Language selection

Search

Patent 2429891 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2429891
(54) English Title: MEMBRANE FUSION PROTEIN
(54) French Title: PROTEINE HYBRIDE DE MEMBRANE
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/62 (2006.01)
  • A61K 47/42 (2017.01)
  • C07K 14/14 (2006.01)
  • C07K 16/46 (2006.01)
  • C07K 19/00 (2006.01)
  • C12N 5/12 (2006.01)
  • C12N 5/16 (2006.01)
(72) Inventors :
  • DUNCAN, ROY (Canada)
(73) Owners :
  • FUSOGENIX INC. (Canada)
(71) Applicants :
  • FUSOGENIX INC. (Canada)
(74) Agent: SMART & BIGGAR
(74) Associate agent:
(45) Issued:
(22) Filed Date: 2003-06-02
(41) Open to Public Inspection: 2004-12-02
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract



A family of membrane fusion proteins and
polynucleotides encoding the proteins have been identified.
The proteins and polynucleotides are derived from the family
Reoviridae. Two membrane fusion proteins have been isolated
from reoviruses isolated from poikilothermic hosts: the p14
protein from reptilian reovirus (RRV) isolated from python,
and the p22 protein from aquareovirus (AQV) isolated from
salmon. The genes encoding these proteins have been cloned
and sequenced. Analysis of the amino acid sequences of
these proteins show that both lack the typical fusion
peptide motif found in other membrane fusion proteins.
Expression of these proteins in cells results in cell-cell
fusion.


Claims

Note: Claims are shown in the official language in which they were submitted.




CLAIMS:
1. An isolated protein:
(a) which is a membrane fusion protein;
(b) whose amino acid sequence is free of fusion peptide
motif I; wherein the fusion peptide motif I:
(i) is an amino acid sequence 17 to 28 residues long;
(ii) has a hydrophobicity value from 0.6 to 0.7; and
(iii) has a content of alanine plus glycine from 29 to
43%;
(c) whose amino acid sequence is free of fusion peptide
motif II; wherein the fusion peptide motif II:
(i) is an amino acid sequence 16 to 28 residues long;
(ii) has a hydrophobicity value from 0.3 to 0.4;
(iii) has a content of alanine plus glycine from 29 to
43%; and
(iv) contains a heptad repeat;
(d) which comprises an amino acid sequence which has at
least 33% identity overall to SEQ ID NO:6; and
(e) which comprises a transmembrane domain whose amino
acid sequence has at least 60% amino acid sequence identity to
the sequence 5' LAICCCCCICCTGGLYLV 3' of SEQ ID NO:6.
2. The protein of claim 1 which is encoded by the genome
of a reovirus selected from the genus consisting of
Orthoreovirus and Aquareovirus,
62



3. The protein of claim 1 which is encoded by the genome
of a reovirus which naturally infects a poikilothermic host.
4. The protein of any one of claims 1 to 3 which has an
isoelectric point equal to or greater than 7.
5. The protein of any one of claims 1 to 4 further
comprising a positive cluster; wherein the positive cluster
consists of at least three positively charged amino acid
residues within a contiguous sequence of at mast 25 residues,
wherein the contiguous sequence is within at most 100 residues
flanking the transmembrane domain at the C-terminal side.
6. The protein of any one of claims 1 to 5 further
comprising a fatty acylation sequence.
7. An isolated protein comprising SEQ ID NO:6.
8. An isolated polynucleotide encoding the protein
according to any one of claims 1 to 8.
9. An isolated polynucleotide comprising SEQ ID NO:5, or
degenerate variations thereof which encode the same amino acid
sequences, or splice variant nucleotide sequences thereof.
10. The isolated polynucleotide according to claim 8 or 9
operatively associated with a promoter.
11. The polynucleotide of claim 10 wherein the promoter
is inducible.
12. A cell expressing the isolated protein according to
any one of claims 1 to 7.
13. A call expressing the polynucleotide according to any
one of claims 8 to 11.
63




14. A liposome containing the protein according to any
one of claims 1 to 7.
15. A liposome containing the polynucleotide according to
any one of claims 8 to 11.
16. An antibody against the protein according to any one
of claims 1 to 7.
17. A method to promote fusion between two or more
membranes, comprising contacting the membranes to be fused with
an effective amount of the protein according to any one of
claims 1 to 7.
18. The method according to claim 17 wherein the
membranes are selected from the group consisting of cell
membranes, liposome membranes and proteoliposome membranes.
19. The method according to claim 17 wherein the
membranes are membranes of cells, and wherein heterokaryons are
produced.
20. The method according to claim 17 wherein the
membranes are of an immortalized cell and a primary B cell or T
cell, for producing a hybridoma cell.
21. The method according to claim 20 wherein the
hybridoma cell produces a substance selected from: monoclonal
antibodies, cytokines and immune modulators.
22. The method according to claim 21 wherein the
hybridoma cells produce monoclonal antibodies, and wherein the
membranes are of an immortalized cell and an antibody-
synthesizing cell.
23. The method according to claim 22 wherein said
immortalized cell is a human or mouse B cell myeloma cell or a
64


T cell myeloma cell, and wherein said antibody-synthesizing
cell is an isolated spleen cell from an immunized mammal.
24. The method according to claim 17, wherein the
membranes are selected from the group consisting of: liposome
membranes, liposome membrane and cell membrane, and
proteoliposome membrane and cell membrane.
25. The method according to claim 24 wherein the liposome
or proteoliposome contains a bioactive drug.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02429891 2003-06-18
Membrane Fusion Proteins Derived from Reovirus
FIELD OF THE INVENTION
The present invention relates to a novel class of
proteins that promote membrane fusion. The proteins and the
polynucleotides encoding them are derived from the family
Reoviridae.
BACKGROUND OF THE INVENTION:
Membrane fusion reactions are common in eukaryotic
cells. Membranes are fused intracellularly in processes
including endocytosis, organelle formation, inter-organelle
traffic, and constitutive and regulated exocytosis.
Intercellularly, membrane fusion occurs during sperm-egg fusion
and myoblast fusion.
Membrane fusion has been induced artificially by the
use of liposomes, in w:h:ich the cell membrane is fused with the
liposomal membrane, and by various chemicals or lipids, which
induce cell-cell fusion to produce heterokaryons. Naturally
occurring proteins shown to induce fusion of biological
membranes are mainly fusion proteins of enveloped viruses.
20~ In :Liposome-based delivery systems, liposomes are
used to encapsulate b.i.oactive molecules inside lipid vesicles
for delivery into the cell. However, the polar lipid
headgroups oriented on both surfaces of the lipid bilayer,
along with an associated water layer, make spontaneous membrane
fusion thermodynamically unfavorable.
Various chemicals or lipids have been used to promote
membrane fusion. However, these reagents usually exhibit
cytotoxic effects (see, for example, Iwamoto et al., in Biol.
Pharm. Bull. 19:860-863 (1996) and Mizuguchi et al., in
3U Biochem. Biophys. Res. Commun., 218:402-407 (1996)).
1


CA 02429891 2003-06-18
It is generally believed that membrane fusion under
physiological conditions is protein-mediated. This has led to
the development of liposomes that contain fusion-promoting
proteins (proteoliposomes), with decreased cytotoxicity (see,
for example, Cheng, Hum. Gene Ther. 7:275-282 (1996); Hara et
al., Gene 159:167-174 (1995); and ~'indeis et al., Trends
Biotechnol., 11:202-205 (1993)).
The only proteins conclusively shown to induce
intercellular fusion of biological membranes are those of
enveloped viruses and two proteins from nonenveloped viruses.
All enveloped viruses encode proteins responsible for fusion of
the viral envelope with the cell membrane. These viral fusion
proteins are essential for infection of susceptible cells. The
mechanism of action of fusion proteins from enveloped viruses
have served as a paradigm for protein-mediated membrane fusion
(see, for example, White, Ann. Rev'. Physiol., 52:675-697
(1990) ; and White, Science, 258:917-924 (1992) ) .
Most enveloped virus fusion proteins are relatively
large, multimE=ric, type I membrane proteins, as typified by the
influenza virus HA protein, a Iow pH-activated fusion protein,
and the Senda:i virus F~ protein, which functions at neutral pH.
These are structural proteins of the virus with the majority of
the fusion protein oriented on the external surface of the
virion to facilitate interactions between the virus particle
2;i and the cell membrane.
According to the mechanism of action of fusion
proteins from. enveloped; viruses, fusion of the viral envelope
with the cell membrane is mediated by an amphipathic alpha-
helical region, referred to as a fusion peptide motif, that is
present in the viral fusion protein. This type of fusion
peptide motif is typically 17 to 28 residues long, hydrophobic
(average hydrophobicity of about 0.6 ~ 0.1), and contains a
2


CA 02429891 2003-06-18
high content of glycine and alanine, typically 36% ~ 7% (White,
Annu. Rev. Physiol.., 52:675-697 (1990).
All of the erxv~eloped virus fusion proteins are
believed to function va_a. extensive conformational changes that,
by supplying the energy to overcome the thermodynamic barrier,
promote membrane fusion. These conformational changes are
frequently mediated by heptad repeat regions that form coiled
coil structures (see Sh;e:hel and Wiley, Cell, 95:871-874
(1998)). Recognition of the importance of fusion peptide
motifs in triggering membrane fusion has resulted in the use of
small peptides containing fusion peptide motifs to enhance
liposome-cell fusion (see, for example, Muga et al.,
Biochemistry 33:4444-4119:8 (1994) ) .
Enveloped virus fusion proteins also trigger cell-
cell fusion, resulting ~_n the formation of polykaryons
(syncytia). Synthesis of the viral fusion protein inside the
infected cell results :i.n transport of the fusion protein
through the endoplasmic reticulum and Golgi transport system to
the cell membrane, an essential step in the assembly and
budding of infectious progeny virus particles from the :infected
cell (Petterson, Curr. Top. Micro. Immunol., 170:67-106
(1991)). The synthesis, transport, and folding of the fusion
protein is facilitated by a variety of components, including
signal peptides to target the protein to the intracellular
transport pathway, glycosylation signals for N-linked
carbohydrate addition to the protein, and a transmembrane
domain to anchor the protein in the cell membrane. These
proteins have been used in reconstituted proteoliposomes
('virosomes') for enhanced, protein-mediated liposome-cell
fusion in both cell culture and in vivo (see, for example,
Ramani et al.,. FEBS Lett., 404:164-168 (1997); Scheule et al.,
Am. J. Respir. Cell Mal. Biol., 13:330-343 (1995); and
Grimaldi, Res. Virol., 146:289-293 (1995)).
3


CA 02429891 2003-06-18
Unlike enveloped viruses, nonenveloped viruses
generally do not encode fusion proteins since the absence of a
viral membrane preclude; membrane fusion-mediated entry.
Because progeny virus ,particles of nonenveloped viruses do not
need to acquire a lipid envelope, these viruses usually do not
bud from infected cells but, rather, are released by cell
lysis. As a result, nonenveloped viruses do not express fusion
proteins on th.e surfacE~ of infected cells and, hence, generally
do not induce syncytiurn formation.
Selected members of the family Reoviridae, however,
do induce syncytium formation (see Duncan et al., Virology,
212:752-756 (1995), Dunc:an, Virology, 260:316-328 (1999), and
references therein). Re~aviridae are a family of nonenveloped
viruses containing segmented double-stranded RNA (dsRNA)
genomes (see, for examp7_e, Nibert et al., Reoviruses and their
replication, I.n: Fundamental Virology, 3rd Edition, B. N.
Fields, D. M. Knipe and P. M. Howley (Eds), Lippincott-Raven
Press, NY (1996)).
Of the family Reoviridae, the genus Orthoreovirus
contains two distinct subgroups typified by the prototypical
avian and the mammalian reoviruses (see Duncan, Virology,
260:316-328 (7.999)). 'The avian reoviruses (ARV) are all
fusogenic and induce rapid and extensive cell-cell fusion,
resulting in ~~yncytium :Formation in infected cell cultures (see
Robertson and Wilcox, Vet. Bull., 56:726-733 (1986)).
Mammalian reo~Jiruses generally are not fusogenic.
However, there: are at least two exceptions. One was isolated
from a flying fox and i;s named Nelson Bay virus (NBV) (see Gard
and Compans, :T. Virol., 6:100-106 (1970)). The other was
isolated from a baboon .and is referred to as Baboon Reovirus
(BRV) (see Duncan et al., Virology, 212:752-756 (1995)).
4


CA 02429891 2003-06-18
Fusogenic reoviruses have also been isolated from
poikilothermic hosts. Two strains of reptilian reoviruses
(RRV, a member of the C~rthoreovirus genus) that induce
syncytium formation have been isolated from snakes (see Ahne et
al., Arch. Virol. 94:1x5-139 (1987), and Vieler et al., Arch.
Virol. 138:341-344 (1994)). In addition, members of the genus
Aquareovirus (AQV) that: infect exclusively piscine (fish) host
species, also induce cell-cell fusion (see Samal et al., J.
Virol. 64:5235-5240 (1990)). These viruses together represent
the few known examples of nonenveloped viruses capable of
inducing membrane fusion.
To date, two membrane fusion proteins have been
identified and sequenced from nonenveloped viruses: the pl0
protein from t:wo strai.n;s of ARV and from NBV, and the p15
1~ protein from BRV. The p10 proteins share 33% amino acid
identity between ARV and NBV and are clearly homologous
proteins. The p15 protein from BRV is not homologous to p10
and appears to belong to a different class. The amino acid
sequences of both the pl0 and p15 proteins contain fusion
peptide motifs (residues 9-24 of ARV and NBV and residues 68-87
of BRV, W099/24582, published May 20, 1999; the p10 protein is
referred to as "p11~~ in W099/24582). The fusion peptide motif
from the p10 protein ~;Shmulevitz and Duncan, EMBO J., 19:902-
912 (2000)), however, is atypical in that it is much less
2:5 hydrophobic than is observed in typical fusion motifs from
enveloped viruses. The hydrophobicity of the fusion motif from
pl0 is estimated to be about 0.3 to 0.4, in contrast to the
typical values of 0.6 1 0.1. Nevertheless, the fusion motif
from p10 still contains the heptad repeats seen in more typical
fusion motifs; i.e. se',ren-residue sequences in which residues
at positions 1 and 4 are apolar; (See Figure 6 which shows the
heptad configuration o:f p10). It is generally thought that the
conserved apalar residues serve to form the hydrophobic face of
5


CA 02429891 2003-06-18
amphipathic helices which are important for membrane-
interactive properties. The presence of heptad repeats in p10
and p15 suggests that these proteins promote membrane fusion by
a mechanism similar to that of membrane fusion proteins from
enveloped viruses.
SUMMARY OF THE INVENTIONf
According to the present invention, a new class of
membrane fusion proteins is described which is derived from
Reoviridae and. whose amino acid sequence is free of fusion
peptide motifs.
In one aspect, members of this class are membrane
fusion proteins which are encoded by the genome of the family
Reoviridae, which comprise at least one transmembrane domain,
and whose amino acid sequence is free of any fusion peptide
motifs. A fusion peptide motif (type I) is an amino acid
sequence typically 17 to 28 residues long, with a
hydrophobicit~r of about 0.6 to 0.'~, and whose content of
alanine plus glycine is about 29 to 43%. A fusion peptide
motif (type II) is 16 to 20 residues long, has a hydrophobicity
2() value about 0.3 to 0.4, has an alanine plus glycine content of
about 29 to 43%; and contains a heptad repeat.
In another aspect, the proteins of the invention are
membrane fusion proteins whose amino acid sequences are free of
any fusion peptide motifs, as defined above, and which are
2~ related to Reoviridae i.n that they comprise an amino acid
sequence which has at least 33% overall identity to the
membrane fusion protein encoded by Reoviridae, and comprise a
transmembrane domain whose amino acid sequence has at least 60%
amino acid sequence identity to the transmembrane domain of the
30 membrane fusion protein encoded by Reovixidae.
6


CA 02429891 2003-06-18
In one embodiment, the proteins of the invention are
encoded by, or related to, the genome of Orthoreovirus or
Aquareovirus, or the genome of a reovirus which naturally
infects a poikilothermic host. Specifically, in one
embodiment, thE: proteins of the invention are proteins
comprising an amino acid sequence of SEQ ID N0:2 or SEQ ID
N0:6. In another embodiment, the proteins of the invention are
membrane fusion proteins whose amino acid sequences are free of
any fusion peptide motif, as defined above, and which are
related to Orthoreovirus or Aquareovirus in that they comprise
an amino acid sequence which has at least 33% overall identity
to the membrane fusion protein encoded by Orthoreovirus or
Aquareovirus, and comprise a transmembrane domain whose amino
acid sequence has at lE::ast 60% amino acid sequence identity to
the transmembrane domain of the membrane fusion protein encoded
by Orthoreovirus or Aquareovirus.
In another embodiment, the proteins of the invention
are membrane fusion proteins whose amino acid sequence is free
of a fusion peptide motif as defined above, which comprise an
amino acid sequence which has at least 33% identity overall to
SEQ ID N0:2, and which comprises a transmembrane domain whose
amino acid sequence has at least 60% amino acid sequence
identity to th.e sequence from residue 3~ to 57 of SEQ ID N0:2.
In another embodiment, the proteins of the invention
are membrane fusion proteins whose amino acid sequence is free
of a fusion peptide motif as defined above, which comprise an
amino acid sequence which has at least 33% identity overall to
SEQ ID N0:6, and which comprises a transmembrane domain whose
amino acid sequence has at least 60% amino acid sequence
identity to the predicted transmembrane sequence
5' LAICCCCCICCTGGLYLV 3' of SEQ ID N0:6,
7


CA 02429891 2003-06-18
In another embodiment, the amino acid sequences
encoded by, or related to, the genome of Reoviridae, comprise
neutral or basic proteins, i.e. they have an isoelectric point
equal to or greater than 7.
In another embodiment, the proteins of the invention
further comprises a positive cluster which consists of at least
three positive:Ly charged amino acid residues within a
contiguous sequence of at most 25 residues, wherein the
contiguous sequence is within at most 100 residues flanking the
transmembrane domain at. the C-terminal side.
In another embodiment, the proteins of the invention,
particularly those related to p14, further comprise a non-
transmembrane domain which comprises a polyproline motif
comprising at least 3 contiguous proline residues.
In another er~~odiment, the proteins of the invention
further comprise a fatty acylation sequence such as a
myristylation consensus sequence.
Another aspect of the invention provides an isolated
polynucleotide encoding the proteins of the invention,
specifically an isolated polynucleotide comprising SEQ ID NO:1
or SEQ ID N0:5, or degenerate variations thereof which encode
the same amino acid sequences, or splice variant nucleotide
sequences thereof. The isolated polynucleotide of the
invention are expressed by being operatively associated with a
promoter, which may be .an inducible promoter.
Another aspect of the invention provides a cell which
contains the proteins or polynucleotides of the invention.
Another aspect of the invention provides a liposome
which contains the proteins or polynucleotides of the
invention. I:n an embodiment where liposomes are used to
8


CA 02429891 2003-06-18
deliver a bioac:tive drug to a cell, the Iiposomes or
proteoliposomes contain both the protein of the invention and
the bioactive drug.
Another aspect of the invention provides an antibody
reactive against the protein of the invention.
Another aspect of the invention provides a method to
promote fusion between two or more membranes, comprising
contacting the membranes to be fused with an effective amount
of the protein of the invention. In one embodiment, the
membranes are cell membranes, liposome membranes or
proteoliposome membranes, depending on whether cell-cell,
Iiposome-liposome, or cell-liposome fusion is required.
In one embodiment, fusion between cells is effected
by expressing the membrane fusion proteins of the invention in
the cells) which are to undergo fusion.
In another embodiment, the fusion method of the
invention is used to produce heterokaryons, particularly
hybridoma cells for the production of monoclonal antibodies,
cytokines, or immune modulators. Where the hybridoma cells
produce monoclonal antibodies, the membranes are from an
immortalized cell and axe antibody-synthesizing cell.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 presents the nucleotide sequence of the
polycistronic AQV genome segment 7 (SEQ ID N0:5), and the
predicted amino acid sequences of the 5'-proximal ORF encoding
the p22 fusion protein (SEQ ID N0:6). The 3'-proximal ORF
encodes the unrelated nonstructural virus protein NS28 (SEQ ID
N0:8).
Figure 2 presents the amino acid sequence of the AQV
;i0 p22 fusion protein (SEQ ID N0:6). The predicted transmembrane
9


CA 02429891 2003-06-18
domain$ is overlined and labeled. The clusters of positively
charged amino acids adrjacent to the transmembrane domains are
labeled with a + symbol..
Figure 3 is a schematic depiction of the apolar amino
acids within t:he fusion peptide motifs of the p10 proteins from
ARV and NBV. The heptad repeats are thought to constitute the
hydrophobic faces of amphipathic helices, which are thought to
be important f:or interaction of this type of membrane fusion
protein with t:he membrane.
DETAILED DESCRIPTION
(A) Proteins
A c7.ass of proteins, exemplified by the p14 protein
from RRV and t:he p22 protein from AQV, have been identified.
The proteins have membrane fusion properties. Accordingly,
this class of proteins, including p14, p22 and related
polypeptides, can be used to promote membrane fusion.
In one aspect of the invention, proteins are provided
which are encoded by they genome of Reoviridae, and whose amino
acid sequence is free of fusion peptide motifs.
The family Rec~viridae includes the genus
Orthoreovirus, which includes avian, mammalian and reptilian
reoviruses, as well as i~he genus Aquareovirus.
Fus~.on peptide motifs, as defined in the present
invention, arE: of two c:Lasses: type T and type II. Type I is
found typically in fusion proteins from enveloped proteins.
Motif type I consists o:E an amino acid sequence 16 to 36
residues long,. preferab:Ly 17 to 28 residues long, with a
hydrophobicity of about 0.5 to 0.8, preferably about 0.6 to
0.7, and has a content of alanine plus glycine of about 24 to
30~ 44%, preferably 29 to 4:3%.


CA 02429891 2003-06-18
Motif type II is an atypical motif. It is found in
the type of fusion protc=_in exemplified by the p10 protein of
ARV and NBV. It consisi~s of an amino acid sequence 15 to 20
residues long, preferab:Ly 16 to 18 residues long, with a
5~ hydrophobicity value about 0.2 to 0.4, preferably about 0.3 to
0.4, has an alanine plus glycine content of about 24 to 44%,
preferably about 29 to ~43%; and contains at least one heptad
repeat.
In accordance with the present invention, a heptad
1C~ repeat is a seven-residue sequence in which residues at
positions 1 and 4 are apolar. The conserved apolar first and
fourth residues are thovught to form part of the hydrophobic
face of amphipathic helices; thus, in proteins containing them,
they are like:Ly to be an essential element for interaction
1E~ between the fusion protein and the membrane.
Hydrophobicity values are determined according to
methods known in the art. According to the present invention,
the values are calculated using the normalized hydrophobicity
scale of Eisenberg. (7.984). Ann. Rev. Biochem. 53:595-623.
2C1 By °'polypeptide" or "protein" is meant any chain of
amino acids, .regardless of length or post-translational
modification (e. g., glycosylation or phosphorylation). Both
terms are used interchangeably in the present application.
In another aspect of the invention, proteins are
2'i provided which are membrane fusion proteins whose amino acid
sequences are free of any fusion peptide motifs, as defined
above, and which are related to Reoviridae in that they
comprise an amina acid. sequence which has at least 33% overall
identity with the membrane fusion protein encoded by
3U Reoviridae. 'The proteins also comprise a transmembrane domain
whose amino acid sequence has at .Least 60% amino acid sequence
11


CA 02429891 2003-06-18
identity to the transmembrane domain of the membrane fusion
protein encoded by Reovi:ridae. The overall percent identity is
also contemplated at at. least 40%, 45%, 50%, 60%,and 75%. The
percent identity with t;he transmembrane domain is also
contemplated at at least 70%, 75%, 80%, 85%, 90% and 95%.
In one aspect., proteins of the invention are related
to Reoviridae in that t~l-.ie proteins comprise an amino acid
sequence which has at ?e:ast 33% overall identity with the
membrane fusion proteizl encoded by Reoviridae. The 33% overall
identity is based on thE: degree of identity between the p10
proteins of ARV (strains. 176 and 138) and of NBV.
As a class, it. is clear that the proteins of the
invention tolerate a grE:at deal of sequence variation while
retaining their membrane fusion properties. The membrane
fusion proteins derived from Reoviridae are thus related by
function and by overal:~ structure, rather than by strict
overall identity. The ~>roteins are functionally defined in
that they have membrane fusion properties which can be readily
determined using a simple test. One such test is that
exemplified in Examples 4 and 8, where the protein which is to
be tested for membrane fusion activity is expressed in cells
and the cells examined under the microscope for syncytium
formation.
The proteins of the invention are structurally
defined in that they are: either encoded by Reoviridae, or are
related to Reoviridae fusion proteins mainly at the
transmembrane domain. Transmembrane domains are readily
recognized in the art. A typical transmembrane domain is
described in White, AriIlla. Rev. Physiol., 52:675-697 (1990), as
a contiguous sequence of: amino acids averaging 29 residues,
with average hydrophobic:ity of 0.7~0.09 and an alanine +
glycine content of 16%~8%.
12


CA 02429891 2003-06-18
In one embodiment, the proteins of the invention are
encoded by, or related to, the genome of Orthoreovirus or
Aquareovirus or the gexlc~rne of a reovirus which naturally
infects a poikilothermic (cold-blooded) host. Orthoreovirus
includes the avian and mammalian reoviruses. Aquareovirus are
reoviruses which naturally infect fish. Poikilothermic hosts
include reptiles such a~; snakes, pythons and fish.
Specifically, one embodiment is directed to proteins
comprising an amino acid sequence of SEQ ID N0:2 or SEQ ID
N0:6. In another embodiment, the proteins of the invention are
membrane fusion proteicm> whose amino acid sequences are free of
any fusion peptide motif:, as defined above, and which are
related to Orthoreoviru~~ or Aquareovirus in that they comprise
an amino acid sequence which has at least 33% overall identity
to the membrane fusion protein encoded by Orthoreovirus or
Aquareovirus, and comprise a transmembrane domain whose amino
acid sequence has at least 60% amino acid sequence identity to
the transmembrane domain of the membrane fusion protein encoded
by Orthoreovirus or Aquareovirus. The overall percent identity
is also contemplated at at least 40%, 45%, 50%, 60%,and 75%.
The percent identity w:it:h the transmembrane domain is
contemplated also at at least 70%, 75%, 80%, 85%, 90% and 95%.
The precise 1~T--terminus of p22 (SEQ ID N0:6) has not
been determined. HowevE:r, the N-terminal residue has been
shown to be located within the first. Z7 residues of SEQ ID
N0:6. Although precise identification of the initiator codon
is not essential to the present invention, the initiator can be
readily determined using methods known in the art. For
example, a stop codon can be inserted systematically into the
first 17 codons, as exp:Lained in Example 5. Alternatively, p22
can be isolated from ce=Lls in which it is expressed, and its N-
terminal region micro-sequenced.
13


CA 02429891 2003-06-18
In another emx>odiment, the membrane fusion proteins
of the invention are neutral or basic; i.e. their isoelectric
point (pI) is equal to or greater than 7, preferably greater
than 8, and more preferably greater than 9. The pI of a
protein can be readily determined using standard methods known
in the art.
In another embodiment, the proteins of the invention
further comprise a positive cluster which consists of at least
three, preferably four, more preferably six, most preferably
seven, positively chargE:d amino acid residues within a
contiguous sequence of at most 25, preferably 23, more
preferably 21, and most preferably 20 residues, wherein the
contiguous sequence is within at most 100, preferably at most
90, and more ~>referably at most 80, residues flanking the
transmembrane domain at the C-terminal side.
In another embodiment, the proteins of the invention
further comprise a non-t:ransmembrane domain which comprises a
polyproline motif comprising at least 3, preferably 4, and more
preferably 5 contiguous proline residues. In a specific
embodiment, the fusion ~?rotein of the invention which comprises
the polyproline motif is related to the p14 protein.
In another emlaodiment, the proteins of the invention
further comprise a fatty acylation sequence, preferably a
myristylation consensus sequence which is: (initiator Met
2C~ removed) Glyl-AA2-AA3-AA.~°AASwAA6-'AA7-Vie- , where
~a,~a,~a.~s~~s are small uncharged residues, AA3 and AA4 are
preferably neutral, where AAS is preferably serine or threonine,
where AA6 is not proline, and where AA7 and AA8 are preferably
basic (Towler et al. Annu. Rev. Biochem. 57:69-99 (1988); Resh.
Biochim. Biophys. Acta. 1451:1-16 (1999)). In a specific
embodiment, the fusion ;protein of the invention which comprises
the fatty acy:Lation sequence is related to the p14 protein.
14


CA 02429891 2003-06-18
As described herein, the invention also encompasses
substantially purified proteins. A "substantially purified
protein" as u:~ed herein is defined as a protein that is
separated from the environment in which it naturally occurs
5~ and/or that is free of the majority of the proteins that are
present in the environr~ient in which it was synthesized. For
example, a substantially purified protein is free from
cytoplasmic proteins. Those skilled in the art would readily
understand that the proteins of the invention may be purified
from a natural source, i.e., a Reoviridae-infected cell, or
produced by recombinant means.
Useful derivatives of the membrane fusion proteins of
the invention,, e.g., fusion-promoting fragments, may be
designed using computer-assisted analysis of amino acid
sequences to identify probable transmembrane regions, or
regions of high positive charge. Parameters such as helix
propensity, heptad repeats and average hydrophobicity can
reveal potential hydraphobi.c, membrane-interacting fragments
which may be used as a basis for selecting useful fusion-
2Q promoting fragments and variants.
One aspect of the invention provides sequences that
are identical or substantially identical to SEQ ID NOs: 2, 4, 6
and 8. By "amino acid sequence substantially identical" is
meant a sequence that is at least 80%, preferably 90%, more
2~a preferably 95% identical to an amino acid sequence of reference
and that preferably differs from the sequence of reference by a
majority of conservative amino acid substitutions.
Conservative amino acid substitutions are
substitutions among amino acids of the same class. These
31) classes include, for example, amino acids having uncharged
polar side chains, such as asparagine, glutamine, serine,
threonine, and tyrosine; amino acids having basic side chains,


CA 02429891 2003-06-18
such as lysine, arginine, and histidine; amino acids having
acidic side chains, such as aspartic acid and glutamic acid;
and amino acids having nonpolar side chains, such as glycine,
alanine, valine, leucine, isoleuci.ne, proline, phenylalanine,
methionine, tryptophan, and cysteine.
Percent identity is measured using sequence analysis
software such as the Sequence Analysis Software Package of the
Genetics Computer Group, University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, WI 53705. Amino acid
1C~ sequences are aligned to maximize identity. Gaps may be
artificially introduced into the sequence to attain proper
alignment. OIlCe the optimal alignment has been set up, the
degree of identity is established by recording all of the
positions in which the amino acids of both sequences are
1:i identical, relative to the total number of positions.
Proteins having a sequence homologous to SEQ ID Nos:
2, 4, 6 or 8 :include naturally-occurring allelic variants, as
well as mutants ar any other non-naturally occurring variants
that retain the membrane fusion properties of the polypeptides
2C) of SEQ ID Nos: 2, 4, 6 or 8. As is known in the art, an
allelic variant is an alternate form of a polypeptide that is
characterized as having a substitution, deletion, or addition
of one or more amino acids that retain the biological function
of the polypeptide, i.e. the membrane-fusion activity.
2;i Homologs and fragments thereof that do not occur
naturally are designed using known methods for identifying
regions of the protein that are likely to tolerate amino acid
sequence changes and/or deletions. As an example, homologous
polypeptides from different species are compared; conserved
3U sequences are identified. The more divergent sequences are the
most likely to tolerate sequence changes. Percent identity and
similarity among sequences may be analyzed using, as an
16


CA 02429891 2003-06-18
example, the BLAST homology searching algorithm of Altschul et
al., Nucleic Acids Res. 25:3389-3402 (1997). Alternatively, a
particular amino acid residue or sequence within the
polypeptide can be mutai~ed in vitro, then the mutant
~~ polypeptides :>creened for their agility to promote membrane
fusion.
A skilled person will understand that by following
the screening process of this invention, it will be determined
without undue experimentation whether a particular protein
encoded by Reoviridae, or related to a protein encoded by
Reoviridae, has membrane fusion activity. The screening
procedure comprises the steps:
(i) Introducing the test protein into a cell
susceptible to syncytia formation, either by delivering the
protein or by expressing the protein from a polynucleotide
encoding it; and
(ii) Observing whether membrane fusion has occurred
by determining whether syncytia have formed, compared to a
negative control where an irrelevant protein is introduced into
the cell.
(B) Antibodies
One aspect of the invention provides antibodies, both
polyclonal and monoclonal. Such antibodies can be employed for
diagnostic applications, therapeutic applications, and the
like. Preferably, for therapeutic applications, the antibodies
employed will. be monoclonal antibodies. Antibodies may be
recombinant, e.g., chimeric (e. g., constituted by a variable
region of murine origin associated with a human constant
region), humanized (a human immunoglobulin constant backbone
together witr4 hypervariable region of animal, e.g., murine,
origin), and~'or single chain. Both polyclonal and monospecific
17


CA 02429891 2003-06-18
antibodies may also be in the form of immunoglobulin fragments,
e.g., F(ab)~2 or Fab fragments. The antibodies of the
invention are of any isotype, e.g., 2gG or IgA, and polyclonal
antibodies are of a single isotype or a mixture of isotypes.
Antibodies against the proteins or fragments of the
invention are generated by immunization of a mammal with a
composition comprising the protein or fragment. Methods to
produce polyclonal or monoclonal antibodies are well known in
the art. For a review, see "Antibodies, A Laboratory Manual,
Cold Spring Harbor Laba:ratory, Eds. E. Harlow and D. Lane
(1988), and D..E. Yeltan et al., 1981. Ann. Rev. Biochem.
50:657-680.
Polyclonal antibodies may be obtained, for example,
by immunizing three manth old male and female white New Zealand
1~'~ rabbits with the synthetic peptide to which Tyr has been added
at the C-terminus in order to couple it, as an antigen, to BSA
by a bisdiazotized benzidine (BDB) linkage by reaction for 2
hours at 4°C. The reaction mixture is dialyzed to remove low
molecular weight material, and the retentate is frozen in
2~ liquid nitrogen and stored at -20°C. Animals are immunized
with the equivalent of 1 mg of the peptide antigen according to
the procedure of Benoit et al. P.N.A.S. USA, 79, 917-921
(1982). At four week intervals, the animals are boosted by
injections of 200 ug of the antigen and bled ten to fourteen
2',~ days later. .After the third boost, antiserum is examined for
its capacity to bind radioiodinated antigen peptide prepared by
the chloramine-T method and then purified by CMC-ion exchange
column chromatography. The antibody molecules are then
collected from the mammal and isolated to the extent desired by
31) well known techniques ~;uch as, for example, by using DEAE-
Sephadex to obtain the IgG fraction.
18


CA 02429891 2003-06-18
For monoclonal. antibodies, see Kohler & Milstein
(1975) Nature 256:495-497. In general, procedures for
preparing monoclonal antibodies involve immunizing an animal
with the protein or fracCment (the 'antigen'). The splenocytes
of such animals are ext:.racted and fused with a suitable myeloma
cell line. After fusion, the resulting hybridoma cells are
selectively maintained i.n HAT medium, and then cloned as
described in Wands et al.. Gastro-enterology. 80:225-232 (1981).
The hybridoma cells obt=ained through such a selection are then
assayed to identify clones which secrete antibodies capable of
binding the antigen.
To enhance thE: specificity of the antibody, the
antibodies may be purified by immunoaffinity chromatography
using solid phase-affixed immunizing polypeptide. The antibody
is contacted with the scalid phase-affixed immunizing
polypeptide for a period of time sufficient for the polypeptide
to immunoreact with the antibody mol.ecuies to form a so:Lid
phase-affixed immunocomplex. The bound antibodies are
separated from the comp7.ex by standard techniques.
A radioimmunoassay is established with the antisera
and serum from subsequent bleeds from the same rabbits. The
native protein is recognized by the antibodies on an equimolar
basis as compared to the synthetic peptide antigen.
The antibody so produced can be used, inter alia, in
diagnostic met=hods and systems to detect the level of fusion
protein present in a test sample. In addition, anti-fusion
protein antibodies can be used in therapeutic methods, e.g.,
blocking the occurrence of undesired fusion processes. The
anti-fusion protein ant=ibodies can. also be used for the
immunoaffinity or affin=ity chromatography purification of such
fusion proteins.
19


CA 02429891 2003-06-18
Accordingly, o:ne aspect of the invention provides a
process for purifying, from a biological sample, a polypeptide
or polypeptide derivative of the invention, which involves
carrying out antibody-based affinity chromatography with the
biological sample, wherein the antibody is a monospecific
ant ibody of thf~ invent 1 on .
For use in a purification process of the invention,
the antibody is either polyclonal or monospecific, and
preferably is of the IgG type. Purified IgG is prepared from
an antiserum using standard methods (see, e.g., Coligan et al.,
Current Protocols in Immunology (1994)John Wiley & Sons, Inc.,
New York, NY.). Conventional chromatography supports, as well
as standard methods for grafting antibodies, are described in,
e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds.
(1988) and outlined below.
Briefly, a b:i.ological sample, such as a cell extract
containing the membrane fusion protein, preferably in a buffer
solution, is applied to a chromatography material, preferably
equilibrated with the x>u.ffer used to dilute the biological
sample so that the polypeptide or polypeptide derivative of the
invention (i.e., the antigen) is allowed to adsorb onto the
material. The chromatography material, such as a gel or a
resin coupled to an antibody of the invention, is in either a
batch form or a column. The unbound components are washed off
and the antigen is then eluted with an appropriate elution
buffer, such as a glyc:~rie buffer or a buffer containing a
chaotropic agent, e.g., guanidine :HC1, or high salt
concentration (e. g., 3M MgCl2). Eluted fractions are recovered
and the presence of the antigen is detected, e.g., by measuring
the absorbance at 280 nm.


CA 02429891 2003-06-18
(C) Polynucleotides, their expression and use:
According to one aspect of the invention, isolated
polynucleotides are provided which encode the membrane fusion
proteins of the invention. In one embodiment, the
polynucleotides are those of SEQ ID NOs: 1 and 5.
The term "isolated polynucleotide" is defined as a
polynucleotide removed from the environment in which it
naturally occurs. For example, a naturally-occurring DNA
molecule present in the genome of a living virus or as part of
a gene bank is not isolated, but the same molecule separated
from the remaining part. of the viral genome, as a result of,
e.g., a cloning event (amplification), is isolated. Typically,
an isolated polynucleot.ide molecule is free from polynucleotide
regions (e. g., coding :regions) with which it is immediately
contiguous at the 5' or 3' end, in the naturally occurring
genome. Such isolated polynucleotides may be part of a vector
or a composition and st:i.ll be defined as isolated in that such
a vector or composition is not part of the natural environment
of such polynucleotide_
The polynucleotide of the invention is either RNA or
DNA (cDNA, genomic DNA, or synthetic DNA), or modifications,
variants, homologs or fragments thereof. The DNA is either
double-stranded or sing7_e-stranded, and, if single-stranded, is
either the coding strand or the non-coding (anti-sense) strand.
Any one of the sequence=_s that encode the proteins of the
invention as :shown in SEQ ID No: 2, 4, 6 and 8 is (a) a coding
sequence, (b) a ribonucleotide sequence derived from
transcription of (a), or (c) a coding sequence which uses the
redundancy or degeneracy of the genetic code to encode the same
polypeptides.
21


CA 02429891 2003-06-18
Homologous polynucleotide sequences are defined in a
similar manner to homologous amino acid sequences. Preferably,
a homologous polynucleotide sequence is one that is at least
45%, more preferably 60%, and most preferably 85% identical to
sequence encoding the proteins of the invention, or to the
coding sequences of SEQ TD NOs 1 and 5, or to the sequence
encoding the proteins of SEQ ID NOs:2, 4, 6 and 8.
Polynucleotides encoding homologous polypeptides or
allelic variants are retrieved by polymerase chain reaction
(PCR) amplification of genomic viral polynucleotides extracted
by conventional methods;' (see Example 2). This involves the
use of synthetic oligonucleotide primers matching upstream and
downstream of the 5' and 3' ends of the coding region.
Suitable primers are designed according to the nucleotide
sequence information provided in SEQ ID Nos:l and 5. The
procedure is as follows: a primer is selected which consists of
10 to 40, preferably 15 to 25 nucleotides. It is advantageous
to select primers containing C and G nucleotides in a
proportion sufficient to ensure efficient hybridization; i.e.,
2C1 an amount of C and G nucleotides of at least 40%, preferably
50% of the total nucleotide content. A standard PCR reaction
contains typically 0.~~ to 5 Units of Taq DNA polymerase per 100
~L, 20 to 200 ~M deoxynucleotide each, preferably at equivalent
concentrations, 0.5 to 2.5 rnM magnesium over the total
deoxynucleotide concentration, 105 to 106 target molecules, and
about 20 pmol of each primer. About 25 to 50 PCR cycles are
performed, with an annealing temperature 15°C to 5°C below the
true Tm of th.e primers. A more stringent annealing temperature
improves discrimination against incorrectly annealed primers
and reduces incorporation of incorrect nucleotides at the 3'
end of primers. A denaturation temperature of 95°C to 97°C is
typical, although higher temperatures may be appropriate for
dematuration of G+C-rich targets. The number of cycles
22


CA 02429891 2003-06-18
performed depends on the starting concentration of target
molecules, though typically more than 40 cycles is not
recommended as non-specific background products tend to
accumulate.
An alternative method for retrieving polynucleotides
encoding homologous po:l.ypeptides or allelic variants is by
hybridization screening of a DNA or RNA library. Hybridization
procedures are well-known in the art and are described in
Ausubel et al., (Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons Inc., 1994), Silhavy et al. {Silhavy
et al. Experiments with Gene Fusions, Cold Spring Harbor
Laboratory Press, 1984), and Davis et al. (Davis et al. A
Manual for Genetic Engineering: Advanced Bacterial Genetics,
Cold Spring Harbor Laboratory Press, 1980)). Important
parameters fox' optimizing hybridization conditions are
reflected in a. formula used to obtain the critical melting
temperature above which two complementary DNA strands separate
from each other (Casey tx Davidson, Nucl. Acid Res. (1977)
4:1539). For polynucleotides of about 600 nucleotides or
larger, this formula is as follows: Tm = 81.5 + 0.41 x (% G+C)
+ 16.6 log (canon ior~ concentration) - 0.63 x (% formamide) -
600/base number. Under appropriate stringency conditions,
hybridization temperature (Th) is approximately 20 to 40°C, 20
to 25°C, or, preferably 30 to 40°C below the calculated Tm.
Those skilled in the art will understand that optimal
temperature and salt conditions can be readily determined.
Polynucleotide molecules according to the invention,
including RNA, DNA, ox' modifications or combinations thereof,
have various applications. A DNA molecule is used, for
example, (i) in a process for producing the encoded polypeptide
in a recombinant host system, (ii) as part of a gene delivery
system, e.g. liposomes, which, upon delivery, becomes expressed
and promote membrane fusion, (iii) operably linked to
23


CA 02429891 2003-06-18
regulatory elements as part of an expression cassette which,
when turned on, expresses the polynucleotide and promote
membrane fusion, and, (iv) as a probe or primer.
Accordingly, nne aspect of the invention encompasses
(i) an expression cassette containing a polynucleotide of the
invention placed under the control of the elements required for
expression, in particular under the control of an appropriate
promoter; (ii) an expression vector containing an expression
cassette of the invention; (iii) a procaryoti.c or eucaryotic
cell transformed ar tra.nsfected with an expression cassette
and/or vector of the invention, as well as (iv) a process for
producing a polypeptide or polypeptide derivative encoded by a
polynucleotide of the :invention, which involves culturing a
procaryotic or eucaryoti.c host cell transformed or transfected
with an expression cassette and/or vector of the invention,
under conditions that allow expression of the DNA molecule of
the invention without t~e~ing toxic to the host cell and,
recovering the encoded polypeptide or polypeptide derivative
from the host cell culture.
A recombinant expression system is selected from
procaryotic arad e~ucaryotic hosts. Since the proteins of the
invention promote membrane fusion, host cells are selected
which can be maintained and which can express the proteins
within tolerable limits of toxicity. Eucaryotic hosts include
2~~ yeast cells (c~.g. , Sacc.haromyces cerevisiae or Pichia
pastoris), plant cells, and cells which preferably have a cell
wall so that i~he integrity of the host cell is not affected by
the fusion activity. A preferred expression system is a
procaryotic host such as E. colt. Bacterial and eucaryotic
cells are available from a number of different sources
including commercial sources to those skilled in the art, e.g.,
the American Type Culture Collection (ATCC; Rockville,
Maryland). Commercial. sources of cells used for recombinant
24


CA 02429891 2003-06-18
protein expression alsc> provide instructions for usage of the
cells.
One ;killed .in the art wauld readily understand that
not all vectors and expression control sequences and hosts
would be expected to express equally well the polynucleotides
of this invention.. Wii:.h the guidelines described below,
however, a selection oi: vectors, expression control sequences
and hosts may 'be made without undue experimentation and without
departing from the scope of this invention.
In selecting a vector, the host must be chosen such
that it is not affected by the fusion activity of the expressed
membrane fusion protein. In addition, a host must be chosen
that is compatible with the vector which is to exist and
possibly replicate in it. Considerations are made with respect
to the vector copy number, the ability to control the copy
number, expression of other proteins such as antibiotic
resistance. I:n selecting an expression control sequence, a
number of variables are considered. Among the important
variables are the relative strength of the sequence (e.g. the
2Q ability to dr~.ve expression under variaus conditions), the
ability to control the sequence's function, compatibility
between the polynuclec>tide to be expressed and the control
sequence (e. g. secondary structures are considered to avoid
hairpin structures which prevent efficient transcription). In
selecting the host, unicellular hosts are selected which are:
compatible with the sE:lected vector, tolerant of any possible
toxic effects of the expressed product, able to express the
product efficiently, able to express the product in the desired
conformation, easily scaled up, and easy to use for purifying
31) the final product .
The choice of the expression cassette depends on the
host system selected a~~ well as the features desired for the


CA 02429891 2003-06-18
expressed polypeptide. Typically, an expression cassette
includes a promoter that is functional in the selected host
system and can be constitutive or inducible; a ribosome binding
site; a start codon (ATG) if necessary; a region encoding a
signal peptide; a polyn.ucleotide of the invention; a stop
codon; and optionally a. 3t terminal region (translation and/or
transcription terminatr~r). The signal peptide encoding region
is adjacent to the polynucleotide of the invention and placed
in proper reading framF~. The signal peptide-encoding region is
homologous or heterologous to the DNA molecule encoding the
mature polypeptide and i.s compatible with the secretion
apparatus of the host rzs~ed for expression. The open reading
frame constituted by the DNA molecule of the invention, solely
or together with the signal peptide, is placed under the
control of the promoter so that transcription and translation
occur in the host system.
Promoters and signal peptide encoding regions are
widely known and available to those skilled in the art and
include, for example, the promoter of Salmonella typhimurium
(and derivatives) that :is inducible by arabinose (promoter
araB) and is functional in Gram-negative bacteria such as E.
coli (as described in U.S. Patent No. 5,028,530 and in Cagnon
et al., (Cagnon et al., Protein Engineering (1991) 4(7):843));
the promoter of the gene of bacteriophage T7 encoding RNA
polymerase, that is functional in a number of E. coli strains
expressing T7 polymerase (described in U.S. Patent
No. 4,952,496); UspA lipidation signal peptide; and RlpB
lipidation signal peptide (Takase et al., J. Bact. (1987)
169:5692).
Promoters contemplated for use herein include
inducible (e. g., minimal CMV promoter, minimal TK promoter,
modified MMLV LTR), constitutive (e. g., chicken alpha-actin
26


CA 02429891 2003-06-18
promoter, MMLV LTR (non-modified), DHFR), and/or tissue
specific promoters .
Inducible promoters contemplated for use in the
practice of the present invention comprise transcription
regulatory regions that function maximally to promote
transcription of mRNA under inducing conditions. Examples of
suitable induc:ible promoters include DNA sequences
corresponding to: the ~'. co.li lac operator responsive to IPTG
(see Nakamura et al., Cell, 18:1109-1117, 1979); the
metallothionein promotEar metal-regulatory-elements responsive
to heavy-metal (e. g., zinc) induction (see Evans et al., U.S.
Patent No. 4,870,009), the phage T7lac promoter responsive to
IPTG (see Studier et a=L., Meth. Enzymol., 185: 60-89, 1990; and
U.S. Patent No. 4,952,96), the heat-shock promoter; the TK
minimal promoter; the CMV minimal promoter; a synthetic
promoter; and the like.
The expression cassette is typically part of an
expression vector, whi:Yl is selected for its ability to
replicate in the chosen expression system. Expression vectors
(e. g., plasmid.s or viral. vectors) can be chosen, for example,
from those described in Pouwels et al. (Cloning Vectors: A
Laboratory Manual 1985, Supp. 1987). Suitable expression
vectors can be: purchased from various commercial sources.
Methods for transforming/transfecting host cells with
expression vectors are well-known in the art and depend on the
host system selected as described in Ausubel et al., (Ausubel
et al., Current Protocols in Molecular Biology, John Wiley &
Sons Inc., 1994).
Upon expression, a recombinant polypeptide of the
3CI invention (or a polypeptide derivative) is produced and remains
in the intracellular compartment, is secreted/excreted in the
27


CA 02429891 2003-06-18
extracellular medium or in the periplasmic space, or is
embedded in the cellular membrane. The polypeptide is
recovered in a substantially purified form from the cell
extract or from the supernatant after centrifugation of the
recombinant cell culture. Typically, the recombinant
polypeptide is purified by antibody-based affinity purification
or by other well-known methods that can be readily adapted by a
person skilled in the a.rt, such as fusion of the polynucleotide
encoding the p~olypepticle or its derivative to a small affinity
binding domain. Antibodies useful for purifying by
immunoaffinity the polypeptides of the invention are obtained
as described below.
The sequence information provided in the present
application enables the design of specific nucleotide probes
and primers that are used for diagnostic purposes.
Accordingly, one aspeci~. of the invention provides a nucleotide
probe or primer having a sequence found in or derived by
degeneracy of the genetic code from a coding sequence shown in
SEQ ID No:1 or 5.
A primer is a prabe of usually about 10 to about
40 nucleotides that is used to initiate enzymatic
polymerization of DNA in an amplification process (e. g., PCR),
in an elongation process, or in a reverse transcription method.
Primers used :in diagnostic methods involving PCR are labeled by
methods known in the art.
In one embodiment, optionally labeled cDNAs encoding
fusion proteins, or fragments thereof, can be employed to probe
library(ies) (e. g., cDNA, genomic, and the like) for additional
sequences encoding novel fusion proteins. Such screening is
31) typically initially c:-~rried out under low-stringency
conditions, which comprise a temperature of less than about
42°C, a formamide concentration of less than about 50%, and a
28


CA 02429891 2003-06-18
moderate to low salt concentration. Presently preferred
screening conditions comprise a temperature of about 37°C, a
formamide concentration of about 20%, and a salt concentration
of about 5X standard saline citrate (SSC; 20X SSC contains 3M
sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions
will allow the identification of sequences which have a
substantial degree of similarity with the probe sequence,
without requiring perfect identity for the identification of a
stable hybrid. The phrase "substantial similarity" refers to
sequences which share at: least 50% identity. Preferably,
hybridization conditions will be selected which allow the
identification of sequences having at least 70% identity with
the probe, while discriminating against sequences which have a
lower degree of identity with the probe.
As used herein, a nucleic acid "probe" is single-
stranded DNA or RNA, or analogs thereof, that has a sequence of
nucleotides that includes at least 14, preferably at least 20,
more preferably at least 50, contiguous bases that are the same
as (or the complement of) any 14 or more contiguous bases set
2C1 forth in any of SEQ Ib N0:1 or 5. Probes may be labeled by
methods well-known in the art, as described hereinafter, and
used in various diagnostic kits.
As used herein, the terms "label" and "indicating
means" in their various grammatical forms refer to single atoms
and molecules that are either directly or indirectly involved
in the production of a detectable signal to indicate the
presence of a complex. Any label or indicating means can be
linked to or incorporated in a nucleic acid probe, an expressed
protein, polypeptide fragment, or antibody molecule that is
part of an antibody or monoclonal antibody composition of the
present invention, or used separately. These atoms or
molecules can. be used alone or in conjunction with additional
29


CA 02429891 2003-06-18
reagents. Such labels <~re themsel.ves well-known in clinical
diagnostic chemistry.
The labeling means can be a fluorescent labeling
agent that chemically binds to antibodies or antigens without
denaturing them to form a fluorochrome (dye) that is a useful
immunofluorescent tracer. Suitable fluorescent labeling agents
are fluorochromes such as fluorescein isocyanate (FIC),
fluorescein i~othiocyant:e (FITC), 5-dimethylamine-1-
naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine
isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl
chloride (RB-200-SC), and the like. A description of
immunofluorescence ana:Lysis techniques is found in DeLuca,
"Immunofluorescence Analysis", in Antibody As a Tool,
Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189-231
(1982) .
In preferred embodiments, the indicating group is an
enzyme, such as horseradish peroxidase (HRP), glucose oxidase,
and the like. In such cases where the principal indicating
group is an enzyme, additional reagents are required to
visualize the fact that a receptor-ligand complex
(immunoreactant) has formed. Such additional reagents for HRP
include hydrogen peroxide and an oxidation dye precursor such
as diaminobenzidine. An additional reagent useful with
glucose oxidase is 2,2'-azino-di-(:3-ethyl-benzthiazoline-G-
sulfonic acid) (ARTS).
Radioactive elements are also useful labeling agents
and are used illustratively herein. An exemplary radiolabeling
agent is a radioactive element that produces gamma ray
emissions. Elements which emit gamma rays, such as 124I, 125I,
126I, 131I and 5lCr, represent one class of radioactive element
indicating groups. Particularly preferred is 1251. Another
group of useful labeling means are those elements such as 11C,


CA 02429891 2003-06-18
18F, 150 and 1.3N which emit positrons. The positrons so
emitted produce gamma :rays upon encounters with electrons
present in the animal's body. Also useful is a beta emitter,
such as 32P, 1.11In or :3H.
The linking of labels to substrate, i.e., labeling of
nucleic acid probes, ant:i.bodies, golypeptides, and proteins, is
well known in the art. For instance, antibody molecules
produced by a hybridoma can be labeled by metabolic
incorporation of radioisotope-containing amino acids provided
as a component. in the ~~ulture medium. see, for example, Galfre
et al., Meth. Enzymol., 73:3-46 (1981). The techniques of
protein conjugation or coupling thraugh activated functional
groups are particularly applicable. See, fox example, Aurameas
et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),
Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Patent No.
4,493,795.
(D) Com ositions_rontaining membrane fusion proteins and
polynucleotides
As used herein, the composition of the invention
contains one or several membrane fusion proteins or derivatives
of the invention.
For use in a composition of the invention, according
to one embodiment, a me=mbrane fusion protein or derivative
thereof is formulated :into or with liposomes, preferably
neutral or anionic liposomes, microspheres, ISCOMS, or virus-
like-particles (VLPs) to facilitate delivery and/or enhance the
immune respon~,e. These compounds are readily available to one
skilled in the art; fox example, see Liposomes: A Practical
Approach, RCP New Ed, IRL press (1990).
Anionic and neutral liposomes are well-known in the
art (see, e.g., Liposome:s: A Practical Approach, RPC New Ed,
31


CA 02429891 2003-06-18
IRL press (1990), for a detailed description of methods for
making liposomes) and are useful for delivering a large range
of products, including polynucleotides.
Cationic lipids are also known in the art and are
commonly used for drug or gene delivery. Such lipids include
LipofectinTM also known as DOTMA (N- [1- (2, 3-dioleyloxy) propyl] -
N,N,N-trimethylammonium chlaride), DOTAP (1,2-bis(oleyloxy)-3-
(trimethylammanio)propane), DDAB (dimethyldioctadecylammonium
bromide), DOGS (dioctadecylamidologlycyl spermine) and
cholesterol derivatives such as DC-Chol (3 beta-(N-(N~,N~-
dimethyl aminamethane)~-carbamoyl) cholesterol). A description
of these cationic lipids can be found in EP 187,702,
WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501,
WO 95/26356, and U.S. F.,atent No. 5,527,928. Cationic lipids
for delivery of polynucleotides are preferably used in
association with a neutral lipid such as DOPE (dioleyl
phosphatidylethanolamine), as described in WO 90/11092 as an
example.
Formulations cantaining cationic liposomes may
optionally contain other transfection-facilitating compounds.
A number of them are described in WO 93/18759,, WO 93/19768, WO
94/25608, and WO 95/02397. They include spermine derivatives
useful for facilitating the transport of DNA through the
nuclear membrane (see, for example, WO 93/18759) and membrane-
permeabilizing compounds such as GALA, Gramicidine S, and
cationic bile salts (see, for example, WO 93/19768).
The ,present invention also contemplates therapeutic
compositions containing a physiologically tolerable carrier
together with a fusion protein, polypeptide fragment thereof,
or anti-fusion protein antibody, as described herein, dissolved
or dispersed therein as an active ingredient.
32


CA 02429891 2003-06-18
As used herein, the terms "pharmaceutically
acceptable", "physiologically tolerable" and grammatical
variations thereof, as they refer to compositions, carriers,
diluents, and reagents, are used interchangeably and represent
that the materials are capable of administration to a mammal
without the production of undesirable physiological effects
such as nausea, dizziness, gastric upset, and the like.
Methods for the preparation of a pharmacological
composition that contains active ingredients dissolved or
dispersed therein is well known in. the art. Typically such
compositions are prepared as injectables either as liquid
solutions or suspensions; however, solid forms suitable for
solution, or suspension, in liquid prior to use can also be
prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients
that are pharmaceutica=LS.y acceptable and compatible with the
active ingredient and in amounts suitable for use in the
therapeutic methods described herein. Suitable excipients are,
for example, water, saline, dextrose, glycerol, ethanol, or the
like, as well as combinations thereof. In addition, if
desired, the composition can contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, and the like, which enhance the effectiveness of the
active ingredient.
The therapeutic composition of the present invention
can include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable nontoxic salts include
the acid addition salts (formed with the free amino groups of
the polypeptide) that are formed with inorganic acids such as,
for example, hydrochloric acid, hydrobromic acid, perchloric
acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric
acid, acetic acid, propionic acid, glycolic acid, lactic acid,
33


CA 02429891 2003-06-18
pyruvic acid, oxalic acid, malonic acid, succinic acid, malefic
acid, fumaric acid, anthranilic acid, cinnamic acid,
naphthalene sulfonic acid, sulfanilic acid, and the like.
Salt=s formed with the free carboxyl groups can also
:i be derived from inorganic bases such as, for example, sodium
hydroxide, ammonium hydroxide, potassium hydroxide, and the
like; and organic bases such as mano-, di-, and tri-alkyl and -
aryl amines (e. g., triethylamine, diisopropyl amine, methyl
amine, dimeth~,rl amine, and the like), and optionally
1C1 substituted et:hanolamines (e. g., ethanolamine, diethanolamine,
and the like).
Physiologically tolerable carriers are well known in
the art. Exemplary liquid carriers are sterile aqueous
solutions than contain no materials other than the active
1~i ingredients and water, or contain a buffer such as sodium
phosphate at physiological pH value, physiological saline, or
both, such as phosphate-buffered saline. Still further,
aqueous carricsrs can contain more than one buffer salt, as well
as salts such as sodium and potassium chlorides, dextrose,
2C1 polyethylene glycol, and other solutes.
Liquid compositions can also contain liquid phases in
addition to and to thE: exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as
cottonseed oi:L, and water-oil emulsions.
2:i A therapeutically effective amount is a predetermined
amount calculated to achieve the desired effect. The required
dosage will v<~ry with the particular treatment and with the
duration of desired treatment; however, it is anticipated that
dosages between about 10 micrograms and about 1 milligram per
3C) kilogram of body weight per day will be used for therapeutic
treatment. In some instances, it may be particularly
34


CA 02429891 2003-06-18
advantageous to administer such compounds in depot or long-
lasting form. A therapeutically effective amount is typically
an amount of a fusion protein according to the invention, or
polypeptide fragment thereof that, when administered in a
~~ physiological:Ly accept.alble composition, is sufficient to
achieve a plasma concenvtration of from about 0.1 ~,g/m1 to about
100 ~g/ml, prE:ferably from about 1.0 ~g/ml to about 50 ~Cg/ml,
more preferably at least about 2 ~tg/ml and usually 5 to 10
~tg/ml. Antibodies are administered in proportionately
appropriate amounts in accordance with known practices in this
art.
(E) Methods of Use
One aspect cf the invention provides methods of using
the proteins, polynucleotides and compositions of the
invention. Ac;cordingl:y,, methods are provided to promote
membrane fusion which ~~omprise contacting the membranes to be
fused with an effective amount of the above-described proteins.
Membranes contemplated for fusion in accordance with
the present invention :include cell membranes, liposome
membranes, proteoliposome membranes, and the like.
In accordance with a still further embodiment of the
present invention, there are provided methods for the
production of heteroka:ryons, such as B cell or T cell hybridoma
cells useful for the production of monoclonal antibodies,
cytokines, and. immune modulators, said methods comprising
contacting, for example, an immortalized myeloma cell and a
primary B cell or T cell in the presence of any one or more of
the above-described proteins. Immortalized cells contemplated
for use herein. include human or mouse B cell myeloma cells, T
cell myelomas, and the like, and antibody-synthesizing cells


CA 02429891 2003-06-18
contemplated i:or use herein include purified spleen cells from
an immunized mammal, and the like.
In accordance with a still further embodiment of the
present invention, there are provided methods for the
production of liposome-liposome fusions or liposome-cell
fusions, said methods comprising contacting lipids suitable for
the formation of liposomes and a suitable cell in the presence
of one or more proteins as described herein.
In accordance with yet another embodiment of the
present invention, there, are provided improved methods for the
intracellular delivery of bioactive compounds employing
liposomes, they improvement comprising incorporating into said
liposomes one or 'more proteins as described herein.
The ability to promote efficient membrane fusion has
broad applicability in clinical, industrial, and basic research
situations. I'he reovirus fusion proteins could be used as
alternatives to chemica7.ly-induced membrane fusion to promote
cell-cell fusion, for example, during the production of
hybridoma cells for monoclonal antibody production. In this
instance, the reovirus fusion proteins would be inducibly
expressed from inside a transiently or permanently transfected
cell population to trigger fusion of these cells with a target
cell population.
The atypical reovirus fusion proteins also have
application in enhancing liposome-cell fusion. Liposomes have
been developed. as a means to introduce nucleic acids, proteins,
and metabolic regulators into cells. Although liposome-cell
fusion has been amply d~;monstrated, the unfavourable
thermodynamics of membrane fusion contribute to variable
efficiencies of fusion and cytotoxicity which lead to the
36


CA 02429891 2003-06-18
development of proteol.iposomes - 7.iposomes containing specific
proteins to promote cell binding and fusion.
Most of the proteoliposome studies reported in the
art relate to the use of various enveloped virus fusion
proteins. In accordancE= with the present invention, it is
possible to take advant<~ge of the novel structural features
associated with the invention reovirus fusion proteins for use
in proteoliposomes to enhance the intracellular delivery of
bioactive compounds (e. g., nucleic acids, proteins or peptides,
pharmacological agents, and the like), both in cell culture and
in vivo.
The reovirus fusion proteins described herein promote
membrane fusion in a diversity of cell types (e. g., fibroblasts
and macrophages) from different species (e.g., avian and
mammalian, including human) suggesting limited cell receptor-
specificity a~~ well as the general applicability of these
proteins. It may also be possible to target reovirus fusion
protein-containing proteoliposomes to specific cell types by
including specific receptor-binding proteins in the liposome
membrane. In this instance, the receptor-binding protein would
confer targeted cell attachment of the liposome followed by
subsequent enhanced lipcasame-cell fusion mediated by the
reovirus fusion protein.
The demonstr<it.ed ability of p14 and p22 to induce
cell-cell fusion indicates their potential use in the
production of heterokaryons, for example, the generation of
hybridomas for monoclonal antibody production. The induction
of cell-cell fusion is usually triggered using the chemical
fusogen polyethylene glycol (PEGS. Although this procedure
does trigger cell-cell fusion, toxic effects on cells hamper
the efficiency of heterokaryon isolation. It is generally
believed that "natural" membrane fusion is mediated by protein-
37


CA 02429891 2003-06-18
lipid interact: ions, therefore, protein-mediated membrane fusion
is likely to be much less cytotoxic than chemically-induced
cell fusion.
The demonstrated ability of the small reovirus fusion
5~ proteins to promote ef:f:icient cell-cell fusion indicates their
potential use as alternatives to chemical-induced cell fusion.
Expression of p14 or g22 in one population of cells, under the
control of a :strong in3r~cible promoter, could trigger fusion
with a second cell population, resulting in decreased
cytotoxicity and more efficient heterokaryon isolation.
The atypical group of nonenveloped virus fusion
proteins described herein represent alternatives to the use of
enveloped virus fusion proteins in the protein-mediated
enhancement of liposome-cell fusion for the intracellular
delivery of bioactive molecules. The potential advantages of
the reovirus fusion proteins relate to their unique structural
and biological features, From a structural perspectives the
small size of the reovirus fusion proteins is the most apparent
advantage offered by this system. The large size, post-
translational glycosylat.ion, and complex tertiary structure of
the enveloped virus fusion proteins makes synthesis and
purification of the functional protein using recombinant DNA
approaches and prokaryot,a.c or eukaryotic expression systems
problematic.
The majority of studies relating to the use of
enveloped virus fusion proteins in proteoliposomes involve the
production of 'virus particles which are subsequently purified,
solubilized with detergent, and the viral envelopes containing
the fusion protein are reconstituted into "virosomes" by
removal of the detergent (see Grimaldi in Res. Virol., 146:289-
293 (1995) and Ramani et al., FEBS Lett., 404:164-168 (1997)).
Unlike most of the enveloped virus fusion proteins, the
38


CA 02429891 2003-06-18
reovirus fusion proteins are small membrane proteins. Their
small size and simple domain organization suggests that these
proteins will be easier and more economical to produce in a
functional foam using a diversity of expression and
purification protocols. It is also likely that the small size
of the reovirus fusion, proteins contributes to less complex
protein folding pathways and tertiary structure required for
correct protean confoi:mation. As a result, an increased
diversity of <;xtraction and solubilization procedures (e. g.,
choice of detergents and denaturants? should be available to
facilitate purification of the functional fusion protein and
incorporation into liposomes.
The attractive biological properties of the reovirus
fusion proteins relate~i~o their pH-independent fusion mechanism
with numerous cell typea. The reavirus fusion proteins
function at nE:utral pH, unlike the influenza virus HA protein,
simplifying their use in cell culture and in vivo under
physiological conditions. Furthermore, the reovirus fusion
proteins fuse numerous types of cells suggesting their broad
applicability as fusogens. This could include such primary cell
types as dendritic cells, neurons, and stem cells which are
difficult to t:ransfect using standard transfection reagents.
Accordingly, the reovirus fusion proteins could be used to
promote liposome-cell fusion and the efficient intracellular
delivery of DNA or other bioactive compounds into a diversity
of cultured cell types, primary cell cultures, tissue explants,
or in vivo.
In order to use reovirus fusion proteins for
heterokaryon production, the proteins will need to be expressed
in a controlled, inducible manner from within cells using
standard recombinant DNA approaches. The utility of this
approach has already been demonstrated in homologous cell-cell
fusion in a non-inducibl.e manner. In a similar fashion, these
39


CA 02429891 2003-06-18
proteins can promote cell-cell fusion between heterologous cell
types in an inc3ucible manner,
The development of reovixus fusion proteins for
enhanced liposome-cell fusion requires the expression and
purification of the functional fusion proteins and their
incorporation :into liposome membranes to produce
proteoliposome;s. The ~:~14 and p22 proteins can be expressed and
purified using standard procedures. Expression can be
accomplished employing a variety of expression systems, e.g.,
baculovirus or yeast eukaryotic expression vectors or from
prokaryotic expression 'vectors, depending on expression levels
and functional activit~~ of the protein. Various detergent
extraction procedures can be used to solubilize the proteins,
which can then be purified as detergent-protein complexes using
standard protein purification protocols. The proteins are
readily soluble in vara.o~us detergents (e. g. 0.8% Triton X100,
0.8% NP40, 0.8% octylglucoside) increasing the diversity of
approaches available for functional protein purification. The
small size of the reovirus fusion proteins suggests that
protein solubilization and purification should be considerably
more simple than simil~~r approaches to purify larger, more
complex membrane proteins.
The detergent-protein complexes can be mixed with
lipids and the detergent: removed by dialysis, chromatography,
or extraction according to standard published procedures,
similar to methods used to generate influenza HA or Sendai
virus F protein-containing virosomes (see Grimaldi, Res.
Virol., 146:289-293 (1995) and Ramani et al., FEBS Lett.,
404:164-168 (1.997)). These procedures will result in the
production of proteoliposomes, lipid vesicles containing the
ARV, NBV, or BRV fusion proteins embedded in the vesicle
membrane. Once again, optimal conditions for proteoliposome
production can be empirically determined as can the lipid


CA 02429891 2003-06-18
composition and size of 'the proteoliposomes which can affect
the efficiency of liposome-cell fusion. Bioactive molecules of
interest (e. g., nucleie: acids, proteins or peptides,
pharmacological compounds, and the like) can be included during
the formation of the proteoliposmes to facilitate packaging of
the molecule within the liposomes. The proteoliposomes can be
purified by centrifugation and used to deliver bioactive
molecules intracellularly, either in cell culture or in vivo,
by protein-enhanced fusion of the proteoliposomes with cell
membranes.
As acknowledged above, the use of liposomes or
proteoliposomes for in;racellular delivery of compounds is
known in the art, and development of such methodology is
proceeding on several fronts. What is unique with the present
system is the use of an atypical, previously unidentified group
of nonenveloped virus i:usion proteins isolated from the only
known fusogenic poikilot;hermic reoviruses. The unusual
structural and. functional properties of this new group of
fusion proteiras suggest that these proteins may circumvent many
of the problems associated with the current development of
protein-mediated membrane fusion.
While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the
spirit and scope of that which is described and claimed.
EXAMPLES
Example 1: Virus growth and purification
Viruses were plaque purified and grown in monkey Vero
cells. Virus particles were isolated and concentrated from
3U infected cell lysates by differential centrifugation, as
41


CA 02429891 2003-06-18
previously described (see Duncan, Virology, 219:179-
189. (1996) ) .
Example 2: Synthesis and cloning of cDNA
The ~riral genomic dsRNA segments were isolated from
concentrated virus stocks pretreated with RNase and DNase to
remove extra-v:irion contaminating cellular nucleic acids.
Virus particles were disrupted using 1~ SDS and the viral dsRNA
isolated by phenol-chloroform extraction and ethanol
precipitation. Aliquots of genomic dsRNA (20 ug) were poly-A-
tailed using E. coli poly-A polymerase, the tailed RNA was
fractionated by agarosE~ gel electrophoresis, and individual
genomic segments were isolated using the RNaid protocol
(Biolol) according to ~;h.e manufacturers specified procedure.
The tailed S class genome segments were used as
templates for reverse t:ranscriptio:n, using Superscript reverse
transcriptase (Life Technologies Inc.) and an oligo-dT primer.
Aliquots of the plus and minus strand cDNAs were used as
templates for PCR amplification using Vent polymerase (New
England Biolabs) and an oligo-dT primer containing a NotI
restriction enzyme site.
The products of the PCR reaction were digested with
NotI, size-fractionated on agarose gels, and products
corresponding to the full length S genome segments were gel-
purified using Geneclean (Bio101). The individual, NotI-
digested, double-stranded cDNAs were cloned into the NotI site
of pBluescript (Stratagene) and used as templates for
sequencing.
Example 3: Sequencing and sequence analysis
The cloned cDa~As were sequenced using an automated
DNA sequences (Licor) at the NRC/Dalhousie Joint Sequencing
42


CA 02429891 2003-06-18
Core Facility. All sequences were determined in their entirety
from both cDNA strands. The full length cDNA sequences were
compiled and analyzed using the GCG sequence analysis software
(see Devereaux et al., Nucleic Acids Res., 12:387-395 (1984)).
Example 4: Identification of genome segments encoding the
fusion proteins of RRV and AQV
The RRV and AQV cDNA clones were subcloned into the
eukaryotic expression vector pcDNA3 (Invitrogen) under the
control of the CMV promoter. Plasmid DNA was isolated and
purified on Qiagen midi columns (Qiagen) according to the
manufacturer '; specific:ations. Plasmid DNA (1 ug) was mixed
with Lipofecta~mine (3 u7~;1 (Life Technologies Inc. ) and used to
transfect sub-confluent cell monolayers grown in 12 well
cluster plates. Transfected cell monolayers were incubated at
37°C for 24-4~ hr before being fixed with methanol and stained
using a water-soluble Wright-Giemsa stain (DiffQuik; VWR-
Canlab), as previously described (see Duncan et al., Virology,
224:453-464 (1996)). Cell fusion was assessed by light
microscopy of stained monolayers and syncytial foci were
photographed at 100x magnification. Using this protocol, the
S1 genome segment of RR'V and genome segment 7 of AQV were
determined to encode t:h~e fusion proteins of these viruses.
Example 5: Identification of p14 and p22 fusion proteins
Two unrelated fusion proteins responsible for the
cell-cell fusion induced by reptilian reovirus (RRV) and
aquareovirus (AQV) have been identified. These proteins are
referred to herein as p1.4 (for RRV) and p22 (for AQV) to
reflect their approximate predicted molecular weights. The
genes encoding p14 and p22 have been cloned and sequenced. The
sequence-predicted structural organization of these proteins
43


CA 02429891 2003-06-18
has been analyzed, and the membrane fusion properties thereof
have been directly demonstrated.
p14 of reptilian reovixws
Sequence analysis determined that the RRV S1 genome
segment is 1501 base pairs long and contains the conserved
terminal nucleotide sequences 5'-GTTA...TCATC-3' present in other
RRV genome segments, indicating the sequence represents the
full-length cD:~TA sequence of the S1 genome segment. This
represents the first nucleotide sequence reported for any
genome segment of any reptilian reovirus.
Sequence ana:l.ysis revealed the presence of two
sequential overlapping ORFs. Based on sequence similarity, the
second ORF spanning nucleotides 386-1432 (including the ATG
start codon and exclud:i.n.g the termination codon) encodes the
349 amino acid homolog of the typical reovirus cell attachment
protein, termed sigma C" for avian reovirus and sigma 1 for
mammalian reovirus (ShaF~ouri et al., J. Gen. Virol., 76:1515-
1520) .
The first ORF, spanning nucleotides 25-399, encodes a
previously unidentified predicted 125 amino acid protein. The
predicted 125 amino acid product of the first ORF had no
homolog amongst the known reovirus proteins, and displayed no
significant sequence s:~milarity to any proteins contained in
the databanks. The fi~_~>t: ORF was subcloned by PCR using
specific primers into pc:DNA3. Expression of the first ORF
alone in transfected cells resulted in cell-cell fusion (see
Example 8 ) ind.icating t~l~iat RRV encodes a novel 14 Kda fusion
protein, termed p14.
p22 of Aquareavirus:_
44


CA 02429891 2003-06-18
The AQV genome segment 7 sequence was found to be
1399 base pairs long a~~d. contained the conserved terminal
nucleotide sequences 5'-GTTT...TCATC-3' present in other AQV
genome segments, indicating the sequence represents the full-
y length cDNA sequence of. genome segment 7.
Sequence anal~~sis revealed a complex arrangement of
ORFs on the AQV genome ~;egment 7. The first methionine start
codon does not occur until 489 nucleotides from the 5'-end of
the gene. This start cc>don is followed by a single ORF
encoding a predicted 2'78 amino acid protein, presumably the
previously identified N~~28 protein, the only known gene product
of this genome segment I;Subramanian et al., virology, 205:75-81
(1994)). In the 5'-terminal 488 nucleotides preceding this
start codon, a.ll 'three reading frames contain small ORFs. The
ORF in reading' frame "a"' spans nucleotides 1-267, the ORF in
reading frame "b" spans nucleotides 8-610, and the ORF in
reading frame "c" spans nucleotides 3-482 (all excluding the
termination codon). Each of these ORFs lack a conventional ATG
start codon, but could conceivably be functional using a codon
other than met.hionine as a start codon, as occurs, for example,
with the C proteins encoded by the P mRNA of Sendai virus (see
Curran and Ko7.akofsky, ~sMBO J., 7:245-251 (1988)). These AQV
ORFs can encode 89, 20:1,, or 160 amino acid proteins for reading
frames a, b, and c, respectively.
The 5'-terminal 485 nucleotides of the AQV genome
segment 7 were' subcloned into pcDNA3 by PCR amplification using
specific primers, expressed in tra.nsfected cells, and found to
induce cell-cell fusion. This result confirms that the AQV
fusion protein was encoded by one of the three predicted small
30~ ORFs present near the 5'-end of the genome segment.
Of t:he three possible gene products encoded by this
region, the potential 201 amino acid product of reading frame


CA 02429891 2003-06-18
b, termed p22, was predicted to be a membrane localized
protein, as would be expected for a protein capable of causing
membrane fusion (a transmembrane region is predicted to exist
in p22, as determined ~m;ing the TMpred and PSORT algorithms;
see Example 7). In order to confirm that p22, encoded by the
second ORF, represents the AQV fusion protein, site-directed
mutagenesis was used to insert a C to T substitution at
position 59. This sub:~t.itution introduces a translation stop
site into reading frame b specifically, which would result in
the premature termination of the p22 ORF after only 17 codons.
Such a substitution eliminated cell fusion, confirming that the
predicted p22 gene product of ORF b represents the AQV fusion
protein. Since the p22 ORF lacks the typical methionine start
codon, the precise stax:t site of p22 is not presently known but
clearly resides in one of the 17 codons that occur upstream of
the nucleotide substitution used to engineer a stop codon at
position 59.
Example 6: Sequence analysis of p14
The RRV p14 fusion protein is one of the smallest
known fusion proteins at only 125 amino acids in length. It
shares no significant sequence similarity with any protein in
the databases, includirig~ those implicated as membrane fusion
proteins such as the enveloped virus fusion proteins, the
cellular SNARE protein:, or even the previously identified
fusion proteins of avian reovirus and Nelson Bay reovirus
(Shmulevitz and Duncan, EMBO J., 19:902-912 (2000), White,
Annu. Rev. Physiol., 52:675-697 (1990), Weber et al., Cell,
92:759-772 (1998)). As such, p14 represents a novel membrane
fusion protein.
The p14 protein contains a single predicted
transmembrane domain, detected using both the TMpred and PSORT
algorithms, located between residues 39-57 (see Figure 4). It
46


CA 02429891 2003-06-18
also contains ,an N-terminal myristylation consensus sequence
(MGXXXS/T) suggesting the protein may be acylated. Sequence
analysis also detected a cluster of basic residues on the C-
proximal side of the transmembrane domain, and a polyproline
motif and N-linked glycosylation consensus sequence (NXS/T)
located near the C-terminus of the protein (see Figure 4).
Interestingly, p14 lacks an obvious fusion peptide, a
small stretch of hydrophobic amino acids located in the
extracellular domain of enveloped virus fusion proteins and
involved in insertion of_ a fusion prote~.n into the target
membrane. Moreover, the p14 protein lacks any apparent heptad
repeats required for the coiled coil rearrangements observed in
enveloped virus fusion proteins. The lack of fusion peptide
and heptad repeat motifs in p14 suggests that p14 may promote
membrane fusion through a mechanism distinct from that proposed
for the well described enveloped virus fusion proteins (see
White, Ann. Rev. Physi.o:l., 52:675-697 (1990)).
Example 7: SE~quence analysis of p22
The AQV p22 fusion protein shares no obvious sequence
homology with either t:he RRV p14 fusion protein, the fusion
proteins of avian reovirus or Nelson Bay reovirus, or any other
protein in the databases. The p22 protein is approximately 201
amino acids i:n size. Th.e precise size is not known and depends
on which of the 17 N-terminal codons present in the p22 ORF
serve as the start codon for this protein. The p22 protein has
a predicted transmembrane domain located approximately between
residues 43-60. As with p14, p22 contains clusters of basic
residues adjacent to the transmembrane domains and p22 also
lacks obvious fusion peptide and heptad repeat motifs.
Example 8: T'he p14 and p22 fusion proteins induce cell-cell
fusion
47


CA 02429891 2003-06-18
The fusion-irlducing potential of these reovirus
proteins has been directly demonstrated by expressing them in
transfected cells in t~.ie absence of any other reovirus
proteins; intracellular expression triggers the induction of
cell-cell fusion and s~~n.cytium formation characteristic of
virus infection by this group of fusogenic reoviruses. Thus,
quail or Vero cell monolayers were mock transfected, or
transfected with plasm:id. DNA expressing the RRV or AQV fusion
proteins. Transfected cells were fixed and the nuclei stained
using a Wright-Giemsa ~~tain at 24 hr post infection and the
stained monolayers were photographed at 100x magnification.
Transfection of plasmids expressing either RRV p14 or
AQV p22 resulted in ext:e:nsive cell fusion and the development
of multinucleated syncvytia Cpolykaryons). The appearance of
polykaryons was evident. when transfected cells were stained to
display the cell nuclei, which clearly showed the clustering of
nuclei within large syxicytial cells. Mock transfected cells
showed no signs of syn.y~tium formation, indicating that cell
fusion was the direct r_e~sult of the expression of the reovirus
proteins within transfected cells. These results conclusively
demonstrate the membrarae~ fusion-inducing capability of the
reovirus fusion protein; of the invention.
Transfection of other reovirus proteins fails to
induce cell fusion, indicating that this is a p14- or p22-
specific event. zn addi.tion, both proteins induce cell fusion
in a variety of cell types of avian or mammalian origin
indicating the general utility of these proteins to induce
membrane fusion.
While the invention has been described in detail with
reference to certain prE:ferred embodiments thereof, it will be
understood that modifications and variations are within the
spirit and scope of that: which is described and claimed.
48


CA 02429891 2003-06-18
Summary of Sequences:
SEQ ID N0:1 is a cDNA nucleotide sequence of the S1
genome segment of reptilian reovirus (RRV). The open reading
frame encoding p14 is shown.
SEQ ID N0:2 is the deduced amino acid sequence of the
p14 protein enc;oded by SEQ ID NO:1.
SEQ ID N0:3 is a cDNA nucleotide sequence of the S1
genome segment of reptilian reovirus (RRV). The open reading
frame encoding the sigma C protein is shown.
SEQ 'ID N0:4 is the deduced amino acid sequence of the
sigma C protein encoded by SEQ ID N0:1 or 3.
SEQ ID N0:5 i.s a cDNA nucleotide sequence of genome
segment 7 of aquareovirus (AQV). 'The apen reading frame
encoding p14 as shown.
SEQ ID N0:6 is the deduced amino acid sequence of the
p22 protein encoded by SEQ ID N0:5. Note that the initiator
codon is one of the first 17 codons; its precise position has
not been determined.
SEQ ID N0:7 is a cDNA nucleotide sequence of genome
segment 7 of aquareovi.rus (AQV). The open reading frame
encoding the NS28 protein is shown.
SEQ ID N0:8 is the deduced amino acid sequence of the
NS28 protein encoded by SEQ TD N0:5 or 7.
49


CA 02429891 2003-06-18
SEQUENCE LISTING
(1) GENERAL INFOF:MATTON:
( i ) APPLICAI'iT : FUSOGENI:K INC .
(ii) TITLE OF' INVENTION: MEMBRANE FUSION PROTEIN
(iii) NUMBER OF SEQUENCES: 8
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: 3Mi'~RT & BIGGAR
O (B) STF:EET: P.O. BO:K 2999, STATION D
( C ) C I7.'Y : OTTAWA
(D) STF~TE: ONT
( E ) COL'INTRY : CAN.~~i~
(F) ZIP: K1P 5Y6
(v) COMPUTEF; READABLE FORM:
(A) MEL)IUM TYPE: Floppy disk
(B) COMPUTER: IBM 1?C compatible
(C) OPE;RATI:NG SYSTEM: PC-DOS/MS-DOS
(D) SOE'TWARE: AaC:CI (text)
ZO (vi) CURRENT APPLICATION DATA:
(A) APPLICATION I~IUMBER:
(B) FILING DATE: 02~-JUNE-2003
(C) CLF,SSIFICATIC)N:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUP4BER:
(B) FILING DATE:
(viii) ATTORNEY'/AGENT INFORMATION:
(A) NAME: SMART tK BIGGAR
(B) REGISTRATION NUMBER:
3O (C) REE'ERENCE/DOC'KET NUMBER: 78973-13
(ix) TELECOMMUNICATION TPdFORMATION:
(A) TEhEPHONE: (61:3)-232-2486
(B) TEhEFAK: (61~~ ) --232-8440
(2) INFORMATION F'OR SEQ ID NO.: 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1501
(B) TYPE: nucleic acid
(C) STRANDEDNES'S:
4O (D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian reovirus
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (25) . . (402)
(xi) SEQUENCE DESCRIPTION: :~EQ ID NO.: 1:
GTTATTTTTT TCCTCC~ATGA AGCC .ATG GGG AGT GGA CCC TCT AAT TTC GTC 51
Meet Gly Ser Gly Pro Ser Asn Phe Val
~ 5
AAT CAC GCA CCT C:IGA GAA GCA ATT GTA ACC GGT TTG GAG AAA GGG GCA 99
Asn His Ala Pro C'ly Glu Ala Ile Val Thr Gly Leu Glu Lys Gly Ala
10 15 20 25
GAT AAA GTA GCT G'~GA ACG ATA 7.."CA CAT ACG ATT TGG GAA GTG ATC GCC 147
Asp Lys Val Ala Gly Thr Ile Ser His Thr Ile Trp Glu Val Ile Ala
30 35 40
50

CA 02429891 2003-06-18
GGA TTA GTA GCC '7.'TG CTG ACr~. 'T'TC TTA GCG TTT GGC TTC TGG TTG TTC 195
Gly Leu Val Ala heu Leu Thr :Phe Leu Ala F~he Gly Phe Trp Leu Phe
45 50 55
AAG TAT CTC CAA AAG AGA AGA GAA AGA AGG AGA CAA CTC ACT GAG TTC 243
Lys Tyr Leu Gln hys Arg Arch Glu Arg Arg Arg Gln Leu Thr Glu Phe
60 65 70
CAA CGGTAT (.TACGG TAC AGGTTGAGT GAGATCCAG AGA 2
AAA AA'.I' 91
~AGC


0 Gln LysArgTyr heuArg Asn.;SerTyr ArgheuSer GluIleGln Arg


75 80 85


CCT ATATCACAG CACGAA TAC_'GAAGAC CCA2'ACGAG CCACCAAGT CGT 339


Pro IleSerGln FiisGlu Tyx Asp ProTyrGlu ProProSer Arg
Glu


90 95 100 105


AGG AAACCACCC CCTCCT CC'I"TATAGC ACATACGTC AACATCGAT AAT 387


Arg LysProPro I?roPro Prc~ Ser ThrTyrVal AsnIleAsp Asn
Tyr


.L 115 12
10 0



GTC TCAGCCATT '.fAGTGATGAGCAA CGGAGGGCCA ATGCTTGGCA
442
TTATTAAGTT


Val SerAlaIle


125


TTTGCTGACGGAGGAACATCAGGA(~CCGATGTAGATGAGTTGATACGTCGCATGGCGGCA502


TTAGAGGTATCGTTA(3TAGAGATAi?.GGCGAGACCTGAC'GGTTCTAGATGGGGATGTAGCC562


TCTGTAATCCGTAGACTACAGGACC~C'TGAGGACGCGATAACGGCATTGTCCAACGCGATG622



CAGGTGGTCCAATCACATATTGAAUAGATAGTTACGCAAGTTCGAAAACAAGTGGAGCAG682


ATAGCGGCTTTGGAGACGGCGGTGF.,C'TCAGAACACGAAGGACATAGATAGTGTGCGTAGC742


ACGGTAACGGATTTAGGATCCTTAGTGAGTGCAGAGAAAGTGAGGTTGGACGGTGTGGCG802


AGAGATGTGTCGACACAGGGACTG'I'C:AATCACTGATTTGCAGGCGCGAGTAGCTAAATTA862


GAAAGGGAAGCTGAACCGACGTCG'I'TCGAATGGCCACTGAGAAAAGATGCGAAGAGTGGA922



TTGCTATCATTGAAC'.CGGGATCCT'I'G~;~TTCTTAGAAACGACTGAAATATTTGGACTCTCA982


TGGGCGCAGTCTGGA(3TTGAGATGGG.AGCCACAACTGGACAAGGAGAATGGCATACACAA1042


AGTGGTGATTACTTG'.CACACCGTGAGCCTTAACTTTAAATTCTACAGATACAGGTCTATG1102


GGAGCCTTTTCACTC'CCAACCGGGAA'rGCGTTGCTGAACGGCCCAAAGGTGGAGCTACGT1162


ATACCATATACCACAGGGGGGACTGGCCTAGAAGGATCTGACCTACAAAACATGACGCCA1222



TCGTCCACCACGAGA'TTTCCGTTGACGTTCGTGACACGAATAACGGTAGGAGGAAGTGAA1282


TATACCATGCCAATTACGGTGACAAT.ACGACGAATTAGTGGTGTGGATACAATCGTGCTA1342


ACTCCAGCGGATTTGCCAGGCGCCAC.AAGCTATCCATGTTATCTGAGGGGGGAGTCGATA1402


TTTTACTACATGAGGGCTAGGCAGATGACGTGATTGCGTGAAGAGGGACTCTCCCCGTAA1462


GGTGAAGCACGATGGGACGTGCGAGG.AAAGCTATTCATC 1501



51


CA 02429891 2003-06-18
(2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 7.25
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptiliaal :reovirus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Met Gly Ser Gly hro Ser Asn ~Phe Val Asn His Ala Pro Gly Glu Ala
1 5 10 15
Ile Val Thr Gly heu Glu Lys Gly Ala Asp Lys Val Ala Gly Thr Ile
25 30
Ser His Thr Ile Trp Glu Val :Ile Ala Gly Leu Val Ala Leu Leu Thr
20 35 40 45
Phe Leu Ala Phe Gly Phe Trp Leu Phe Lys Tyr Leu Gln Lys Arg Arg
50 55 60
Glu Arg Arg Arg Gln Leu Thr c;lu Phe Gln L~ys Arg Tyr Leu Arg Asn
65 70 75 80
Ser Tyr Arg Leu Ser Glu Ile G1n Arg Pro Ile Ser Gln His Glu Tyr
f!5 90 95
Glu Asp Pro Tyr Glu Pro Pro Ser Arg Arg Lys Pro Pro Pro Pro Pro
100 105 110
Tyr Ser Thr Tyr Val Asn Ile Asp Asn Val Ser Ala Ile
115 :120 125
(2) INFORMATION FOR SEQ ID NO.: 3:
(i) SEQUENCE CHARACTERISTIC:3
4O (A) LENGTH: 7.501
(B) TYPE: nucle~.ic acid
(C) STRANDEDNE:~S:
(D) TOPOLOGY:
(ii) MOLECULE TS'PE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian :reovirus
(ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (386)..(14:35)
5O (xi) SEQUENCE DFsSCRIPTION: SEQ ID NO.: 3:
GTTATTTTTT TCCTCCiATGA AGCCATGGGG AGTGGACC'CT CTAATTTCGT CAATCACGCA 60
CCTGGAGAAG CAATTC3TAAC CGGT'.I'TGGAG AAAGGGGC'AG ATAAAGTAGC TGGAACGATA 12 0
TCACATACGA TTTGGC3AAGT GATC(3CCGGA TTAGTAGCCT TGCTGACATT CTTAGCGTTT 180
GGCTTCTGGT TGTTCAAGTA TCTCC'AAAAG AGAAGAGAAA GAAGGAGACA ACTCACTGAG 240
TTCCAAAAAC GGTAT(:TACG GAATAGCTAC AGGTTGAGTG AGATCCAGAG ACCTATATCA 300
CAGCACGAAT ACGAAGACCC ATACGAGCCA CCAAGTCGTA GGAAACCACC CCCTCCTCCT 360
52


CA 02429891 2003-06-18
TATAGCACAT ACGTC31ACAT CGATA :ATG TCT CAG C'.CA TTT AGT GAT GAG CAA 412
Met Ser Gln Pro Phe Ser Asp Glu Gln
1 5
CGG AGG GCC ATT ATT AAG TTA TGC TTG GCA TTT GCT GAC GGA GGA ACA 460
Arg Arg Ala Ile :Cle Lys Leu Cys Leu Ala Phe Ala Asp Gly Gly Thr
15 20 25
'IO TCA GGA GCC GAT (3TA GAT GAG TTG ATA CGT CGC ATG GCG GCA TTA GAG 508
Ser Gly Ala Asp Val Asp Glu Leu Ile Arg Arg Met Ala Ala Leu Glu
:30 35 40
GTA TCG TTA GTA GAG ATA AGG CGA GAC CTG ACG GTT CTA GAT GGG GAT 556
Val Ser Leu Val (31u Ile Arc_( Arg Asp Leu Thr Val Leu Asp Gly Asp
45 50 55
GTA GCC TCT GTA ATC CGT AGA CTA CAG GAC CSCT GAG GAC GCG ATA ACG 604
Val Ala Ser Val :Ile Arg Arg Leu G1n Asp Ala Glu Asp Ala Ile Thr
60 65 70
GCA TTG TCC AAC GCG ATG CAG GTG GTC CAA TCA CAT ATT GAA GAG ATA 652
Ala Leu Ser Asn :Ala Met Gln Val Val Gln Ser His Ile Glu Glu Ile
75 80 85
GTT ACG CAA GTT CGA AAA CAA GTG GAG CAG ATA GCG GCT TTG GAG ACG 700
Val Thr Gln Val .Arg Lys Gl.n Val Glu Gln Ile Ala Ala Leu Glu Thr
90 95 100 105
C~ GCG GTG ACT CAG AAC ACG AAG GAC ATA GAT AGT GTG CGT AGC ACG GTA 748
Ala Val Thr Gln .Asn Thr Lys Asp Ile Asp Ser Val Arg Ser Thr Val
110 115 120
ACG GAT TTA GGA TCC TTA GTG AGT GCA GAG AAA GTG AGG TTG GAC GGT 796
Thr Asp Leu Gly Ser 'Leu Va:l Ser Ala Glu Lys Val Arg Leu Asp Gly
125 130 135
GTG GCG AGA GAT GTG TCG ACA CAG GGA CTG TCA ATC ACT GAT TTG CAG 844
Val Ala Arg Asp Val Ser Thr Gln Gly Leu Ser Ile Thr Asp Leu Gln
140 145 150
GCG CGA GTA GCT AAA TTA GAA AGG GAA GCT GAA CCG ACG TCG TTC GAA 892
Ala Arg Val Ala Lys Leu Glu Arg Glu Ala Glu Pro Thr Ser Phe Glu
155 160 165
4~i
TGG CCA CTG AGA AAA GAT GCG AAG AGT GGA 'TTG CTA TCA TTG AAC TGG 940
Trp Pro Leu Arg Lys Asp Ala Lys Ser Gly Leu Leu Ser Leu Asn Trp
170 175 180 185
5O GAT CCT TGG TTC TTA GAA ACG ACT GAA ATA TTT GGA CTC TC;A TGG GCG 988
Asp Pro Trp Phe Leu Glu Thr Thr Glu Ile 'Phe Gly Leu Ser Trp Ala
190 195 200
CAG TCT GGA GTT GAG ATG GGA GCC ACA ACT GGA CAA GGA GAA TGG CAT 1036
5:i Gln Ser Gly Val Glu Met Gly Ala Thr Thr Gly Gln Gly Glu Trp His
205 210 215
ACA CAA AGT GGT GAT TAC TTG TAC ACC GTG AGC CTT AAC TTT AAA TTC 1084
Thr Gln Ser Gly Asp Tyr Leu Tyr Thr Val Ser Leu Asn Phe Lys Phe
220 225 230
53


CA 02429891 2003-06-18
TAC AGA TAC AGG 7.'CT ATG GGA GCC TTT TCA CTC TCA ACC GGG AAT GCG 1132
Tyr Arg Tyr Arg Ser Met Gly .Ala Phe Ser Leu Ser Thr Gly Asn Ala
235 240 245
TTG CTG AAC GGC (:CA AAG GTGI GAG CTA CGT ATA CCA TAT ACC ACA GGG 1180
Leu Leu Asn Gly I?ro Lys Va:l Glu Leu Arg Ile Pro Tyr Thr Thr Gly
250 255 e:60 265
GGG ACT GGC CTA GAA GGA TC'I' GAC CTA CAA AAC ATG ACG CCA TCG TCC 1228
0 Gly Thr Gly Leu Cslu Gly Sex :Asp Leu Gln Asn Met Thr Pro Ser Ser
:'.70 275 280
ACC ACG AGA TTT C:CG TTG ACCs~ 'TTC GTG ACA CGA ATA ACG GTA GGA GGA 12 7 6
Thr Thr Arg Phe Pro Leu Thr :Phe Val Thr Arg Ile Thr Val Gly Gly
285 290 295
AGT GAA TAT ACC ATG CCA ATT':~1CG GTG ACA F,TA CGA CGA ATT AGT GGT 1324
Ser Glu Tyr Thr Met Pro Ile 'Thr Val Thr I:le Arg Arg Ile Ser Gly
300 305 310
GTG GAT ACA ATC C~TG CTA AC'C' CCA GCG GAT' TTG CCA GGC GCC ACA AGC 1372
Val Asp Thr Ile TTal Leu Thz :Pro Ala Asp Leu Pro Gly Ala Thr Ser
315 320 325
TAT CCA TGT TAT C:TG AGG GGG GAG TCG ATA TTT TAC TAC ATG AGG GCT 1420
Tyr Pro Cys Tyr heu Arg Gly G1u Ser Ile Phe Tyr Tyr Met Arg Ala
330 335 340 345
AGG CAG ATG ACG 7.'GA TTGCGTGA~AG AGGGACTCTC CCCGTAAGGT GAAGCACGAT 1475
Arg Gln Met Thr
GGGACGTGCG AGGAAAGCTA TTCAT'C 1501
(2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CFiARACTERISTIClS
(A) LENGTH: .l49
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TS!PE: polypept:ide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian :reovirus
(xi) SEQUENCE DESCRIPTION: ;SEQ ID NO.: 4:
Met Ser Gln Pro I?he Ser AsI: Glu G1n Arg Arg Ala Ile Ile Lys Leu
1 5 10 15
Cys Leu Ala Phe Ala Asp Gly G1y Thr Ser Gly Ala Asp Val Asp Glu
20 25 30
Leu Ile Arg Arg Met Ala Ala.:Leu Glu Val Ser Leu Va1 Glu Ile Arg
35 ~40 45
Arg Asp Leu Thr Val Leu As~ma:ly Asp Val Ala Ser Val Ile Arg Arg
50 55 60
Leu Gln Asp Ala Glu Asp Als. Ile Thr Ala I,eu Ser Asn Ala Met Gln
70 75 80
54


CA 02429891 2003-06-18
Val VaI Gln Ser Iiis Ile Ghu Glu Ile Val Thr Gln Val Arg Lys Gln
85 90 95
Val Glu Gln Ile Ala Ala Leu Glu Thr Ala val Thr Gln Asn Thr Lys
100 105 110
1 C~
Asp Ile Asp Ser 'Val Arg Ser. Thr Val Thr Asp Leu Gly Ser Leu Val
115 120 125
Ser Ala Glu Lys 'Val Arg Leu Asp Gly Val Ala Arg Asp Val Ser Thr
130 135 140
Gln Gly Leu Ser Ile Thr Asp Leu Gln Ala Arg Val Ala Lys Leu Glu
1~ 145 150 155 160
Arg Glu Ala Glu Pro Thr Ser Phe Glu Trp Pro Leu Arg Lys Asp Ala
165 170 175
2() Lys Ser Gly Leu Leu Ser Leu Asn Trp Asp Pro Trp Phe Leu Glu Thr
180 185 190
Thr Glu Ile Phe Gly Leu Se:r Trp Ala Gln Ser Gly Val Glu Met Gly
195 200 205
2~i
Ala Thr Thr Gly Gln Gly Glu Trp His Thr Gln Ser Gly Asp Tyr Leu
210 215 220
Tyr Thr Val Ser Leu Asn Phe Lys Phe Tyr Arg Tyr Arg Ser Met Gly
3~) 225 230 235 240
Ala Phe Ser Leu Ser Thr Gly Asn Ala Leu Leu Asn Gly Pro Lys Val
245 250 255
37 Glu Leu Arg Ile Pro Tyr Thr Thr Gly Gly Thr G:ly Leu G:Lu Gly Ser
260 265 270
Asp Leu Gln Asn Met Thr Pro Ser Ser Thr Thr Arg Phe Pro Leu Thr
275 ?.80 285
41l
Phe Val Thr Arg Ile Thr Val Gly Gly Ser Glu Tyr Thr Met Pro Ile
290 295 300
Thr Val Thr Ile Arg Arg Ile Ser Gly Val Asp Thr Ile Val Leu Thr
4.5 305 310 315 320
Pro Ala Asp Leu Pro Gly Aaa Thr Ser Tyr Pro Cys Tyr Leu Arg Gly
325 330 335
5~ Glu Ser Ile Phe Tyr Tyr Met Arg Ala Arg Gln Met Thr
340 345
(2) INFORMATION FOR SEQ Ii) NO. : 5:
55 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1399
(B) TYPE: nucleic acid
(C) STRANDEDNESS:
(D) TOPOLOGY.
6O ( i i ) MOLECULE 'TYPE : DNA
(vi) ORIGINAL SOURCE:


CA 02429891 2003-06-18
(A) ORGANISM: aquareov:i.rus
(ix) FEATURE
(A) NAME/KEY: misc_feat.ure
(B) LOCATION: (8). (610)
(C) OTHER INFORMATION: Initiator codon unknown; coding sequence begins
at a position wit:
hin the first 17 codon
(xi) SEQUENCE DESCRIPTION: S:EQ ID NO.: 5:
gttttag tca atc atc ctg gg~~ aat acc atc t:ca aac acc gtt cag tac 49
Ser Ile :Cle Leu G1.~ .Asn Thr Ile Ser Asn Thr Val Gln Tyr
1 5 10
acg gta ctg cag atc gac aga tct tgc tgt atc aaa acc agc ctc acc 97
Thr Val Leu Gln :Cle Asp Arc~~ Ser Cys Cys Ile Lys Thr Ser Leu Thr
15 20 25 30
gcc act tcc gaa gcc act tcc~ tgg gcc atc c:cc cct ctc gca atc tgt 145
Ala Thr Ser Glu Ala Thr SeewTrp Ala Ile Pro Pro Leu Ala Ile Cys
35 40 45
tgc tgc tgt tgc atc tgc tgt .acc ggc gga cta tat ctc gtt cat tct 193
Cys Cys Cys Cys :Cle Cys Cyc,'Thr Gly Gly Leu Tyr Leu Val His Ser
50 55 60
gga cgt ttt cca ggc ctc ag4: cga agg ttg gac gtg ctc gga ggt tcg 241
Gly Arg Phe Pro Gly Leu Sex .~3.rg Arg Leu Asp Val Leu Gly Gly Ser
65 70 75
ggg tca acc cca aaa cac tcc~ ctg cgt agc cac cgg cac cca aag cca 289
Gly Ser Thr Pro Lys His Ser :Leu Arg Ser His Arg His Pro Lys Pro
80 85 90
cgt gta cat cgc c~tt agc ttc .agt gat tct agt gac tct agt gat atc 337
Arg Val His Arg Val Ser Phe Ser Asp Ser Ser Asp Ser Ser Asp Ile
95 100 1.05 110
tct gat ctg gaa t:tg cct cgg cac ggg tct cat cct ctg gcg cat tca 385
Ser Asp Leu Glu l~eu Pro Arg :His Gly Ser His Pro Leu Ala His Ser
:C15 120 125
ttc agg cct gaa c3tc gat cgc~ cac cgc cct cgt ccc tca acg caa gtc 433
Phe Arg Pro Glu Val Asp Arg :His Arg Pro Arg Pro Ser Thr Gln Val
130 135 140
cag cag aca tct i:tc atc ccg ctc gta cca ctc agg tcc gga tca agt 481
Gln Gln Thr Ser 1?he Ile Pro :Leu Val Pro Leu Arg Ser Gly Ser Ser
145 150 155
tta gac gat ggg atc gta cgc tct caa ccc t.ca cgg gat tcg cgg ccc 529
Leu Asp Asp Gly :Cle Val Arg Ser Gln Pro Ser Arg Asp Ser Arg Pro
160 16~~ 170
cac gag caa ttt gag gat tgg ctt caa caa gca cat ctc cta cga cca 577
His Glu Gln Phe c3lu Asp Trp Leu Gln Gln Ala His Leu Leu Arg Pro
175 180 1.85 190
gga cga gtt tcc gga tct acc aac ccc ttc acc tgaccacatt cccgactggg 630
Gly Arg Val Ser Gly Ser Thz- .Asn Pro Phe Thr
:L95 200
tgacggatca tgacaagttc aacggtcatc ccctccccct cgtctacgat ggacgtctga 690
56


CA 02429891 2003-06-18
cacccatcac gggtcctcac catcttt:ggg agcctgacag ttatgtagag tggcagacct 750
gggggtgcct ccgacccttc tctcctt;t:ca gcgtttggcc accaacggta ccgaactggt 810
tcagccgtaa ggtcctccac gtcttcagca acatgtcccc gtacgcctgc gctgctgaga 870
agagtcccaa tccccttccc tactggcgtt tgaatgatca gggtcgtgac tggagcgtat 930
tctgggactt aatttggcga tgtgctcaga cacgtggtgc tcgcatctgt tttgcgaaga 990
cccccttcat ccagacgatg ctacgcc:tga ctgacgatca gctgtcccgt cttccatccg 1050
ctgaggatcc aatcagtctc ttaaacatcg caggatggga cgcccttctt ctcaacggtc 1110
ttccccctaa cctggtgcga gcatt.gatga ggtcccctcc aaacccagag gtcgttgagc 1170
tggatctgct cgtctcctgg ttcgatc~t.cg tgattcgtat tccctatgac gtgcaacacc 1230
ccctaggcct tggtttcagc cctgatc:aat tttggactca tccgttcgtc gtcctgtgct 1290
acctgcgctg gcgtttgttg ggagqtc3acg actaggatgg cgtccgcgac agttgaggcc 1350
tgggcctcgg ggatttagtc ccctgtc:gcc agcgtgactg ctattcatc 1399
(2) INFORMATION F'OR SEQ ID NO.: 6:
(i) SEQUENCE CHARACTERISTTCS
(A) LENGTH: 201
(B) TYPE: amino acid
(C) STRANDEDNES~S:
(D) TOPOLOGY:
( ii ) MOLECULE T~i.'PE : polypept: ide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: aquareovirus
(ix) FEATURE
(A) NAME/KEY: misc_featu~re
(B) LOCATION: (8). (610)
(C) OTHER INFORMATION: Initiator codon unknown; coding sequence begins
at a position within the fwrst 17 codon
(xi) SEQUENCE DESCRTPTION SEQ ID NO.: 6:
Ser Ile Ile Leu Gly Asn Thr :Ile Ser Asn Thr Val Gln Tyr Thr Val
10 15
Leu Gln Ile Asp Arg Ser Cys Cys Ile Lys Thr Ser Leu Thr Ala Thr
20 25 30
Ser Glu Ala Thr Ser Trp Ala :Ile Pro Pro Leu Ala Ile Cys Cys Cys
35 ~40 45
Cys Cys Ile Cys C:ys Thr Gly Gly Leu Tyr Leu val His Ser Gly Arg
50 55 60
Phe Pro Gly Leu Ser Arg Arg :Leu Asp Val Leu Gly Gly Ser Gly Ser
70 75 80
Thr Pro Lys His Ser Leu Arg Ser His Arg His Pro Lys Pro Arg Val
85 90 95
His Arg Val Ser 1?he Ser Asp Ser Ser Asp Ser Ser Asp Ile Ser Asp
57

CA 02429891 2003-06-18
loo los llo
Leu Glu Leu Pro Arg His Gly ~~er His Pro Leu Ala His Ser Phe Arg
115 1.20 125
Pro Glu Val Asp Arg His Arg F°ro Arg Pra Ser Thr Gln Va1 Gln Gln
130 135 140
Thr Ser Phe Ile Pro Leu Val Pro Leu Arg Ser G1y Ser Ser Leu Asp
0 145 150 155 160
Asp Gly Ile Val Arg Ser Gln. Pro Ser Arg Asp Ser Arg Pro His Glu
165 170 175
Gln Phe Glu Asp Trp Leu Gln Gl.n A1a His Leu Leu Arg Pro Gly Arg
180 185 190
Val Ser Gly Ser Thr Asn Pro F>he Thr
19 5 2 C~ 0
(2) INFORMATION FOR SEQ ID 7:
NO.:


(i) SEQUENCE CHARACTERISTICS


(A) LENGTH: 1398


(B) TYPE: nucleic acid


(C) STRANDEDNESS:


(D) TOPOLOGY:


(ii) MOLECULE TYPE: DNA


(vi) ORIGINAL SOURCE:


(A) ORGANISM: aquareovi.rus


(ix) FEATURE


(A) NAME/KEY: CDS


(B) LOCATION: (488) . . (1.3'<'4)


(xi) SEQUENCE DESCRIPTION: ID NO.:
SEQ 7:


GTTTTAGTCA ATCATC'CTGG GGAATACCATCTCAAACACCGTTCAGTACA CGGTACTGCA60


GATCGACAGA TCTTGCTGTA TCAAAACC~AGCCTCACCGCCACTTCCGAAG CCACTTCCTG120


GGCCATCCCC CCTCTC'GCAA TCTGTTGCTGCTGTTGCATCTGCTGTACCG GCGGACTATA180



TCTCGTTCAT TCTGGP,CGTT TTCCAGGCCTCAGCCGAAGGTTGGACGTGC TCGGAGGTTC240


GGGGTCAACC CCAAAP,CACT CGCTGCGTAGCCACGGCACCCAAAGCCACG TGTACATCGC300


f


GTTAGCTTCA GTGATT'CTAG TGACTC'.CAGTGATATCTCTGATCTGGAATT GCCTCGGCAC360


GGGTCTCATC CTCTGGCGCA TTCATTCAGGCCTGAAGTCGATCGCCACCG CCCTCGTCCC420


TCAACGCAAG TCCAGC.'AGAC ATCTTTCATCCCGCTCGTACCACTCAGCTC CGGATCAAGT480



TTAGACG ATG GGA TCG TAC GCT AAC CCT GGG ATT CGC GGC CCC 529
CTC CAC


Met Gly Ser Tyr Ala :Leu Asn Pro Gly Ile Arg Gly Pro
His


1 5 10


ACG AGC AAT TTG AGG ATT GGt:AAC AAG ATC TCC TAC GAC CAG 577
'rTC CAC


Thr Ser Asn Leu Arg Tle Gly Asn Lys Ile Ser Tyr Asp Gln
Phe His


15 20 25 30


GAC GAG TTT CCG GAT CTA CCA CCT TCA GAC CAC ATT CCC GAC 625
.ACC CCT


Asp Glu Phe Pro Asp heu Pro Pro Ser Asp His Ile Pro Asp
'Thr Pro


a5 40 45


58




CA 02429891 2003-06-18
TGG GTG ACG GAT C:AT GAC AAG 'rTC AAC GGT C:AT CCC CTC CCC CTC GTC 673
Trp Val Thr Asp His Asp Lys :Phe Asn Gly His Pro Leu Pro Leu Val
50 55 60
TAC GAT GGA CGT C:TG ACA CCC .ATC ACG GGT CCT CAC CAT CTT TGG GAG 721
Tyr Asp Gly Arg heu Thr Prr_~ I.le Thr Gly Pro His His Leu Trp Glu
65 70 75
O CCT GAC AGT TAT GTA GAG TGG ~~AG ACC TGG GGG TGC CTC CGA CCC TTC 769
Pro Asp Ser Tyr Val Glu Trp G:ln Thr Trp Gly Cys Leu Arg Pro Phe
80 85 90
TCT CCT TTC AGC GTT TGG CCA CCA ACG GTA CCG AAC TGG TTC AGC CGT 817
Ser Pro Phe Ser Val Trp Prc~ Pro Thr Val F'ro Asn Trp Phe Ser Arg
95 100 105 110
AAG GTC CTC CAC GTC TTC AGC' ;?SAC ATG TCC C'CG TAC GCC TGC GCT GCT 865
Lys Val Leu His Val Phe Se:~ ;4sn Met Ser Pro Tyr Ala Cys Ala Ala
7.15 120 125
GAG AAG AGT CCC AAT CCC CT'I' CCC TAC TGG C'GT TTG AAT GAT CAG GGT 913
Glu Lys Ser Pro Asn Pro Leu.:Pro Tyr Trp Arg Leu Asn Asp Gln Gly
130 135 140
CGT GAC TGG AGC C~TA TTC TGG GAC TTA ATT TGG CGA TGT GCT CAG ACA 961
Arg Asp Trp Ser Val Phe Trp ;Asp Leu Ile Trp Arg Cys Ala Gln Thr
145 150 155
3O CGT GGT GCT CGC ATC TGT TT'I' GCG AAG ACC C'CC TTC ATC CAG ACG ATG 10 0 9
Arg Gly Ala Arg Ile Cys Phe ;Ala Lys Thr Pro Phe Ile Gln Thr Met
160 165 170
CTA CGC CTG ACT GAC GAT CAG'~CTG TCC CGT CTT CCA TCC GCT GAG GAT 1057
Leu Arg Leu Thr Asp Asp G1n :Leu Ser Arg Leu Pro Ser Ala Glu Asp
175 180 185 190
CCA ATC AGT CTC 7.'TA AAC ATC GCA GGA TGG GAC GCC CTT CTT CTC AAC 1105
Pro Ile Ser Leu heu Asn Ile ;Ala Gly Trp Asp Ala Leu Leu Leu Asn
195 200 205
GGT CTT CCC CCT AAC CTG GTC: CGA GCA TTG A,TG AGG TCC CCT CCA AAC 1153
Gly Leu Pro Pro Asn Leu Val :4rg Ala Leu Met Arg Ser Pro Pro Asn
210 215 220
CCA GAG GTC GTT GAG CTG GA'I'CTG CTC GTC TCC TGG TTC GAT GTC GTG 1201
Pro Glu Val Val C~lu L~eu Asr~:Leu Leu Val Ser Trp Phe Asp Val Val
225 230 235
5O ATT CGT ATT CCC TAT GAC GTG CAA CAC CCC CTA GGC CTT GGT TTC AGC 1249
Ile Arg Ile Pro Tyr Asp Val G1n His Pro Leu Gly Leu Gly Phe Ser
240 24!~ 250
CCT GAT CAA TTT 7.'GG ACT CA'.I' CCG TTC GTC GTC CTG TGC TAC CTG CGC 1297
Pro Asp Gln Phe .'rp Thr His Pro Phe Val Val Leu Cys Tyr Leu Arg
255 260 265 270
TGG CGT TTG TTG GGA GGT GAC!GAC TAG GATGGCGTCC GCGACAGTTG 1344
Trp Arg Leu Leu Gly Gly Asp .Asp
275
59


CA 02429891 2003-06-18
AGGCCTGGGC CTCGGGGATT TAGTCCC'CTG TCGCCAGCGT GACTGCTATT CATC 1398
(2)
INFORMATION
FOR
SEQ
ID
NO.:
8:


(i) SEQUENCE CHARACTERISTICS


(A) LENGTH: 278


(B) TYPE: amino acid


(C) STRANDEDNESS:


(D) TOPOLOGY:


(ii) MOLECULE TYPE: polype:pt:ide


(vi) ORIGINAL SGURCE:


(A) ORGANISM: aquareovi.rus


(xi) SEQUENCE DESCRIPTION: SEQ ID
NO.: 8:



Met Gly Ser Tyr Ala Leu Asn Pro His Gly Ile Arg Gly Pro Thr Ser
1 5 10 15
Asn Leu Arg Ile Cily Phe Asn 7~ys His Ile Ser Tyr Asp Gln Asp Glu
20 25 30
Phe Pro Asp Leu Pro Thr Pra Ser Pro Asp His Ile Pro Asp Trp Val
35 ~40 45
Thr Asp His Asp hys Phe Asn G:ly His Pro Leu Pro Leu Val Tyr Asp
50 55 60
Gly Arg Leu Thr Pro Ile Thr Gly Pro His His Leu Trp Glu Pro Asp
65 70 75 80
Ser Tyr Val Glu '.~rp Gln Thr Trp Gly Cys Leu Arg Pro Phe Ser Pro
85 90 95
Phe Ser Val Trp '.Pro Pro Thr Val Pro Asn Trp Phe Ser Arg Lys Val
100 105 110
Leu His Val Phe Ser Asn Met Ser Pro Tyr Ala Cys Ala Ala G1u Lys
115 120 125
40'
Ser Pro Asn Pro Leu Pro Tyr Trp Arg Leu Asn Asp Gln Gly Arg Asp
130 135 140
Trp Ser Val Phe Trp Asp Leu Ile Trp Arg Cys Ala Gln Thr Arg Gly
145 150 255 160
Ala Arg Ile Cys Phe Ala Lys Thr Pro Phe Ile Gln Thr Met Leu Arg
165 170 175
Leu Thr Asp Asp Gln Leu Se:r Arg Leu Pro Ser Ala Glu Asp Pro Ile
180 185 190
Ser Leu Leu Asn Ile Ala Gly Trp Asp Ala Leu Leu Leu Asn Gly Leu
195 200 205
Pro Pro Asn Leu Val Arg Ala Leu Met Arg Ser Pro Pro Asn Pro Glu
210 27.5 220
Val Val Glu Leu Asp Leu Leu Val 5er Trp Phe Asp Val Val Ile Arg
225 230 235 240


CA 02429891 2003-06-18
Ile Pro Tyr Asp Val GIn His Pro Leu Gly Leu Gly Phe Ser Pro Asp
2!E5 250 255
Gln Phe Trp Thr H:is Pro Phe val val Leu Cys Tyr Leu Arg Trp Arg
~J 260 265 270
Leu Leu Gly Gly Asp Asp
275
61

Representative Drawing

Sorry, the representative drawing for patent document number 2429891 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date Unavailable
(22) Filed 2003-06-02
(41) Open to Public Inspection 2004-12-02
Dead Application 2008-06-02

Abandonment History

Abandonment Date Reason Reinstatement Date
2007-06-04 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2003-06-02
Registration of a document - section 124 $100.00 2003-07-22
Maintenance Fee - Application - New Act 2 2005-06-02 $100.00 2005-06-02
Maintenance Fee - Application - New Act 3 2006-06-02 $100.00 2006-06-02
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
FUSOGENIX INC.
Past Owners on Record
DUNCAN, ROY
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2003-06-02 1 23
Description 2003-06-02 61 3,062
Claims 2003-06-02 4 134
Drawings 2003-06-02 3 79
Description 2003-06-18 61 2,962
Claims 2003-06-18 4 122
Abstract 2003-06-18 1 22
Drawings 2003-06-18 3 76
Cover Page 2004-11-09 1 28
Correspondence 2003-06-26 1 28
Assignment 2003-06-02 4 136
Correspondence 2003-06-18 70 3,219
Correspondence 2003-08-05 1 28
Prosecution-Amendment 2003-06-02 1 67
Correspondence 2003-07-22 1 36
Assignment 2003-07-22 2 60
Prosecution-Amendment 2003-08-19 1 26
Fees 2005-06-02 1 35
Fees 2006-06-02 1 34

Biological Sequence Listings

Choose a BSL submission then click the "Download BSL" button to download the file.

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.

Please note that files with extensions .pep and .seq that were created by CIPO as working files might be incomplete and are not to be considered official communication.

BSL Files

To view selected files, please enter reCAPTCHA code :