCA TCCGCGCCGTGACCATCGACCGCGTGACCAGCTACCTGAACGCCACCTGA660
<210> 8
<211> 660
20 <212> nucleic acid
<400> 8
ATGTGCCCCG CCCGCAGCCTGCTGCTCGTGGCCACCCTGGTGCTCCTGGACCACCTCAGC60
CTGGCCCGCA ACCTCCCCGTGGCCACCCCAGACCCCGGCATGTTCCCATGCCTGCACCAC120
2 5 AGCCAGAACCTGCTGGCGGCCGTGAGCAACATGCTGCAGAAGGCCGCGCAGACCCTGGAG180
TTCTACCCCT GCACCAGCGAGGAGATCGACCACGAGGACATCACCAAGGACAAGACCAGC240
ACCGTGGAGG CCTGCCTGCCCCTCGAGTTAACCAAGAACGAGAGCTGCCTCAACAGCCGC300
GAGACCTCCT TCATCACCAACGGCACTTGCCTGGCCTCCCGCAAGACCAGCTTCATGATG360
GCCCTGTGCC TGAGCTCCATCTACGAGGACCTGAAGATGTACCAGGTGGAGTTCAAGACC920
3 O ATGAACGCCAAGCTCCTGATGGACCCCAAGCTCCAGATCTTCCTGGACCAGAACATGCTG480
GCCGTGATCG ACGAGCTGATGCAGGCCCTGAACTTCAACAGCGAGACCGTGCCCCAGAAG540
AGCAGCCTGG AGGAGCCCGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGCTGCAC600
GCCTTCCGCA TCCGGGCCGTGACCATCGACCGCGTGACCAGCTACCTGAACGCCACGTGA660
35 <210> 9
<211> 188
<212> amino acid
<400> 9
40 Met. Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys
1 5 10 15
Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu
20 25 30
Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser
35 40 45
Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu
50 55 60
Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His
65 70 75 80
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Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
85 90 95
Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
100 105 110
Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val
115 120 125
Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys
130 135 140
Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro
145 150 155 160
Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
165 170 175
Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
180 185
<210> 10
<211> 567
<212> nucleic acid
<400> 10
ATGGCCTTGA CCTTTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAA GTCAAGCTGC60
TCTGTGGGCT GTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGAC CTTGATGCTC120
3 O CTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACA TGACTTTGGA180
TTTCCCCAGG AGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCC TGTCCTCCAT240
3AGATGATCC AGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGC TGCTTGGGAT300
GAGACCCTCC TAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGA CCTGGAAGCC360
TGTGTGATAC AGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGA CTCCATTCTG420
3 S GCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAA ATACAGCCCT980
TGTGCCTGGG AGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTC AACAAACTTG540
CAAGAAAGTT TAAGAAGTAAGGAATGA 567
<210> 11
40 <211> 567
<212> nucleic acid
<400> 11
ATGGCCCTGA CCTTCGCCCTGCTGGTGGCCCTGCTGGTGCTGAGCTGCAA 60
GAGCAGCTGC
4 S TCCGTGGGGTGCGACCTGCCCCAGACCCACAGCCTGGGGAGCCGGCGGACCCTGATGCTG120
CTGGCCCAGA TGCGGCGGATCAGCCTGTTCAGCTGCCTGAAGGACCGGCACGACTTCGGG180
TTCCCCCAGG AGGAGTTCGGGAACCAGTTCCAGAAGGCCGAGACCATCCCCGTGCTGCAC290
GAGATGATCC AGCAGATCTTCAACCTGTTCAGCACCAAGGACAGCAGCGCCGCCTGGGAC300
GAGACCCTGC TGGACAAGTTCTACACCGAGCTGTACCAGCAGCTGAACGACCTGGAGGCC360
5 O TGCGTGATCCAGGGGGTGGGGGTGACCGAGACCCCCCTGATGAAGGAGGACAGCATCCTG920
GCCGTGCGGA AGTACTTCCAGCGGATCACCCTGTACCTGAAGGAGAAGAAGTACTCCCCC980
TGCGCCTGGG AGGTGGTGCGGGCCGAGATCATGCGGAGCTTCAGCCTGAGCACCAACCTG540
CAGGAGAGCC TGCGGAGCAAGGAGTGA 567
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<210> 12
<211> 567
<212> nucleic acid
<223> "Y" stands for C or T; "R" stands for A or G;
"W" stands for T or A; "S" stands for C or G; "N"
stands for any base.
<400> 12
ATGGCNYTNA CNTTYGCNYT NYTNGTNGCNYTNYTNGTNY TNWSNTGYAA RWSNWSNTGY60
1 WSNGTNGGNT GYGAYYTNCC NCARACNCAYWSNYTNGGNW SNMGNMGNAC NYTNATGYTN120
O
YTNGCNCARA TGMGNMGNAT HWSNYTNTTYWSNTGYYTNA ARGAYMGNCA YGAYTTYGGN180
TTYCCNCARG ARGARTTYGG NAAYCARTTYCARAARGCNG ARACNATHCC NGTNYTNCAY240
GARATGATHC ARCARATHTT YAAYYTNTTYWSNACNAARG AYWSNWSNGC NGCNTGGGAY300
GARACNYTNY TNGAYAARTT YTAYACNGARYTNTAYCARC ARYTNAAYGA YYTNGARGCN360
1 TGYGTNATHC ARGGNGTNGG NGTNACNGARACNCCNYTNA TGAARGARGA YWSNATHYTN420
5
GCNGTNMGNA ARTAYTTYCA RMGNATFIACNYTNTAYYTNA ARGARAARAA RTAYWSNCCN480
TGYGCNTGGG ARGTNGTNMG NGCNGARATHATGMGNWSNT TYWSNYTNWS NACNAAYYTN540
CARGARWSNY TNMGNWSNAA RGARTRR 567
20 <210> 13
<211> 191
<212> nucleic acid
<400> 13
2 5 GGGTGGCATC CCTGTGACCC CTCCCCAGTG CCTCTCCTGG CCCTGGAAGT TGCCACTCCA 60
GTGCCCACCA GCCTTGTCCT AATAAAATTA AGTTGCATCA TTTTGTCTGA CTAGGTGTCC 120
TTCTATAATA TTATGGGGTG GAGGGGGGTG GTATGGAGCA AGGGGCAAGT TGGGAAGACA 180
ACCTGTAGGG C 191
30 <210> 14
<211> 58
<212> nucleic acid
<400> 14
3 5 AAGCTTACTC AACACAATAA CAAACTTACT TACAATCTTA ATTAACAGGC CACCATGG 58
<210> 15
<211>~ 15
<212> nucleic acid
40 <400> 15
CAGGTAAGTG TCTTC 15
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<210> 16
<211> 30
<212> nucleic acid
<400> 16
TACTAACGGT TCTTTTTTTC TCTTCACAGG 30
<210> 17
<211> 271
<212> nucleic acid
<400> 17
TCTAGAGCAT TTTTCCCTCT GCCAAAAATT ATGGGGACAT CATGAAGCCC CTTGAGCATC 60
TGACGTCTGG CTAATAAAGG AAATTTATTT TCATTGCAAT AGTGTGTTGG AATTTTTTGT 120
1 5 GTCTCTCACT CGGTACTAGA GCATTTTTCC CTCTGCCAAA AATTATGGGG ACATCATGAA 180
GCCCCTTGAG CATCTGACGT CTGGCTAATA AAGGAAATTT ATTTTCATTG CAATAGTGTG 240
TTGGAATTTT TTGTGTCTCT CACTCGGTAC C 271
<210> 18
<211> 5686
<212> nucleic acid
<400> 18
CCGGCCACAGTCGATGAATC GCCATTTTCCACCATGATATTCGGCAAGCA60
CAGAAAAGCG
2 GGCATCGCCATGCGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGC120
5
GAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAG180
ACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGG240
GCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTT300
CTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAG360
3 CCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGT420
O
GGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTC480
GGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGA540
GCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGG600
AGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTG660
3 ATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTAC720
5
TTTGCAGGGCTTCCCAACCTTACCAGAGGGCGAATTCGAGCTTGCATGCCTGCAGGTCGT780
TACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC840
GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG900
GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAG960
4 TACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT1020
O
GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT1080
GGTGATGC~GTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT1140
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGA1200
CTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACG1260
4 GTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCA1320
5
TCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGA1380
ACGGTGCATTGGAACGCGGATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCTGTT1440
TCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTGTATTTTTGC1500
TAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACCATGGGTCCAGCGCGCA1560
5 GCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCC1620
O
CCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGA1680
GGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTT1790
CTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTT1800
TACCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAA1860
5 CTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTA1920
5
GTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTC1980
TGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGC2040
CA 02323604 2000-09-18
WO 99/47678 PCTNS99/05394
9
TGATGCAGGC CTGTGCCACA CTTGAAGAAC2100
CCTGAATTTC AAAATCCTCC
AACAGTGAGA
CGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGG2160
CAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCTAACAATTCTAGAAAAGCC2220
GAATTCTGCAGGAATTGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCT2280
S GGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTT2340
GTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGG2900
GCAAGTTGGGAAGACAACCTGTAGGGCTCGAGGGGGGGCCCGGTACGGTCGTTACATAAC2460
TTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAA2520
TGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT2580
Z ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCC2640
O
CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTAT2700
GGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGC2760
GGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTC2820
TCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAA2880
Z AATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGG2990
S
TCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCT3000
GTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCA3060
TTGGAACGCGGATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCTGTTTCCTTCCC3120
CTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTGTATTTTTGCTAGAATTG3180
Z TACTAACGGTTCTTTTTTTCTCTTCACAGGCTTAAGTCATGGGTCACCAGCAGTTGGTCA3240
O
TCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAGA3300
AAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCC3360
TCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGG3420
TCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGT3480
2 ACACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGG3540
AAGATGGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCT3600
TTCTAAGATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAA3660
TCAGTACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGG3720
TGACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATG3780
3 AGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCA3890
O
TTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCT3900
TCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCATTAAAGA3960
ATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTACTCCACATTCCT4020
ACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGAGAAAAGAAAGATA4080
3 GAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCAGCATTAGCG4140
5
TGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCTGTGCCCTGCA4200
GTTAGACGCGCTAGAAAAGCCGAATTCTGCAGGAATTGGGTGGCATCCCTGTGACCCCTC4260
CCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAAT4320
AAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAG4380
4 GGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCTCGAGGGGGGGC4440
O
CCGGTACCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAATCATGGT4500
CATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCG4560
GAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGT4620
TGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCG4680
4 GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTG4740
S
ACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA9800
TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC9860
AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCC9920
CTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTAT4980
S AAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC5040
O
CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT5100
CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG5160
AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACC5220
CGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA5280
S GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA5340
5
GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA5900
GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC5460
AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG5520
ACGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGG5580
6 CGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATAT5640
O
CACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAG 5686
CA 02323604 2000-09-18
WO 99/47678 PCTNS99/05394
<210> 19
<211> 3426
<212> nucleic acid
<400> 19
5
GATCCATGGCTAGGCTCTGTGCTTTCCTCATGATCCTAGTAATGATGAGCTACTACTGGT60
CAGCCTGTTCTCTAGGATGTGACCTGCCTCACACTTATAACCTCGGGAACAAGAGGGCCT120
TGACAGTCCTGGAAGAAATGAGAAGACTCCCCCCTCTTTCCTGCCTGAAGGACAGGAAGG180
ATTTTGGATTCCCCTTGGAGAAGGTGGATAACCAACAGATCCAGAAGGCTCAAGCCATCC240
1 TTGTGCTAAGAGATCTTACCCAGCAGATTTTGAACCTCTTCACATCAAAAGACTTGTCTG300
O
CTACTTGGAATGCAACTCTCCTAGACTCATTCTGCAATGACCTCCATCAGCAGCTCAATG360
ATCTCAAAGCCTGTGTGATGCAGGAACCTCCTCTGACCCAGGAAGACTCCCTGCTGGCTG420
TGAGGACATACTTCCACAGGATCACTGTGTACCTGAGAAAGAAGAAACACAGCCTCTGTG480
CCTGGGAGGTGATCAGAGCAGAAGTCTGGAGAGCCCTCTCTTCCTCAACCAACTTGCTGG540
1 CAAGACTGAGTGAGGAGAAGGAGTGATCTAGAAAGCCGAATTCTGCAGGAATTTGGCATC600
5
CCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCA660
GCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATA720
TTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCCCAAGTTGGGAAGACAACCTGTAG780
GGCTCGAGGGGGGGCCCGGTACCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCT840
2 TGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCAC900
O
ACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC960
TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGC1020
TGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCG1080
CTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTC1140
2 ACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT1200
5
GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC1260
ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAA1320
ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC1380
CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG1440
3 CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC1500
O
TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC1560
GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA1620
GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT1680
ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCG1740
3 GAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTT1800
5
TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT1860
TTTCTACGGGGTCTGACGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCT1920
GCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAA1980
GCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCA2040
4 GCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGC2100
O
AGGCATCGCCATGCGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGG2160
CGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAA2220
GACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATG2280
GGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTT2340
4 TCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCA2400
5
GCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCG2460
TGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGT2520
CGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAG.CCGGAACACGGCGGCATCAG2580
AGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCG2640
5 GAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTT2700
O
GATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTA2760
CTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGAATTCGAGCTTGCATGCCTGCAGGTCG2820
TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGA2880
CGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT2990
5 GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA3000
5
GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACA3060
TGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA3120
TGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT3180
TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG3240
6 ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC3300
O
GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC3360
ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAG3420
CTAGAG
3426
CA 02323604 2000-09-18
WO 99/47678 PCT/US99/05394
11
<210> 20
<211> 5966
<212> nucleic acid
<400> 20
AGCTTCGAGGGGGGGCCCGGTACCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGC60
TTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCA120
CACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA180
CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG240
1 CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC300
O
GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT360
CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG420
TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC480
CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA540
1 AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT600
5
CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG660
GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG720
CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT780
CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAAC840
2 AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAAC900
O
TACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTC960
GGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT1020
TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC1080
TTTTCTACGGGGTCTGACGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGC1190
2 TGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCA1200
S
AGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCC1260
AGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAG1320
CAGGCATCGCCATGCGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTG1380
GCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACA1940
3 AGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAAT1500
O
GGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACT1560
TTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGC1620
AGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTC1680
GTGGCCAGCCACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGG1740
3 TCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCA1800
S
GAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCC1860
GGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCT1920
TGATCAGATCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTT1980
ACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTT2040
4 GCTGTCCATAAAACCGCCCAGTCTAGCAACTGTTGGGAAGGGCGGGGCTGCAGGAATTCG2100
O
AGCTTGCATGCCTGCAGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC2160
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG2220
GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC2280
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG2390
4 CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG2400
5
TATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT2460
AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT2520
TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGC2580
AAATGGGCGGTAGGCGTGTA.CGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACC2640
S GTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACC2700
O
GATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGTTAATTAAC2760
AGGTAAGTGTCTTCCTCCTGTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAA2820
ACTGCAGTATCTGTATTTTTGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAG2880
GCCACGATGTGTCAATCACGCTACCTCCTCTTTTTGGCCACCCTTGCCCTCCTAAACCAC2940
5 CTCAGTTTGGCCAGGGTCATTCCAGTCTCTGGACCTGCCAGGTGTCTTAGCCAGTCCCGA3000
5
AACCTGCTGAAGACCACAGATGACATGGTGAAGACGGCCAGAGAAAAACTGAAACATTAT3060
TCCTGCACTGCTGAAGACATCGATCATGAAGACATCACACGGGACCAAACCAGCACATTG3120
AAGACCTGTTTACCACTGGAACTACACAAGAACGAGAGTTGCCTGGCTACTAGAGAGACT3180
TCTTCCACAACAAGAGGGAGCTGCCTGCCCCCACAGAAGACGTCTTTGATGATGACCCTG3240
E>OTGCCTTGGTAGCATCTATGAGGACTTGAAGATGTACCAGACAGAGTTCCAGGCCATCAAC3300
GCAGCACTTCAGAATCACAACCATCAGCAGATCATTCTAGACAAGGGCATGCTGGTGGCC3360
ATCGATGAGCTGATGCAGTCTCTGAATCATAATGGCGAGACTCTGCGCCAGAAACCTCCT3420
GTGGGAGAAGCAGACCCTTACAGAGTGAAAATGAAGCTCTGCATCCTGCTTCACGCCTTC3480
AGCACCCGCGTCGTGACCATCAACAGGGTGATGGGCTATCTGAGCTCCGCCTTAACTAGT3590
CA 02323604 2000-09-18
WO 99/47678 PCT/US99/05394
12
GAAAGCTCAA CACAGCGCCCTCCTCACACAGATAGGAATTCTGCAGATCC3600
GGCCCTCTGC
TAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC3660
CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA3720
TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG3780
GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG3840
CTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGAAATTCGAGCTTGCATGCCT3900
GCAGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG3960
CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG4020
ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA4080
1 TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC4140
O
CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC4200
TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTC9260
ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAA4320
TCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAG9380
1 GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTG9440
5
GAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCG4500
CGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGTTAATTAACAGGTAAGTGTCTT9560
CCTCCTGTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTG4620
TATTTTTGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACGATGTGTC9680
2 CTCAGAAGCTAACCATCTCCTGGTTTGCCATCGTTTTGCTGGTGTCTCCACTCATGGCCA4790
O
TGTGGGAGCTGGAGAAAGACGTTTATGTTGTAGAGGTGGACTGGACTCCCGATGCCCCTG4800
GAGAAACAGTGAACCTCACCTGTGACACGCCTGAAGAAGATGACATCACCTGGACCTCAG4860
ACCAGAGACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTC4920
TAGATGCTGGCCAGTACACCTGCCACAAAGGAGGCGAGACTCTGAGCCACTCACATCTGC9980
2 TGCTCCACAAGAAGGAAAATGGAATTTGGTCCACTGAAATTTTAAAAAATTTCAAAAACA5040
5
AGACTTTCCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTGG5100
TGCAAAGAAACATGGACTTGAAGTTCAACATCAAGAGCAGTAGCAGTTCCCCTGACTCTC5160
GGGCAGTGACATGTGGAATGGCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGG5220
ACTATGAGAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGA5280
3 CCCTGCCCATTGAACTGGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCA5340
O
CCAGCTTCTTCATCAGGGACATCATCAAACCAGACCCGCCCAAGAACTTGCAGATGAAGC5400
CTTTGAAGAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCC5460
ATTCCTACTTCTCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGG5520
AGACAGAGGAGGGGTGTAACCAGAAAGGTGCGTTCCTCGTAGAGAAGACATCTACCGAAG5580
3 TCCAATGCAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTACAATTCCTCAT5640
5
GCAGCAAGTGGGCATGTGTTCCCTGCAGGGTCCGATCCTAGAGCTCGCTGATCAGCCTCG5700
ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACC5760
CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT5820
CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT5880
4 TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAA5940
O
AGAACCAGCTGGGGCTCGAGCATGCA 5966
<210> 21
<211> 3589
45 <212> nucleic acid
<400> 21
CGTTACATAA TGGCTGACCGCCCAACGACCCCCGCCCATT60
CTTACGGTAA
ATGGCCCGCC
GACGTCAATATGACGTATGA GGGACTTTCCATTGACGTCA120
TTCCCATAGTAACGCCAATA
5 ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC180
O
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA240
CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC300
CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG360
ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACG420
5 GGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT480
5
ACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACG540
CCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCG600
GGAACGGTGCATTGGAACGCGGATTCCCCGTGTTAATTAACAGGTAAGTGTCTTCCTCCT660
GTTTCCTTCCCCTGCTATTCTGCTCAACCTTCCTATCAGAAACTGCAGTATCTGTATTTT720
6 TGCTAGCAGTAATACTAACGGTTCTTTTTTTCTCTTCACAGGCCACCATGGCCTTGACCT780
O
TTGCTTTACTGGTGGCCCTCCTGGTGCTCAGCTGCAAGTCAAGCTGCTCTGTGGGCTGTG840
ATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTCCTGGCACAGATGA900
GGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGATTTCCCCAGGAGG960
AGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGC1020
CA 02323604 2000-09-18
WO 99/47678 PCTNS99/05394
13
AGATCTTCAA ACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAG1080
TCTCTTCAGC
ACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGG1140
GGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAAT1200
ACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGGGAGG1260
S TTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAA1320
GAAGTAAGGAATGAATCTAGAAAAGCCGAATTCTGCAGGAATTGGGTGGCATCCCTGTGA1380
CCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGT1990
CCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGG1500
GTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGAGAACCTGTAGGGCTCGAGG1560
1 GGGGGCCCGGTACCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTTCGAGCTTGGCGTAAT1620
O
CATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATAC1680
GAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAA1790
TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT1800
GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC1860
1 TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG1920
CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAG1980
GCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCC2040
GCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG2100
GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA2160
2 CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC2220
O
ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG2280
TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT2340
CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA2400
GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA2460
2 CTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG2520
5
TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCA2580
AGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGG2640
GGTCTGACGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGG2700
GAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAG2760
3 CAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCAC2820
O
AGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGC2880
CATGCGTCACGACGAGATCCTCGCCGTCGGGCATGCGCGCCTTGAGCCTGGCGAACAGTT2940
CGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTT3000
CCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAG3060
3 CCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAG3120
5
GAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCC3180
TTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCC3240
ACGATAGCCGCGCTGCCTCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGA3300
CAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGA3360
4 TTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTG3420
O
CGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGATC3480
TTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGG3540
GCTTCCCAACCTTACCAGAGGGCGAATTCGAGCTTGCATGCCTGCAGGT 3589
45 <210> 22
<211> 567
<212> nucleic acid
<400> 22
S O ATGGCCTTGA CCTTTGCTTT ACTGGTGGCC CTCCTGGTGC TCAGCTGCAA GTCAAGCTGC 60
TCTGTGGGCT GTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCTC120
CTGGCACAGA TGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGACATGACTTTGGA180
TTTCCCCAGG AGGAGTTTGGCAACCAGTTCCAAAAGGCTGAAACCATCCCTGTCCTCCAT240
GAGATGATCC AGCAGATCTTCAATCTCTTCAGCACAAAGGACTCATCTGCTGCTTGGGAT300
S 5 GAGACCCTCCTAGACAAATTCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCC360
TGTGTGATAC AGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTG420
GCTGTGAGGA AATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCT980
TGTGCCTGGG AGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTG540
CAAGAAAGTT TAAGAAGTAAGGAATGA 567
60
CA 02323604 2000-09-18
WO 99/47678 PCT/US99/05394
14
<210> 23
<211> 16
<212> nucleic acid
<223> "Y" stands for C or T; "N" stands for any base.
<400> 23
YYYYYYYYYY YNYAGG 16
<210> 24
<211> 660
<212> nucleic acid
<223> "Y" stands for C or T; "R" stands for A or G;
"W" stands for T or A; "S" stands for C or G; "N"
stands for any base.
<400> 24
ATGTGYCCNG CNMGNWSNYTNYTNYTNGTN GCNACNYTNG YCAYYTNWSN60
TNYTNYTNGA
YTNGCNMGNA AYYTNCCNGTNGCNACNCCN GAYCCNGGNA YYTNCAYCAY120
TGTTYCCNTG
WSNCARAAYY TNYTNMGNGCNGTNWSNAAY ATGYTNCARA RACNYTNGAR180
ARGCNMGNCA
2 O TTYTAYCCNTGYACNWSNGARGARATHGAY CAYGARGAYA YAARACNWSN290
THACNAARGA
ACNGTNGARG CNTGYYTNCCNYTNGARYTN ACNAARAAYG NAAYWSNMGN300
ARWSNTGYYT
GARACNWSNT TYATHACNAAYGGNWSNTGY YTNGCNWSNM NTTYATGATG360
GNAARACNWS
GCNYTNTGYY TNWSNWSNATHTAYGARGAY YTNAARATGT RTTYAARACN420
AYCARGTNGA
ATGAAYGCNA ARYTNYTNATGGAYCCNAAR MGNCARATHT RAAYATGYTN480
TYYTNGAYCA
2 5 GCNGTNATHGAYGARYTNATGCARGCNYTN AAYTTYAAYW NCCNCARAAR540
SNGARACNGT
WSNWSNYTNG ARGARCCNGAYTTYTAYAAR ACNAARATHA HYTNYTNCAY600
ARYTNTGYAT
GCNTTYMGNA THMGNGCNGTNACNATHGAY MGNGTNACNW YGCNWSNTRR660
SNTAYYTNAA
30 <210> 25
<211> 987
<212> nucleic acid
<223> "Y" stands for C or T: "R" stands for A or G; "W"
stands for T or A; "S" stands for C or G: "N"
35 stands for any base.
<400> 25
ATGTGYCAYC ARCARYTNGTNATHWSNTGGTTYWSNYTNG TNTTYYTNGCNWSNCCNYTN60
GTNGCNATHT GGGARYTNAARAARGAYGTNTAYGTNGTNG ARYTNGAYTGGTAYCCNGAY120
4 GCNCCNGGNG ARATGGTNGTNYTNACNTGYGAYACNCCNG ARGARGAYGGNATHACNTGG180
O
ACNYTNGAYC ARWSNWSNGARGTNYTNGGNWSNGGNAARA CNYTNACNATHCARGTNAAR240
GARTTYGGNG AYGCNGGNCARTAYACNTGYCAYAARGGNG GNGARGTNYTNWSNCAYWSN300
YTNYTNYTNY TNCAYAARAARGARGAYGGNATHTGGWSNA CNGAYATHYTNAARGAYCAR360
AARGARCCNA ARAAYAARACNTTYYTNMGNTGYGARGCNA ARAAYTAYWSNGGNMGNTTY920
4 ACNTGYTGGT GGYTNACNACNATHWSNACNGAYYTNACNT TYWSNGTNAARWSNWSNMGN480
5
GGNWSNWSNG AYCCNCARGGNGTNACNTGYGGNGCNGCNA CNYTNWSNGCNGARMGNGTN540
MGNGGNGAYA AYAARGARTAYGARTAYWSNGTNGARTGYC ARGARGAYWSNGCNTGYCCN600
GCNGCNGARG ARWSNYTNCCNATHGARGTNATGGTNGAYG CNGTNCAYAARYTNAARTAY660
GARAAYTAYA CNWSNWSNTTYTTYATHMGNGAYATHATHA ARCCNGAYCCNCCNAARAAY720
5 YTNCARYTNA ARCCNYTNAARAAYWSNMGNCARGTNGARG TNWSNTGGGARTAYCCNGAY780
O
ACNTGGWSNA CNCCNCAYWSNTAYTTYWSNYTNACNTTYT GYGTNCARGTNCARGGNAAR840
CA 02323604 2000-09-18
WO 99/47678 PCT/US99/05394
WSNAARMGNG ARAARAARGA YMGNGTNTTY ACNGAYAARA CNWSNGCNAC NGTNATHTGY 900
MGNAARAAYG CNWSNATHWS NGTNMGNGCN CARGAYMGNT AYTAYWSNWS NWSNTGGWSN 960
GARTGGGCNW SNGTNCCNTG YWSNTRR 987
