U.S. patent application number 10/118079 was filed with the patent office on 2003-06-05 for fusion proteins.
Invention is credited to McKerracher, Lisa.
Application Number | 20030103957 10/118079 |
Document ID | / |
Family ID | 27171502 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030103957 |
Kind Code |
A1 |
McKerracher, Lisa |
June 5, 2003 |
Fusion proteins
Abstract
The Rho family GTPases regulates axon growth and regeneration.
Inactivation of Rho with C3, a toxin from Clostridium botulinum,
can stimulate regeneration and sprouting of injured axons. The
present invention provides novel chimeric C3-like Rho antagonists.
These new antagonists are a significant improvement over C3
compounds because they are 3-4 orders of magnitude more potent to
stimulate axon growth on inhibitory substrates than recombinant C3.
The invention further provides evidence that these compounds
promote repair when applied to the injured mammalian central
nervous system.
Inventors: |
McKerracher, Lisa; (Ile des
Soeurs, CA) |
Correspondence
Address: |
Ronald S. Kosie
BROUILLETTE KOSIE
25th Floor
1100 Rene-Levesque Boulevard West
Montreal
QC
H3B 5C9
CA
|
Family ID: |
27171502 |
Appl. No.: |
10/118079 |
Filed: |
April 9, 2002 |
Current U.S.
Class: |
424/94.5 |
Current CPC
Class: |
A61P 25/16 20180101;
A61P 25/28 20180101; A61P 9/12 20180101; A61P 27/06 20180101; A61P
9/00 20180101; A61K 47/6425 20170801; A61P 43/00 20180101; A61P
35/00 20180101; A61P 25/00 20180101; A61P 25/02 20180101 |
Class at
Publication: |
424/94.5 |
International
Class: |
A61K 038/51 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 12, 2001 |
CA |
2,342,970 |
Nov 13, 2001 |
CA |
2,362,004 |
Jan 15, 2002 |
CA |
2,367,636 |
Claims
I claim:
1. A drug delivery construct comprising at least one transport
agent region and an active agent region, wherein the transport
agent region is able to facilitate the uptake of the active agent
region into a cell, and wherein the active agent region is an
active therapeutic agent region able to facilitate axon growth, and
an analogue thereof.
2. A drug delivery construct as defined in claim 1 wherein the
active agent region is selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogue
thereof.
3. A drug conjugate consisting of a transport polypeptide moiety
covalently linked to an active cargo moiety wherein the transport
polypeptide moiety is able to facilitate the uptake of the active
cargo moiety into a mammalian tissue or cell and wherein the active
cargo moiety is an active therapeutic moiety able to facilitate
axon growth.
4. A drug conjugate as defined in claim 3, wherein the transport
polypeptide moiety is selected from the group consisting of a
transport subdomain of HIV Tat protein, a homeodomain of
antennapedia, a Histidine tag and analogues thereof and wherein the
active cargo moiety is selected from the group consisting of C3
protein able to facilitate axon growth.
5. A drug conjugate as defined in claim 4 wherein the C3 protein is
selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues thereof.
6. A drug conjugate as defined in claim 3 wherein the transport
polypeptide moiety includes an active contiguous amino acid
sequence as described herein.
7. The use of a drug delivery construct as defined in claim 1, and
analogues thereof for suppressing the inhibition of neuronal axon
growth.
8. The use of a drug delivery construct as defined in claim 2, and
analogues thereof for suppressing the inhibition of neuronal axon
growth.
9. The use of a drug conjugate as defined in claim 3 and analogues
thereof for suppressing the inhibition of neuronal axon growth.
10. The use of a drug conjugate as defined in claim 4 and analogues
thereof for suppressing the inhibition of neuronal axon growth.
11. The use of a drug conjugate as defined in claim 5 and analogues
thereof for suppressing the inhibition of neuronal axon growth.
12. The use of a drug conjugate as defined in claim 6 and analogues
thereof for suppressing the inhibition of neuronal axon growth.
13. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug delivery construct as defined in claim 1 and
analogues thereof.
14. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug delivery construct as defined in claim 2 and
analogues thereof.
15. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug conjugate as defined in claim 3 and analogues
thereof.
16. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug conjugate as defined in claim 4 and analogues
thereof.
17. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug conjugate as defined in claim 5 and analogues
thereof.
18. A pharmaceutical composition, the pharmaceutical composition
comprising a pharmaceutically acceptable carrier and an effective
amount of a drug conjugate as defined in claim 6 and analogues
thereof.
19. The use of a drug delivery construct as defined in claim 1 and
analogues thereof for the manufacture of a pharmaceutical
composition.
20. The use of a drug delivery construct as defined in claim 2 and
analogues thereof for the manufacture of a pharmaceutical
composition.
21. The use of a drug conjugate as defined in claim 3 and analogues
thereof for the manufacture of a pharmaceutical composition.
22. The use of a drug conjugate as defined in claim 4 and analogues
thereof for the manufacture of a pharmaceutical composition.
23. The use of a drug conjugate as defined in claim 5 and analogues
thereof for the manufacture of a pharmaceutical composition.
24. The use of a drug conjugate as defined in claim 6 and analogues
thereof for the manufacture of a pharmaceutical composition.
25. A method for preparing a drug delivery construct as defined in
claim 1 comprising: cultivating a host cell under conditions which
provide for the expression of the drug delivery construct within
the cell; and recovering the drug delivery construct by a
purification step.
26. A method for preparing a drug delivery construct as defined in
claim 2 comprising: cultivating a host cell under conditions which
provide for the expression of the drug delivery construct within
the cell; and recovering the drug delivery construct by a
purification step.
27. A method for preparing a drug conjugate as defined in claim 3,
comprising: cultivating a host cell under conditions which provide
for the expression of the drug conjugate within the cell; and
recovering the drug conjugate by a purification step.
28. A method for preparing a drug conjugate as defined in claim 4,
comprising: cultivating a host cell under conditions which provide
for the expression of the drug conjugate within the cell and
recovering the drug conjugate by a purification step.
29. A method for preparing a drug conjugate as defined in claim 5,
comprising: cultivating a host cell under conditions which provide
for the expression of the drug conjugate within the cell and
recovering the drug conjugate by a purification step.
30. A method for preparing a drug conjugate as defined in claim 6,
comprising: cultivating a host cell under conditions which provide
for the expression of the drug conjugate within the cell; and
recovering the drug conjugate by a purification step.
31. A pharmaceutical composition comprising; a) a polypeptide
selected from the group consisting of SEQ ID NO. :4, SEQ ID NO.:6,
SEQ ID NO.:14, SEQ ID NO.:18, SEQ ID NO.: 20, SEQ ID NO.:25, SEQ ID
NO.:30, SEQ ID NO.:35, SEQ ID NO.:37, and SEQ ID NO.: 43 and; b) a
pharmaceutically acceptable carrier.
32. A pharmaceutical composition according to claim 31 further
comprising a biological adhesive.
33. A pharmaceutical composition according to claim 31 further
comprising fibrin.
34. A pharmaceutical composition comprising; a) a polypeptide
comprising at least one transport agent region and an active agent
region, said active agent region being selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues, and; b) a pharmaceutically acceptable
carrier.
35. A pharmaceutical composition according to claim 34 further
comprising a biological adhesive.
36. A pharmaceutical composition according to claim 34 further
comprising fibrin.
37. A pharmaceutical composition according to claim 34, wherein the
transport agent region is selected from the group consisting of a
basic amino acid rich region and a proline rich region.
38. A pharmaceutical composition according to claim 37, wherein the
basic amino acid rich region is selected from the group consisting
of a subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
39. A pharmaceutical composition according to claim 37, wherein the
basic amino acid region is selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues
thereof.
40. A pharmaceutical composition according to claim 37, wherein the
proline rich region is selected from the group consisting of SEQ ID
NO.: 48 and analogues thereof.
41. A polypeptide selected from the group consisting of SEQ ID
NO.:4, SEQ ID NO.:6, SEQ ID NO.:14, SEQ ID NO.:18, SEQ ID NO.: 20,
SEQ ID NO.:25, SEQ ID NO.:30, SEQ ID NO.:35, SEQ ID NO.:37, and SEQ
BD NO.: 43, and analogues thereof.
42. A polypeptide comprising at least one transport agent region
and an active agent region, said active agent region being selected
from the group consisting of ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues.
43. A polypeptide according to claim 42, wherein the transport
agent region is selected from the group consisting of a basic amino
acid rich region and a proline rich region.
44. A polypeptide according to claim 42, wherein the transport
agent region is at the carboxy-terminal end of said polypeptide and
the active agent region is at the amino terminal end of said
polypeptide.
45. A polypeptide according to claim 43, wherein the basic amino
acid rich region is selected from the group consisting of a
subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
46. A polypeptide according to claim 43, wherein the basic amino
acid region is selected from the group consisting of SEQ ID NO.:
21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ ID NO.: 45,
SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues thereof.
47. A polypeptide according to claim 43, wherein the proline rich
region is selected from the group consisting of SEQ ID NO.: 48 and
analogues thereof.
48. The use of a polypeptide selected from the group consisting of
SEQ ID NO.:4, SEQ ID NO.:6, SEQ ID NO.:14, SEQ ID NO.:18, SEQ ID
NO.: 20, SEQ ID NO.:25, SEQ ID NO.:30, SEQ ID NO.:35, SEQ ID
NO.:37, and SEQ ID NO.: 43 for the manufacture of a pharmaceutical
composition.
49. The use of a polypeptide for the manufacture of a
pharmaceutical composition, wherein said polypeptide comprises at
least one transport agent region and an active agent region, and
wherein said active agent region is selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues.
50. The use of a polypeptide according to claim 49, wherein the
transport agent region is at the carboxy-terminal end of said
polypeptide and wherein the active agent region is at the
amino-terminal end of said polypeptide.
51. The use of a polypeptide according to claim 49, wherein the
transport agent region is selected from the group consisting of a
basic amino acid rich region and a proline rich region.
52. The use of a polypeptide according to claim 51, wherein the
basic amino acid rich region is selected from the group consisting
of a subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
53. The use of a polypeptide according to claim 51, wherein the
basic amino acid region is selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues
thereof.
54. The use of a polypeptide according to claim 51, wherein the
proline rich region is selected from the group consisting of SEQ ID
NO.: 48 and analogues thereof.
55. A method of suppressing the inhibition of neuronal axon growth
comprising delivering a polypeptide comprising at least one
transport agent region and an active agent region selected from the
group consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues directly at a central nervous system
lesion site or a peripheral nervous system lesion site, in an
amount effective to counteract said inhibition.
56. A method according to claim 55, wherein said transport agent
region is selected from the group consisting of a basic amino acid
rich region and a proline rich region.
57. A method according to claim 56, wherein the basic amino acid
rich region is selected from the group consisting of a subdomain of
HIV Tat protein, a homeodomain of antennapedia, a Histidine tag,
and analogues thereof.
58. A method according to claim 56, wherein the basic amino acid
region is selected from the group consisting of SEQ ID NO.: 21, SEQ
ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ ID NO.: 45, SEQ ID
NO.: 46, SEQ ID NO.: 47, and analogues thereof.
59. A method according to claim 56, wherein the proline rich region
is selected from the group consisting of SEQ ID NO.: 48 and
analogues thereof.
60. A method of facilitating axon growth comprising delivering a
polypeptide comprising at least one transport agent region and an
active agent region selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogues
directly at a central nervous system lesion site or a peripheral
nervous system lesion site, in an amount effective to facilitate
said growth.
61. A method according to claim 60, wherein said transport agent
region is selected from the group consisting of a basic amino acid
rich region and a proline rich region.
62. A method according to claim 61, wherein the basic amino acid
rich region is selected from the group consisting of a subdomain of
HIV Tat protein, a homeodomain of antennapedia, a Histidine tag,
and analogues thereof.
63. A method according to claim 61, wherein the basic amino acid
region is selected from the group consisting of SEQ ID NO.: 21, SEQ
ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ ID NO.: 45, SEQ ID
NO.: 46, SEQ ID NO.: 47, and analogues thereof.
64. A method according to claim 61, wherein the proline rich region
is selected from the group consisting of SEQ ID NO.: 48 and
analogues thereof.
65. A method of treating nerve injury comprising delivering a
polypeptide comprising at least one transport agent region and an
active agent region selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogues
directly at a central nervous system lesion site or a peripheral
nervous system lesion site.
66. A method according to claim 65, wherein said transport agent
region is selected from the group consisting of a basic amino acid
rich region and a proline rich region.
67. A method according to claim 66, wherein the basic amino acid
rich region is selected from the group consisting of a subdomain of
HIV Tat protein, a homeodomain of antennapedia, a Histidine tag,
and analogues thereof.
68. A method according to claim 66, wherein the basic amino acid
region is selected from the group consisting of SEQ ID NO.: 21, SEQ
ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ ID NO.: 45, SEQ ID
NO.: 46, SEQ ID NO.: 47, and analogues thereof.
69. A method according to claim 66, wherein the proline rich region
is selected from the group consisting of SEQ ID NO.: 48 and
analogues thereof.
70. A method of treating ischemic damage related to stroke
comprising delivering a polypeptide comprising at least one
transport agent region and an active agent region selected from the
group consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues directly at a central nervous system
lesion site.
71. A method according to claim 70, wherein said transport agent
region is selected from the group consisting of a basic amino acid
rich region and a proline rich region.
72. A method according to claim 71, wherein the basic amino acid
rich region is selected from the group consisting of a subdomain of
HIV Tat protein, a homeodomain of antennapedia, a Histidine tag,
and analogues thereof.
73. A method according to claim 71, wherein the basic amino acid
region is selected from the group consisting of SEQ ID NO.: 21, SEQ
ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ ID NO.: 0.45, SEQ
ID NO.: 46, SEQ ID NO.: 47, and analogues thereof.
74. A method according to claim 71, wherein the proline rich region
is selected from the group consisting of SEQ ID NO.: 48 and
analogues thereof.
75. A method for preparing a polypeptide comprising at least one
transport agent region and an active agent region, wherein said
transport agent region being selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, SEQ ID NO.: 48 and
analogues thereof, and wherein said active agent region being
selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues, said method comprising:
cultivating a host cell under conditions which provide for the
expression of the polypeptide within the cell; and recovering the
polypeptide by a purification step.
76. The use of a polypeptide comprising at least one transport
agent region and an active agent region, said active agent region
being selected from the group consisting of ADP-ribosyl transferase
C3 and ADP-ribosyl transferase C3 analogues for the manufacture of
a medicament for suppressing the inhibition of neuronal axon
growth.
77. The use of a polypeptide according to claim 76, wherein the
transport agent region is selected from the group consisting of a
basic amino acid rich region and a proline rich region.
78. The use of a polypeptide according to claim 77, wherein the
basic amino acid rich region is selected from the group consisting
of a subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
79. The use of a polypeptide according to claim 77, wherein the
basic amino acid region is selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues
thereof.
80. The use of a polypeptide according to claim 77, wherein the
proline rich region is selected from the group consisting of SEQ ID
NO.: 48 and analogues thereof.
81. The use of a polypeptide comprising at least one transport
agent region and an active agent region, said active agent region
being selected from the group consisting of ADP-ribosyl transferase
C3 and ADP-ribosyl transferase C3 analogues for the manufacture of
a medicament for facilitating axon growth.
82. The use of a polypeptide according to claim 81, wherein the
transport agent region is selected from the group consisting of a
basic amino acid rich region and a proline rich region.
83. The use of a polypeptide according to claim 82, wherein the
basic amino acid rich region is selected from the group consisting
of a subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
84. The use of a polypeptide according to claim 82, wherein the
basic amino acid region is selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues
thereof.
85. The use of a polypeptide according to claim 82, wherein the
proline rich region is selected from the group consisting of SEQ ID
NO.: 48 and analogues thereof.
86. The use of a polypeptide comprising at least one transport
agent region and an active agent region, said active agent region
being selected from the group consisting of ADP-ribosyl transferase
C3 and ADP-ribosyl transferase C3 analogues for the manufacture of
a medicament for treating nerve injury.
87. The use of a polypeptide according to claim 86, wherein the
transport agent region is selected from the group consisting of a
basic amino acid rich region and a proline rich region.
88. The use of a polypeptide according to claim 87, wherein the
basic amino acid rich region is selected from the group consisting
of a subdomain of HIV Tat protein, a homeodomain of antennapedia, a
Histidine tag, and analogues thereof.
89. The use of a polypeptide according to claim 87, wherein the
basic amino acid region is selected from the group consisting of
SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ ID NO.: 44, SEQ
ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, and analogues
thereof.
90. The use of a polypeptide according to claim 87, wherein the
proline rich region is selected from the group consisting of SEQ ID
NO.: 48 and analogues thereof.
91. The use of a polypeptide selected from the group consisting of
SEQ ID NO.:4, SEQ ID NO.:6, SEQ ID NO.:14, SEQ ID NO.:18, SEQ ID
NO.: 20, SEQ ID NO.:25, SEQ ID NO.:30, SEQ ID NO.:35, SEQ ID
NO.:37, and SEQ ID NO.: 43 for the manufacture of a pharmaceutical
composition for suppressing the inhibition of neuronal axon
growth.
92. The use of a polypeptide selected from the group consisting of
SEQ ID NO.:4, SEQ ID NO.:6, SEQ ID NO.: 14, SEQ ID NO.: 18, SEQ ID
NO.: 20, SEQ ID NO.:25, SEQ ID NO.:30, SEQ ID NO.:35, SEQ ID
NO.:37, and SEQ ID NO.: 43 for the manufacture of a pharmaceutical
composition for facilitating axon growth.
93. The use of a polypeptide selected from the group consisting of
SEQ ID NO.:4, SEQ ID NO.:6, SEQ ID NO.: 14, SEQ ID NO.: 18, SEQ ID
NO.: 20, SEQ ID NO.:25, SEQ ID NO.:30, SEQ ID NO.:35, SEQ ID
NO.:37, and SEQ ID NO.: 43 for the manufacture of a pharmaceutical
composition for treating nerve injury.
94. An isolated polynucleotide comprising at least the
polynucleotide sequence selected from the group consisting of SEQ
ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 13, SEQ ID NO.: 17, SEQID
NO.: 19, SEQ ID NO.: 24, SEQ ID NO.: 29, SEQ ID NO.: 34, SEQ ID
NO.: 36, and SEQ ID NO.: 42.
95. A cell transformed with an isolated polynucleotide comprising
at least the polynucleotide sequence selected from the group
consisting of SEQ ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 13, SEQ ID
NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 24, SEQ ID NO.: 29, SEQ ID
NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 42.
96. A delivery agent consisting of a cargo moiety in combination
with a transport moiety, wherein the transport moiety is selected
from the group consisting of SEQ ID NO: 48 and analogues
thereof.
97. The agent of claim 96, wherein the cargo moiety retains
biological activity following transport moiety-dependent
intracellular delivery.
98. The agent of claim 97, wherein the agent is a fusion protein
having an amino-terminal that is the cargo moiety and having a
carboxy-terminal that is a transport moiety.
99. The agent of claim 97, wherein the cargo moiety is selected
from the group consisting of analytical molecules, therapeutic
molecules, prophylactic molecules, and diagnostic molecules.
100. The agent of claim 97, wherein the cargo moiety is selected
from the group consisting of ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues thereof.
101. The polypeptide set forth in SEQ ID NO: 48 and analogues
thereof.
102. A polypeptide as set forth in SEQ ID NO: 48 and analogues
thereof, wherein said polypeptide and analogues are able to act as
transport agent for the intracellular delivery of a cargo agent
selected from the group consisting of analytical molecules,
therapeutic molecules, prophylactic molecules, and diagnostic
molecules.
103. The polypeptide of claim 102, wherein said cargo agent is
selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to conjugate or fusion type
proteins (polypeptides) comprising, for example, C3 (see below)
(i.e., C3-like protein, C3 chimeric proteins). Although, in the
following, fusion-type proteins of the present invention, will be
particularly discussed in relation to the use to facilitate
regeneration of axons and neuroprotection, it is to be understood
that the fusion proteins may be exploited in other contexts.
[0002] The present invention in particular pertains to the field of
mammalian nervous system repair (e.g. repair of a central nervous
system (CNS) lesion site or a peripheral nervous system (PNS)
lesion site), axon regeneration and axon sprouting, neurite growth
and protection from neurodegeneration and ischemic damage.
[0003] The Rho family GTPases regulates axon growth and
regeneration. Inactivation of Rho with Clostridium botulinum C3
exotransferase (hereinafter simply referred to as C3) can stimulate
regeneration and sprouting of injured axons; C3 is a toxin purified
from Clostridium botulinum (see Saito et al., 1995, FEBS Lett
371:105-109; Wilde et al 2000. J. Biol. Chem. 275:16478). Compounds
of the C3 family from Clostridium botulinum inactivate Rho by
ADP-ribosylation and thus act as antagonists of Rho effect or
function (Rho antagonists).
[0004] The present invention in particular relates to a means of
intracellular delivery of C3 protein (e.g. C3 itself or other
active analogues such as C3-like transferases--see below) or other
Rho antagonists to repair damage in the nervous system, to prevent
ischemic cell death, and to treat various disease where the
inactivation of Rho is required. The means of delivery may take the
form of chimeric (i.e. conjugate) C3-like Rho antagonists. These
conjugate antagonists provide a significant improvement over C3
compounds (alone) because they are 3 to 4 orders of magnitude more
potent with respect to the stimulation of axon growth on inhibitory
substrates than recombinant C3 alone. Examples of these Rho
antagonists have been made as recombinant proteins created to
facilitate penetration of the cell membrane (i.e. to enhance cell
uptake of the antagonists), improve dose-response when applied to
neurons to stimulate growth on growth inhibitory substrates, and to
inactivate Rho. Examples of these conjugate Rho antagonists are
described below in relation to the designations C3APL, C3APLT,
C3APS, C3-TL, C3-TS, C3Basic1, C3Basic2 and C3Basic3.
BACKGROUND OF THE INVENTION
[0005] Traumatic injury of the spinal cord results in permanent
functional impairment. Most of the deficits associated with spinal
cord injury result from the loss of axons that are damaged in the
central nervous system (CNS). Similarly, other diseases of the CNS
are associated with axonal loss and retraction, such as stroke,
human immunodeficiency virus (HIV) dementia, prion diseases,
Parkinson's disease, Alzheimer's disease, multiple sclerosis and
glaucoma. Common to all of these diseases is the loss of axonal
connections with their targets, and cell death. The ability to
stimulate growth of axons from the affected or diseased neuronal
population would improve recovery of lost neurological functions,
and protection from cell death can limit the extent of damage. For
example, following a white matter stroke, axons are damaged and
lost, even though the neuronal cell bodies are alive, and stroke in
grey matter kills many neurons and non-neuronal (glial) cells.
Treatments that are effective in eliciting sprouting from injured
axons are equally effective in treating some types of stroke
(Boston life sciences, Sep. 6, 2000 Press release). Neuroprotective
agents often tested as potential compounds that can limit damage
after stroke. Compounds which show both growth-promotion and
neuroprotection are especially good candidates for treatment of
stroke and neurodegenerative diseases. Similarly, although the
following discussion will generally relate to delivery of Rho
antagonists, etc. to a traumatically damaged nervous system, this
invention may also be applied to damage from unknown causes, such
as during stroke, multiple sclerosis, HIV dementia, Parkinson's
disease, Alzheimer's disease, prion diseases or other diseases of
the CNS were axons are damaged in the CNS environment. Also, Rho is
an important target for treatment of cancer and metastasis (Clark
et al (2000) Nature 406:532-535), and hypertension (Uehata et al.
(1997) Nature 389:990) and RhoA is reported to have a
cardioprotective role (Lee et al. FASEB J. 15:1886-1884).
Therefore, the new C3-like proteins are expected to be useful for a
variety of diseases were inhibition of Rho activity is
required.
[0006] It has been proposed to use various Rho antagonists as
agents to stimulate regeneration of (cut) axons, i.e. nerve
lesions; please see, for example, Canadian Patent application nos.
2,304,981 (McKerracher et al) and 2,300,878 (Strittmatter). These
patent application documents propose the use of known Rho
antagonists such as for example C3, chimeric C3 proteins, etc. (see
blow) as well as substances selected from among known trans-4-amino
(alkyl)-1-pyridylcarbamoylcyclohexane compounds (also see below) or
Rho kinase inhibitors for use in the regeneration of axons. C3
inactivates Rho by ADP-ribosylation and is fairly non-toxic to
cells (Dillon and Feig (1995) Methods in Enzymology: Small GTPases
and their regulators Part. B.256:174-184).
[0007] While the following discussion will generally relate or be
directed at repair in the CNS, the techniques described herein may
be extended to use in many other diseases including, but not
restricted to, cancer, metastasis, hypertentension, cardiac
disease, stroke, diabetic neuropathy, and neurodegenerative
disorders such as stroke, Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS). Treatment with Rho antagonists
would be used to enhance the rate of axon growth of peripheral
nerves and thereby be effective for repair of peripheral nerves
after surgery, for example after reattaching severed limbs. Also,
treatment with our fusion compounds (proteins) is expected to be
effective for the treatment of various peripheral neuropathies
because of their axon growth promoting effects.
[0008] As mentioned above, traumatic injury of the spinal cord
results in permanent functional impairment. Axon regeneration does
not occur in the adult mammalian CNS because substrate-bound growth
inhibitory proteins block axon growth. Many compounds, such as
trophic factors, enhance neuronal differentiation and stimulate
axon growth in tissue culture. However, most factors that enhance
growth and differentiation are not able to promote axon
regenerative growth on inhibitory substrates. To demonstrate that a
compound known to stimulate axon growth in tissue culture most
accurately reflects the potential for therapeutic use in axon
regeneration in the CNS, it is important for the cell culture
studies to include the demonstration that a compound can permit
axon growth on growth inhibitory substrates. An example of trophic
and differentiation factors that stimulate growth on permissive
substrates in tissue culture, are neurotrophins such as nerve
growth factor (NGF) and brain-derived growth factor. NGF, however,
does not promote growth on inhibitory substrates (Lehmann, et al.
(1999) 19: 7537-7547) and it has not been effective in promoting
axon regeneration in vivo. Brain derived neurotrophic factor (BDNF)
is not effective to promote regeneration in vivo either
(Mansour-Robaey, et al. J. Neurosci. (1994) 91: 1632-1636). BDNF
does not promote neurite growth on growth inhibitory substrates
(Lehmann et al supra).
[0009] Targeting intracellular signaling mechanisms involving Rho
and the Rho kinase for promoting axon regeneration has been
proposed (see, for example, the above-mentioned Canadian Patent
application nos. 2,304,981 (McKerracher et al)). For demonstration
that inactivation of Rho promotes axon regeneration on growth
inhibitory substrates, recombinant C3, a protein that inactivates
Rho by ADP ribosylation of the effector domain was used. While such
a C3 protein can effectively promote regeneration, it has been
noted that such a C3 protein does not easily penetrate into cells,
and high doses must therefore be applied for it to be effective.
The high dose of recombinant C3 needed to promote functional
recovery presents a practical constraint or limitation on the use
of C3 in vivo to promote regeneration (Lehmann, et al. (1999) J.
Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995) Methods
in Enzymology, Vol 256 part B, pg.196-206. In tissue culture
studies, it has, for example, been determined that the minimum
amount of C3 that can be used to induce growth on inhibitory
substrates is 25 ug/ml (Lehmann, et al. (1999) J. Neurosci. 19:
7537-7547; Morii, N and Narumiya, S. (1995) Methods in Enzymology,
Vol 256 part B, pg.196-206. If the cells are not triturated, even
this dose is ineffective. It has been estimated, for example, that
at least 40 .mu.g of C3 per 20 g mouse needs to be applied to
injured mouse spinal cord or rat optic nerve (McKerracher, Canadian
patent application No.: 2,325,842). Calculating doses that would be
required to treat an adult human on an equivalent dose per weight
scale up used for rat and mice experiments, it would be necessary
to apply 120 mg/kg of C3 (i.e. alone) to the injured human spinal
cord. The large amount of recombinant C3 protein needed creates
significant problems for manufacturing, due to the large-scale
protein purification and cost. It also limits the dose ranging that
can be tested because of the large amount of protein needed for
minimal effective doses.
[0010] Another related limitation with respect to the use of C3 to
promote repair in the injured CNS is that it does not easily
penetrate the plasma membrane of living cells. In tissue culture
studies when C3 is applied to test biological effects it has been
microinjected directly into the cell (Ridley and Hall (1992) Cell
70: 389-399), or applied by trituration of the cells to break the
plasma membrane (Lehmann, et al. (1999) J. Neurosci.19: 7537-7547,
Jin and Strittmatter (1997) J. Neurosci. 17: 6256-6263). In the
case of axon injury in vivo, the C3 protein is likely able to enter
the cell because injured axons readily take up substances from
their environment. However, C3-like proteins of the present
invention are likely to act also on surrounding undamaged neurons
and help them make new connections as well, thus facilitating
recovery. After incomplete SCI, there is plasticity of motor
systems attributed to cortical and subcortical levels, including
spinal cord circuitry (Raineteau, O., and Schwab, M. E. (2001) Nat
Rev Neurosci 2: 263-73). This plasticity may be attributed to
axonal or dendritic sprouting of collaterals and synaptic
strengthening or weakening. Additionally, it has been shown that
sparing of a few ventrolateral fibers may translate into
significant differences in locomotor performance since these fibers
are important in the initiation and control of locomotor pattern
through spinal central pattern generators (Brustein, E., and
Rossignol, S. (1998) J Neurophysiol 80: 1245-67). It is well
documented that reorganization of spared collateral cortical spinal
fibers occurs after spinal cord injury and this contributes to
functional recovery (Weidner et al, 2001 Proc. Natl. Acad. Sci. 98:
3513-3518). The process of reorganization and sprouting of spared
fibers would be enhanced by treatment with C3-like proteins able to
enter non-injured neurons. This would enhances spontaneous
plasticity of axons and dendritic remodeling known to help
functional recovery.
[0011] Other methods of delivery of C3 in vitro have been to make a
recombinant protein that can be taken up by a receptor-mediated
mechanism (Boquet, P. et al. (1995) Meth. Enzymol. 256: 297-306).
The disadvantage of this method is that the cells needing treatment
must express the necessary receptor. Lastly, addition of a C2II
binding protein to the tissue culture medium, along with a C21N-C3
fusion toxin allows uptake of C3 by receptor-mediated endocytosis
(Barthe et al. (1998) Infection and Immunity 66:1364). The
disadvantage of this system is that much of the C3 in the cell will
be restrained within a membrane compartment. More importantly, two
different proteins must be added separately for transport to occur
(Wahl et al. 2000. J. Cell Biol. 149:263), which make this system
difficult to apply to for treatment of disease in vivo.
SUMMARY OF THE INVENTION
[0012] In accordance with the present invention a conjugate, drug
delivery construct, or fusion protein comprising a therapeutically
active agent is provided whereby the active agent may be delivered
across a cell wall membrane, the conjugate or fusion protein
comprising at least a transport subdomain(s) or moiety(ies) (i.e.,
transport agent region) in addition to an active agent moiety(ies)
(i.e., active agent region). More particularly, as discussed
herein, in accordance with the present invention a conjugate or
fusion protein is provided wherein the therapeutically active agent
is one able to facilitate (for facilitating) axon (or dendrite, or
neurite) growth (e.g. regeneration) i.e. a conjugate or fusion
protein in the form of a conjugate Rho antagonist.
[0013] The present invention in accordance with an aspect thereof
provides a drug delivery construct or conjugate [e.g. able to (for)
suppress(ing) the inhibition of neuronal axon growth at a central
nervous system (CNS) lesion site or a peripheral nervous system
(PNS) lesion site] comprising at least one transport agent region
and an active agent region not naturally associated with the active
agent region, wherein the transport agent region is able to
facilitate (i.e. facilitates) the uptake of the active agent region
into a mammalian (i.e. human or animal) tissue or cell, and wherein
the active agent region is an active therapeutic agent region able
(i.e. has the capacity or capability) to facilitate axon growth for
example on growth inhibitory substrates (e.g. regeneration), either
in vivo (in a mammal (e.g., human or animal)) or in vitro (in cell
culture), including a derivative or homologue thereof (i.e.
pharmaceutically acceptable chemical equivalents
thereof--pharmaceuticall- y acceptable derivative or
homologue).
[0014] In accordance with the present invention the active agent
region may be an ADP-ribosyl transferase C3 region. In accordance
with the present invention the ADP-ribosyl transferase C3 may be
selected from the group consisting of ADP-ribosyl transferase
(e.g., ADP-ribosyl transferase C3) derived from Clostridium
botulinum and a recombinant ADP-ribosyl transferase (e.g.,
recombinant ADP-ribosyl transferase C3) that includes the entire C3
coding region, or only a part (fragment) of the C3 coding region
that retains the ADP-ribosyl transferase activity, or analogues
(derivatives) of C3 that retains the ADP-ribosyl transferase
activity, or enough of the C3 coding region to be able to
effectively inactivate Rho. The active agent could also be selected
from other known ADP-ribosyl transferases that act on Rho (Wilde et
al. 2000 J. Biol. Chem. 275-16478-16483; Wilde et al 2001. J. Biol.
Chem. 276:9537-9542).
[0015] In accordance with another aspect the present invention
provides a drug conjugate consisting of a transport polypeptide
moiety (e.g. rich in basic amino acids e.g. arginine, lysine,
histidine, asparagine, glutamine) covalently linked to an active
cargo moiety (e.g. by a peptide bond or a labile bond (i.e. a bond
readily cleavable or subject to chemical change in the interior
target cell environment)) wherein the transport polypeptide moiety
is able to or has the capability to facilitate(s) the uptake of the
active cargo moiety into a mammalian (e.g. human or animal) tissue
or cell (for example, a transport subdomain of HIV (e.g., HIV-1)
Tat protein, a homeoprotein transport sequence (referred also as a
transport homeoprotein) (e.g. the homeodomain of antennapedia), a
Histidine tag (ranging in length from 4 to 30 histidine repeat) or
a variation derivative or homologue thereof, (i.e. pharmaceutically
acceptable chemical equivalents thereof)) [by a receptor
independent process] and wherein the active cargo moiety is an
active therapeutic moiety able (i.e. has the capacity or
capability) to facilitate (i.e. for facilitating) axon growth (e.g.
regeneration, budding) or neuroprotection (prevention of cell
death) either in vivo (in a mammal (e.g., human or animal)) or in
vitro (in cell culture).
[0016] In accordance with the present invention the transport
polypeptide moiety may be selected from the group consisting of SEQ
ID NO.: 48, a transport subdomain of HIV (e.g., HIV-1) Tat protein
such as for example SEQ ID NO.: 46, SEQ ID NO.:47, a homeodomain of
antennapedia, such as for example SEQ ID NO.: 44, SEQ ID NO.: 45, a
Histidine tag and a functional derivative and analogues thereof
(e.g., SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31) [i.e. by the
addition of polyamine, or any random sequence enriched in basic
amino acids]--[i.e. pharmaceutically acceptable chemical
equivalents thereof] and wherein the active cargo moiety is
selected from the group consisting of C3 protein able (i.e. has the
capacity or capability) to facilitate (i.e. for facilitating) axon
growth (e.g. regeneration, budding) or neuroprotection (prevention
of cell death) either in vivo (in a mammal (e.g., human or animal))
or in vitro (in cell culture).
[0017] In accordance with the present invention the C3 protein may
be selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogue. In accordance with the
present invention the ADP-ribosyl transferase C3 may be selected
from the group consisting of ADP-ribosyl transferase (e.g.,
ADP-ribosyl transferase C3) derived from Clostridium botulinum and
a recombinant ADP-ribosyl transferase (e.g., recombinant
ADP-ribosyl transferase C3). The ADP-ribosyl transferase may be a
protein with a C3-like activity, such as that derived from
Staphylococcus aureus (Wilde et al 2001. J. Biol. Chem.
276:9537-9542). The ADP-ribosyl transferase may be any other
transferase that acts to inactivate RhoA, RhoB and/or RhoC such as
those derived from Clostridium limosum, and Bacillus cereus (Wilde
et al 2000. J. Biol. Chem. 275:16478-16483). In accordance with the
present invention the transport polypeptide moiety may include an
active contiguous amino acid sequence as described herein.
[0018] In accordance with an additional aspect the present
invention provides a fusion protein (polypeptide) [e.g. able to
(for) suppress(ing) the inhibition of neuronal axon growth at a
central nervous system (CNS) lesion site or a peripheral nervous
system (PNS) lesion site] consisting of a carboxy terminal active
cargo moiety and an amino terminal transport moiety, wherein the
amino terminal transport moiety is selected from the group
consisting of a transport subdomain of HIV (e.g., HIV-1) Tat
protein, homeoprotein transport sequence (referred also as a
transport homeoprotein) (e.g. the homeodomain of antennapedia), a
Histidine tag and a functional derivatives and analogues thereof
(i.e. pharmaceutically acceptable chemical equivalents thereof) and
wherein the active cargo moiety consists of a C3 protein.
[0019] The present invention in particular provides a fusion
protein (polypeptide) (e.g. able to (for) suppressing the
inhibition of neuronal axon growth at a central nervous system
(CNS) lesion site or a peripheral nervous system (PNS) lesion site)
consisting of a carboxy terminal active cargo moiety and an amino
terminal transport moiety, wherein the amino terminal transport
moiety consists of the homeodomain of antennapedia and the active
cargo moiety consists of a C3 protein (i.e. as described herein).
The present invention also in particular provides a fusion protein
(polypeptide) (e.g. able to (for) suppressing the inhibition of
neuronal axon growth at a central nervous system (CNS) lesion site
or a peripheral nervous system (PNS) lesion site) consisting of a
carboxy terminal active cargo moiety and an amino terminal
transport moiety, wherein the amino terminal transport moiety
consists of a transport subdomain of (e.g., HIV-1) Tat protein and
the active cargo moiety consists of a C3 protein (i.e. as described
herein).
[0020] In accordance with the present invention the C3 protein may
be selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues. In accordance with the
present invention the ADP-ribosyl transferase C3 is selected from
the group consisting of ADP-ribosyl transferase (e.g., ADP-ribosyl
trans ferase C3) derived from Clostridium botulinum and a
recombinant ADP-ribosyl transferase (e.g., recombinant ADP-ribosyl
transferase C3).
[0021] In accordance with an additional aspect the present
invention provides a fusion protein (polypeptide) [e.g. able to
(for) suppress(ing) the inhibition of neuronal axon growth at a
central nervous system (CNS) lesion site or a peripheral nervous
system (PNS) lesion site] consisting of an amino terminal active
cargo moiety and a carboxy terminal transport moiety, wherein the
carboxy terminal transport moiety is selected from the group
consisting of a transport subdomain of HIV Tat protein, a
homeoprotein transport sequence (referred also as a transport
homeoprotein) (e.g. the homeodomain of antennapedia), a Histidine
tag and a functional derivatives and analogues thereof (i.e.
pharmaceutically acceptable chemical equivalents thereof) and
wherein the active cargo moiety consists of a C3 protein.
[0022] The present invention in particular provides a fusion
protein (polypeptide) (e.g. able to (for) suppressing the
inhibition of neuronal axon growth at a central nervous system
(CNS) lesion site or a peripheral nervous system (PNS) lesion site)
consisting of an amino terminal active cargo moiety and a carboxy
terminal transport moiety, wherein the carboxy terminal transport
moiety consists of the homeodomain of antennapedia and the active
cargo moiety consists of a C3 protein (i.e. as described
herein).
[0023] The present invention also in particular provides a fusion
protein (polypeptide) (e.g. able to (for) suppressing the
inhibition of neuronal axon growth at a central nervous system
(CNS) lesion site or a peripheral nervous system (PNS) lesion site)
consisting of an amino terminal active cargo moiety and a carboxy
terminal transport moiety, wherein the carboxy terminal transport
moiety consists of a transport subdomain of HIV Tat protein and the
active cargo moiety consists of a C3 protein (i.e. as described
herein).
[0024] In accordance with the present invention the C3 protein may
be selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues. In accordance with the
present invention the ADP-ribosyl transferase C3 is selected from
the group consisting of ADP-ribosyl transferase C3 derived from
Clostridium botulinum and a recombinant ADP-ribosyl transferase
C3.
[0025] The present invention in a further aspect provides for the
use of a member selected from the group consisting of a drug
delivery construct as described herein, a drug conjugate as
described herein and a fusion protein (polypeptide) as described
herein (e.g. including pharmaceutically acceptable chemical
equivalents thereof) for suppressing the inhibition of neuronal
axon growth.
[0026] The present invention in a further aspect relates to a
pharmaceutical composition (e.g. for suppressing the inhibition of
neuronal axon growth), the pharmaceutical composition comprising a
pharmaceutically acceptable diluent or carrier and an effective
amount of an active member selected from the group consisting of a
drug delivery construct as described herein, a drug conjugate as
described herein, and a fusion protein (polypeptide) as described
herein (e.g. including pharmaceutically acceptable chemical
equivalents thereof).
[0027] The present invention further provides for the use of a
member selected from the group consisting of a drug delivery
construct as described herein, a drug conjugate as described
herein, and a fusion protein (polypeptide) as described herein
(e.g. including pharmaceutically acceptable chemical equivalents
thereof) for the manufacture of a pharmaceutical composition (e.g.
for suppressing the inhibition of neuronal axon growth).
[0028] The present invention also relates to a method for preparing
a drug delivery construct, a conjugate or fusion protein
(polypeptide) as defined above comprising
[0029] cultivating a host cell (bacterial or eukaryotic) under
conditions which provide for the expression of the drug delivery
construct, the conjugate or fusion protein (polypeptide) within the
cell; (the drug delivery construct, conjugate or fusion protein
(polypeptide), could also be expressed to be produced in an
animals, such as, for example, the production of recombinant
proteins in the milk of farm animals) and,
[0030] recovering the drug delivery construct, conjugate or fusion
protein (polypeptide) by a purification step.
[0031] The purification of the drug delivery construct, conjugate
or fusion protein (polypeptide) may be done by affinity methods,
ion exchange chromatography, size exclusion chromatography,
hydrophobicity or any other purification technique typically used
for protein purification. Preferably, the purification step would
be performed under non-denaturating conditions. On the other hand,
if a denaturating step is required, the protein may be renatured
using techniques known in the art.
[0032] The present invention also relates to the expression of the
drug delivery construct, conjugate or fusion protein (polypeptide)
in a mammalian cell, which when used with a signal sequence, will
allow expression and secretion of the fusion protein into the
extracellular milieu. Other system of expression (yeast cells,
bacterial cells, insect cells, etc.) may be suitable to express
(produce) the drug delivery construct, conjugate or fusion protein
(polypeptide) of the present invention as discussed herein.
[0033] The present invention in particular provides a fusion
protein (polypeptide) selected from the group consisting of C3APL
(SEQ ID NO.: 4), C3APLT (SEQ ID NO.: 37), C3APS (SEQ ID NO.:6),
C3-TL (SEQ ID NO.:14), C3-TS (SEQ ID NO.: 18), C3Basic1 (SEQ ID
NO.:25), C3Basic2 (SEQ ID NO.: 30), C3Basic3 (SEQ ID NO.:35), SEQ
ID NO.: 20, and SEQ ID NO.: 43 and pharmaceutically acceptable
chemical equivalents thereof.
[0034] In accordance with an additional aspect, the present
invention provides a pharmaceutical composition comprising a
polypeptide selected from the group consisting of C3APL (SEQ ID
NO.:4), C3APLT (SEQ ID NO.:37), C3APS (SEQ ID NO.:6), C3-TL (SEQ ID
NO.: 14), C3-TS (SEQ ID NO.:18), C3Basic1 (SEQ ID NO.:25), C3Basic2
(SEQ ID NO.:30), C3Basic3 (SEQ ID NO.:35), SEQ ID NO.: 20 and SEQ
ID NO.: 43, and a pharmaceutically acceptable carrier.
[0035] In accordance with the present invention, the pharmaceutical
composition may further comprise a biological adhesive, such as,
for example, fibrin (fibrin glue).
[0036] In a further aspect the present invention provides a
pharmaceutical composition comprising a polypeptide comprising at
least one (one or more) transport agent region and an active agent
region, said active agent region being selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues, and a pharmaceutically acceptable
carrier.
[0037] In accordance with the present invention, the transport
agent region may be at the carboxy-terminal end of said polypeptide
and the active agent region may be at the amino terminal end of
said polypeptide.
[0038] In accordance with the present invention, the pharmaceutical
composition may further comprise a biological adhesive, such as,
for example, fibrin (fibrin glue).
[0039] In an additional aspect, the present invention provides a
polypeptide comprising at least one (one or more) transport agent
region and an active agent region, said active agent region being
selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues (wherein the transport
agent region is able to facilitate the uptake of the active agent
region into (inside the cell or in the cell membrane) a cell).
[0040] In an additional aspect, the present invention provides a
polypeptide consisting of a carboxy-terminal active agent moiety
and an amino-terminal transport moiety region (wherein the
transport agent region is able to facilitate the uptake of the
active agent region into (inside the cell or in the cell membrane)
a cell) and wherein said carboxy-terminal active agent moiety may
be selected from the group consisting of ADP-ribosyl transferase C3
and ADP-ribosyl transferase C3 analogues thereof.
[0041] In accordance with the present invention, the
carboxy-terminal transport moiety region may be selected from the
group consisting of a basic amino acid rich region and a proline
rich region.
[0042] In a further aspect, the present invention relates to a
polypeptide consisting of an amino-terminal active agent moiety and
a carboxy-terminal transport moiety region, wherein said
amino-terminal active agent moiety may be selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues thereof.
[0043] In accordance with the present invention, the
carboxy-terminal transport moiety region may be selected from the
group consisting of a basic amino acid rich region and a proline
rich region.
[0044] In yet a further aspect, the present invention relates to a
conjugate comprising at least one transport agent region (including
one, two, three or more transport agent region) and an active agent
region, said active agent region being selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues, wherein said transport agent region is
covalently linked to said active agent region.
[0045] In accordance with the present invention, the transport
agent region may be cross-linked (e.g., chemically cross-linked, UV
cross -linked) to the active agent region (C3-like proteins of the
present invention and analogues thereof).
[0046] In accordance with the present invention, the transport
agent region may be fused to ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues according to recombinant DNA
technology (e.g., cloning the DNA sequence of the transport agent
region in frame with the DNA sequence of the ADP-ribosyl
transferase C3 or an ADP-ribosyl transferase C3 analogue comprising
or not a spacer DNA sequence (multiple cloning site, linker) or any
other DNA sequence that would not interfere with the activity of
the C3-like protein once expressed).
[0047] In an additional aspect, the present invention relates to
the use of a polypeptide selected from the group consisting of
C3APL (SEQ ID NO.: 4), C3APLT (SEQ ID NO.:37), C3APS (SEQ ID
NO.:6), C3-TL (SEQ ID NO.:14), C3-TS (SEQ ID NO.:18), C3Basic1 (SEQ
ID NO.:25), C3Basic2 (SEQ ID NO.:30), C3Basic3 (SEQ ID NO.:35), SEQ
ID NO.: 20 and SEQ ID NO.: 43, for the manufacture of a
pharmaceutical composition.
[0048] In other aspects, the present invention relates to the use
of a polypeptide comprising at least one (one or more) transport
agent region and an active agent region, for the manufacture of a
pharmaceutical composition, or to facilitate (for facilitating)
axon growth or for treating (in the treatment of) nerve injury
(e.g., nerve injury arising from traumatic nerve injury or nerve
injury caused by disease), or for preventing (diminishing,
inhibiting (partially or totally)) cell apoptosis (cell death, such
as following ischemia in the CNS), or for suppressing (diminishing)
the inhibition of neuronal axon growth, or for the treatment of
ischemic damage related to stroke, or for suppressing (diminishing)
Rho activity, or to regenerate (for regenerating) injured axon
(helping injured axon to recover, partially or totally, their
function), or to help (for helping) neurons to make new connections
(developing axon, dendrite, neurite) with other (surrounding) cells
(neuronal cells), in a mammal, (e.g., human, animal), wherein said
active agent region being selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3
analogues.
[0049] In accordance with the present invention, the transport
agent region may be at the amino-terminal end of the polypeptide
(i.e., protein) and the ADP-ribosyl transferase C3 or ADP-ribosyl
transferase C3 analogue may be at the carboxy-terminal end of the
polypeptide (i.e., protein).
[0050] In accordance with the present invention, the transport
agent region may be at the carboxy-terminal end of the polypeptide
(i.e., protein) and the ADP-ribosyl transferase C3 or ADP-ribosyl
transferase C3 analogue may be at the amino-terminal end of the
polypeptide (i.e., protein).
[0051] In a further aspect, the present invention provides a method
of (for) suppressing the inhibition of neuronal axon growth (e.g.,
in a mammal, (e.g., human, animal)) comprising administering (e.g.,
delivering) a member selected from the group consisting of a drug
delivery construct, a drug conjugate, a fusion protein and a
polypeptide (e.g. including pharmaceutically acceptable chemical
equivalents thereof), said polypeptide comprising at least one (one
or more) transport agent region and an active agent region selected
from the group consisting of ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues (directly) at (to) a central
nervous system (CNS) lesion site or a peripheral nervous system
(PNS) lesion site (of a patient), in an amount effective to
counteract said inhibition. Such application could be useful for
treatment of a wide variety of peripheral neuropathies, such as
diabetic neuropathy.
[0052] The present invention, for example, provides recombinant Rho
antagonists comprising C3 enzymes with basic stretches of amino
acids (e.g., a basic amino acid rich region) or a proline rich
region added to the C3 coding sequence to facilitate the uptake
thereof into tissue or cells for the repair and/or promotion of
repair or promotion of growth in the CNS, even in the lack of
traumatic axon damage. Examples of basic amino acid rich regions
and proline rich regions are given below.
[0053] In yet a further aspect, the present invention provides a
method of (for) facilitating axon growth (e.g., in a mammal, (e.g.,
human, animal)) comprising delivering a polypeptide or conjugate
comprising at least one transport agent region and an active agent
region selected from the group consisting of ADP-ribosyl
transferase C3 and ADP-ribosyl transferase C3 analogues directly at
a central nervous system (CNS) lesion site or a peripheral nervous
system (PNS) lesion site, in an amount effective to facilitate said
growth.
[0054] In an additional aspect, the present invention provides a
method of (for) treating nerve injury (e.g., in a mammal, (e.g.,
human, animal)) comprising delivering a polypeptide or conjugate
comprising at least one transport agent region and an active agent
region selected from the group consisting of ADP-ribosyl
transferase C3 and ADP-ribosyl transferase C3 analogues directly at
(to) a central nervous system (CNS) lesion site or a peripheral
nervous system (PNS) lesion site.
[0055] In yet an additional aspect, the present invention provides
a method of (for) preventing cell apoptosis (e.g., in a mammal,
(e.g., human, animal)) comprising delivering a polypeptide or
conjugate comprising at least one transport agent region and an
active agent region selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogues
directly at a central nervous system (CNS) lesion site or a
peripheral nervous system (PNS) lesion site.
[0056] In another aspect, the present invention provides a method
of (for) treating ischemic damage related to stroke (e.g., in a
mammal, (e.g., human, animal)) comprising delivering a polypeptide
or conjugate comprising at least one transport agent region and an
active agent region selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogues
directly at a central nervous system (CNS) lesion site (to said
mammal).
[0057] In yet another aspect, the present invention provides a
method of (for) suppressing Rho activity comprising delivering a
polypeptide or conjugate comprising at least one transport agent
region and an active agent region selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues directly at a central nervous system (CNS)
lesion site or a peripheral nervous system (PNS) lesion site, in an
amount effective to suppress said activity.
[0058] In accordance with an additional aspect, the present
invention provides a method of (for) regenerating injured axon
(e.g., in a mammal, (e.g., human, animal)) comprising delivering a
polypeptide or conjugate comprising at least one transport agent
region and an active agent region selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues directly at a central nervous system (CNS)
lesion site or a peripheral nervous system (PNS) lesion site (e.g.,
in a mammal), in an amount effective to regenerate said injured
axon.
[0059] In accordance with a further aspect, the present invention
provides a method of (for) helping neurons to make new cell
connection (developing axon, dendrite, neurite with other
(surrounding) cells (neuronal cells) comprising delivering a
polypeptide or conjugate comprising at least one transport agent
region and an active agent region selected from the group
consisting of ADP-ribosyl transferase C3 and ADP-ribosyl
transferase C3 analogues directly at a central nervous system (CNS)
lesion site or a peripheral nervous system (PNS) lesion site.
[0060] In an additional aspect, the present invention provides a
method for (of) preparing a polypeptide comprising at least one
(one or more) transport agent region and an active agent region,
wherein said transport agent region may be selected from the group
consisting of SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.: 31, SEQ
ID NO.: 44, SEQ ID NO.: 45, SEQ ID NO.: 46, SEQ ID NO.: 47, SEQ ID
NO.: 48 and analogues thereof, and wherein said active agent region
may be selected from the group consisting of ADP-ribosyl
transferase C3 and ADP-ribosyl transferase C3 analogues, said
method comprising:
[0061] cultivating a host cell under conditions which provide for
the expression of the polypeptide within the cell; and
[0062] recovering the polypeptide by a purification step.
[0063] In accordance with the present invention, the purification
of polypeptide may be done by affinity methods, ion exchange
chromatography, size exclusion chromatography, hydrophobicity or
any other purification technique typically used for protein
purification. Preferably, the purification step would be performed
under non-denaturating conditions. On the other hand, if a
denaturating step is required, the protein may be renatured using
techniques known in the art.
[0064] In another aspect, the present invention provides a
polypeptide consisting of a basic amino acid rich region and an
active agent region, wherein, amino acids from said basic rich
region comprises amino acids selected from the group consisting of
Histidine, Asparagine, Glutamine, Lysine and Arginine and wherein
the active agent region is ADP-ribosyl transferase C3.
[0065] In yet another aspect, the present invention relates to the
use of a polypeptide comprising at least one transport agent region
and an active agent region, said active agent region being selected
from the group consisting of ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues for the manufacture of a
medicament (or a pharmaceutical composition) for suppressing the
inhibition of neuronal axon growth.
[0066] In accordance with the present invention, the polypeptide
may be selected from the group consisting of C3APL (SEQ ID NO.: 4),
C3APL (SEQ ID NO.:37), C3APS (SEQ ID NO.:6), C3-TL (SEQ ID NO.:14),
C3-TS (SEQ ID NO.:18), C3Basic1 (SEQ ID NO.:25), C3Basic2 (SEQ ID
NO.:30), C3Basic3 (SEQ ID NO.:35), SEQ ID NO.: 20 and SEQ ID NO.:
43.
[0067] In a further aspect, the present invention relates to the
use of a polypeptide comprising at least one transport agent region
and an active agent region, said active agent region being selected
from the group consisting of ADP-ribosyl transferase C3 and
ADP-ribosyl transferase C3 analogues for the manufacture of a
medicament (or pharmaceutical composition) for facilitating axon
growth.
[0068] In accordance with the present invention, the polypeptide
may be selected from the group consisting of C3APL (SEQ ID NO.: 4),
C3APLT (SEQ ID NO.:37), C3APS (SEQ ID NO.:6), C3-TL (SEQ ID
NO.:14), C3-TS (SEQ ID NO.:18), C3Basic1 (SEQ ID NO.:25), C3Basic2
(SEQ ID NO.:30), C3Basic3 (SEQ ID NO.:35), SEQ ID NO.: 20 and SEQ
ID NO.: 43.
[0069] In yet a further aspect the present invention relates to the
use of a polypeptide comprising at least one (one or more)
transport agent region and an active agent region, said active
agent region being selected from the group consisting of
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3 analogues
for the manufacture of a medicament (or pharmaceutical composition)
for treating nerve injury (e.g., in a mammal, (e.g., human,
animal)).
[0070] In accordance with the present invention, the polypeptide
may be selected from the group consisting of C3APL (SEQ ID NO.: 4),
C3APLT (SEQ ID NO.:37), C3APS (SEQ ID NO.:6), C3-TL (SEQ ID
NO.:14), C3-TS (SEQ ID NO.:18), C3Basic1 (SEQ ID NO.:25), C3Basic2
(SEQ ID NO.:30), C3Basic3 (SEQ ID NO.:35), SEQ ID NO.: 20 and SEQ
ID NO.: 43.
[0071] In accordance with the present invention, the transport
agent region discussed herein may be selected from the group
consisting of a basic amino acid rich region (region comprising
basic amino acid (e.g., arginine, lysine, histidine, glutamine,
and/or asparagine)) and a proline rich region (e.g. region
comprising prolines).
[0072] In accordance with the present invention, the basic amino
acid rich region discussed herein may be selected from the group
consisting of SEQ ID NO.: 48, a subdomain of HIV Tat protein (e.g.,
SEQ ID NO.: 46, SEQ ID NO.: 47, or any other subdomain of Tat, that
could act as a transport sequence), a homeodomain of antennapedia
(e.g., SEQ ID NO.: 44, SEQ ID NO.: 45, or any other domain of
antennapedia, that could act as a transport sequence), a
homeoprotein transport sequence, a Histidine tag, and analogues
thereof (e.g., SEQ ID NO.: 21, SEQ ID NO.: 26, SEQ ID NO.:31).
[0073] In accordance with the present invention, the basic amino
acid region discussed herein may be selected from the group
consisting of SEQ ID NO.: 21(Basic1), SEQ ID NO.: 26 (Basic2), SEQ
ID NO.: 31 (Basic3), SEQ ID NO.: 44 (APL), SEQ ID NO.: 45 (APS) SEQ
ID NO.: 46 (TL), SEQ ID NO.: 47 (TS), and analogues thereof.
[0074] In accordance with the present invention, the proline rich
region discussed herein may be selected from the group consisting
of SEQ ID NO.: 48 (APLT) and analogues thereof.
[0075] In another aspect, the present invention provides an
isolated polynucleotide comprising at least the polynucleotide
sequence (for example the polynucleotide sequence disclosed herein
in addition with (or in some cases without) a suitable (DNA)
backbone (e.g., plasmid, viral vector)) selected from the group
consisting of SEQ ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 13, SEQ ID
NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 24, SEQ ID NO.: 29, SEQ ID
NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 42.
[0076] In yet another aspect, the present invention provides a cell
transformed (transfected, transduced, infected, electroporated,
micro-injected, etc.) with an isolated polynucleotide comprising at
least the polynucleotide sequence (for example the polynucleotide
sequence disclosed herein in addition with (or in some cases
without) a suitable backbone (e.g., plasmid, viral vector))
selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 5,
SEQ ID NO.: 13, SEQ ID NO.: 17, SEQ ID NO.: 19, SEQ ID NO.: 24, SEQ
ID NO.: 29, SEQ ID NO.: 34, SEQ ID NO.: 36, and SEQ ID NO.: 42.
[0077] In a further aspect, the present invention provides a
delivery agent consisting of a cargo moiety in combination with a
transport moiety, wherein the transport moiety is selected from the
group consisting of SEQ ID NO: 48 and analogues thereof. SEQ ID NO:
48 and analogues thereof act as a transport moiety which facilitate
penetration of the cell membrane. Any cargo moiety (e.g., protein,
chemicals) linked (e.g. attached) to SEQ ID NO: 48 or to some
analogues thereof are encompassed by the present invention. For
example, SEQ ID NO: 48 and analogues thereof may be fused to an
anticancer agent, a therapeutic agent, an apoptotic agent, an
anti-apoptotic agent, a reporter protein, an antibody, an antibody
fragment, a dye, a probe, a marker etc.
[0078] In accordance with the present invention, the cargo moiety
may retain biological activity following transport moiety-dependent
intracellular delivery. Biological activity may include for
example, biological properties (e.g. enzymatic activity) as well as
its immunological properties. The cargo moiety may have a direct
biological effect on the cell, such as for example killing the cell
following its internalization or may have an indirect biological
effect, for example, the cargo moiety may be a pro-drug that is
inactive by itself but becomes active following modification (e.g.,
cleavage, phosphorylation, etc.) or when a second molecules is
introduced inside the cell. The cargo moiety may also be a
biologically inactive (i.e., inert) compound such as a labeling
molecule (e.g., chemicals, proteins), an imaging molecule etc.
[0079] In accordance with the present invention, the agent may be a
fusion protein having an amino-terminal that is the cargo moiety
and having a carboxy-terminal that is the transport moiety.
[0080] In accordance with the present invention the cargo moiety
may be selected from the group consisting of analytical molecules
(e.g., molecules used in tissue culture experiments, markers,
probes, dyes, reporter proteins) therapeutic molecules (e.g.,
toxin, drug, pro-drug), prophylactic molecules and diagnostic
molecules (i.e., molecules used in in vivo or in vitro detection of
a specific condition, metabolite, other molecule). Examples of
analytical molecules, therapeutic molecules, prophylactic molecules
and diagnostic molecules includes proteins (e.g., enzymes (e.g.,
nucleases, proteases, kinases, etc.), cytokines, chemokines,
antigen, antibodies, antibody fragments, reporter proteins such as
horseradish peroxidase, beta-galactosidase, fluorescent proteins
(e.g., green fluorescent protein)), nucleic acids, polysaccharides,
dyes, isotopes (e.g., radioisotope), markers, probes, and other
types of chemicals. Transport polypeptides of the present invention
may be advantageously attached to cargo molecules by chemical
cross-linking or by genetic fusion.
[0081] In accordance with the present invention the cargo moiety
may be selected from the group consisting of ADP-ribosyl
transferase C3 and ADP-ribosyl transferase C3 analogues
thereof.
[0082] In an additional aspect, the present invention relates to
the polypeptide set forth in SEQ ID NO: 48 and analogues
thereof.
[0083] In yet an additional aspect, the present invention provides
a polypeptide as set forth in SEQ ID NO: 48 and analogues thereof,
wherein said polypeptide and analogues may be able to act as a
transport agent for the intracellular delivery of a cargo agent
selected from the group consisting of analytical molecules,
therapeutic molecules, prophylactic molecules, and diagnostic
molecules.
[0084] In accordance with the present invention, the cargo agent
may be selected from the group consisting of ADP-ribosyl
transferase C3 and ADP-ribosyl transferase C3 analogues
thereof.
[0085] The transport of a cargo moiety across the cellular membrane
(intracellular delivery) may be facilitated (increased) when linked
(e.g., genetically fused, chemically cross-linked, etc.) to SEQ ID
NO: 48 and analogues thereof. Therefore it is an object of the
present invention to provide a method for the intracellular
delivery of a cargo moiety, the method comprising exposing the cell
to a delivery agent comprising a cargo moiety and a transport
moiety, said transport moiety being selected from the group
consisting of SEQ ID NO: 48 and analogues thereof and wherein said
transport moiety enables the delivery agent to be delivered inside
the cell (i.e., across cellular membranes). An example of a cargo
moiety that may be delivered across the cell membrane is
ADP-ribosyl transferase C3 and analogues thereof. Other examples of
a cargo moiety are mentionned herein. The method also comprise
bringing the delivery agent comprising a cargo moiety and a
transport moiety (SEQ ID NO: 48 and analogues thereof) in the
surrounding of a target cell in a manner (e.g., concentration)
sufficient to permit the uptake of the delivery agent by the cell.
For example, in the case of in vitro (e.g., cell culture) delivery,
the delivery agent (in a pharmaceutically acceptable carrier,
diluent, excipient, etc.) may be added directly to the
extracellular milieu (e.g., cell culture media) of adherent cells
(i.e., cell lines or primary cells) or cells in suspension.
Alternatively, cells may be harvested and concentrated before being
put in contact with the delivery agent. Intracellular delivery may
be monitored by techniques known in the art, such as for example,
immunofluorescence, immunohistochemistry or by the intrinsic
properties of the cargo moiety (e.g., its enzymatic activity).
[0086] In vivo delivery (in a mammal) may be performed for example,
by exposing (i.e., contacting) a tissue, a nerve injury site, an
open wound, etc. with the delivery agent (in a pharmaceutically
acceptable carrier, diluent, excipient, fibrin gel etc.) of the
present invention in an amount sufficient to promote the biological
effect of the cargo moiety (e.g., recovery, healing of the wounded
tissue, etc.). In addition, in vivo delivery may be performed by
other methods known in the art such as for example, injection via
the intramuscular (IM), subcutaneous (SC), intra-dermal (ID),
intra-venous (IV) or intra-peritoneal (IP) routes or administration
at the mucosal membranes including the oral and nasal cavity
membranes using any suitable means. Alternatively, cells may be
isolated from a mammal and treated (exposed) ex-vivo (e.g., in gene
therapy techniques) with the delivery agent of the present
invention before being re-infused in the same individual or in a
compatible individual.
[0087] The term "Rho antagonists" as used herein includes, but is
not restricted to, (known ) C3, including C3 chimeric proteins, and
like Rho antagonists.
[0088] The term "C3 protein" refers to ADP-ribosyl transferase C3
isolated from Clostridium botulinum or a recombinant ADP-ribosyl
transferase.
[0089] The term "C3-like protein", "ADP-ribosyl transferase C3-like
protein", "ADP-ribosyl transferase C3 analogue", "C3-like
transferase" or "C3 chimeric proteins" as used herein refers to any
protein (polypeptide) having a biological activity similar (e.g.,
the same, substantially similar), to ADP-ribosyl transferase C3.
Examples of such C3-like protein include, for example, but are not
restricteed to C3APL, C3APLT, C3APS, C3-TL, C3-TS, C3Basic1,
C3Basic2 and C3Basic3 and the protein defined in SEQ ID NO.:
20.
[0090] The term "nerve injury site" refers to a site of traumatic
nerve injury or nerve injury caused by disease. The nerve injury
site may be a single nerve (eg sciatic nerve) or a nerve tract
comprised of many nerves (eg. damaged region of the spinal cord).
The nerve injury site may be in the central nervous system or
peripheral nervous system or in any region needing repair. The
nerve injury site may form as a result of damage caused by stroke.
The nerve injury site may be in the brain as a result of surgery,
brain tumour removal or therapy following a cancerous lesion. The
nerve injury site may result from stroke, Parkinson's disease,
Alzheimer's disease, amyotrophic lateral sclerosis (ALS), diabetes
or any other type of neurodegenerative disease.
[0091] The term "cargo" refers to a molecule other than the
transport moiety and that is either (1) not inherently capable of
entering a cell (e.g., cell compartment) or (2) not inherently
capable of entering a cell (e.g., cell compartment) at a useful
rate. The term "cargo" as used herein refers either to a molecule
per se, i.e., before conjugation, or to the cargo moiety of a
transport polypeptide-cargo conjugate. Examples of "cargo" include,
but are not limited to, small molecules and macromolecules such as
polypeptides, nucleic acids (polynucleotides), polysaccharides and
chemicals.
[0092] As used herein, the term "delivery agent" relates to an
agent comprising a cargo moiety and a transport moiety. Examples of
cargo moiety are discussed above and includes for example
ADP-ribosyl transferase C3 and ADP-ribosyl transferase C3
analogues. Examples of transport moiety comprise for example SEQ ID
NO: 48 and analogues thereof.
[0093] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is a mixture of single- and double-stranded regions,
hybrid molecules comprising DNA and RNA that may be single-stranded
or, more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" includes but
is not limited to linear and end-closed molecules. "Polynucleotide"
also embraces relatively short polynucleotides, often referred to
as oligonucleotides.
[0094] "Polypeptides" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds (i.e., peptide isosteres). "Polypeptide"
refers to both short chains, commonly referred as peptides,
oligopeptides or oligomers, and to longer chains generally referred
to as proteins. As described above, polypeptides may contain amino
acids other than the 20 gene-encoded amino acids.
[0095] As used herein the term "analogues" relates to mutants,
variants, chimeras, fusions, deletions, additions and any other
type of modifications made relative to a given polypeptide. The
term "analogue" is synonym of homologue, derivative and chemical
equivalent or biological equivalent.
[0096] As used herein, the term "homologous" sequence relates to
nucleotide or amino acid sequence derived from the DNA sequence or
polypeptide sequence of C3APL, C3APLT, C3APS, C3-TL, C3-TS,
C3Basic1, C3Basic2 and C3Basic3.
[0097] As used herein, the term "heterologous" sequence relates to
DNA sequence or amino acid sequence of a heterologous polypeptide
and includes sequence other than that of C3APL, C3APLT, C3APS,
C3-TL, C3-TS, C3Basic1, C3Basic2 and C3Basic3.
[0098] As used herein the term "basic amino acid rich region"
relates to a region of a protein with a high content of the basic
amino acids such as Arginine, Histidine, Asparagine, Glutamine,
Lysine (Lys). A "basic amino acid rich region" may have, for
example 15% or more (up to 100%) of basic amino acids. In some
instance, a "basic amino acid rich region" may have less than 15%
of basic amino acids and still function as a transport agent
region. More preferably, a basic amino acid region will have 30% or
more (up to 100%) of basic amino acids.
[0099] As used herein the term "proline rich region" refers to a
region of a protein with 5% or more (up to 100%) of proline in its
sequence. In some instance a "proline rich region" may have between
5% and 15% of prolines. Additionally, a "proline rich region"
refers to a region, of a protein containing more prolines than what
is generally observed in naturally occurring proteins (e.g.,
proteins encoded by the human genome). "Proline rich region" of the
present invention function as a transport agent region.
[0100] As used herein the term "to help neuron make new connections
with other cells" or "helping neurons to make new cell connection"
means that upon treatment of cells (e.g., neuron(s)) or tissue with
a drug delivery construct, a conjugate, a fusion-protein, a
polypeptide or a pharmaceutical compositions of the present
invention, neurons may grow (develop) for example new dendrite, new
axon or new neurite (i.e., cell bud), or already existing
dendrite(s), axon or neurite (i.e., cell bud) are induce to grow to
a greater extent.
[0101] As used herein, the term "vector" refers to an autonomously
replicating DNA or RNA molecule into which foreign DNA or RNA
fragments are inserted and then propagated in a host cell for
either expression or amplification of the foreign DNA or RNA
molecule. The term "vector" comprises and is not limited to a
plasmid (e.g., linearized or not) that can be used to transfer DNA
sequences from one organism to another.
[0102] The term "pharmaceutically acceptable carrier" or "adjuvant"
and "physiologically acceptable vehicle" and the like are to be
understood as referring to an acceptable carrier or adjuvant that
may be administered to a patient, together with a compound of this
invention, and which does not destroy the pharmacological activity
thereof. Further, as used herein "pharmaceutically acceptable
carrier" or "pharmaceutical carrier" are known in the art and
include, but are not limited to, 0.01-0.1 M and preferably 0.05 M
phosphate buffer or 0.8% saline. Additionally, such
pharmaceutically acceptable carriers may be aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's
or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers such as those based on
Ringer's dextrose, and the like. Preservatives and other additives
may also be present, such as, for example, antimicrobials,
antioxidants, collating agents, inert gases and the like.
[0103] As used herein, "pharmaceutical composition" means
therapeutically effective amounts (dose) of the agent together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvant and/or carriers. A "therapeutically effective
amount" as used herein refers to that amount which provides a
therapeutic effect for a given condition and administration
regimen. Such compositions are liquids or lyophilized or otherwise
dried formulations and include diluents of various buffer content
(e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,
additives such as albumin or gelatin to prevent absorption to
surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile
acid salts). Solubilizing agents (e.g., glycerol, polyethylene
glycerol), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol,
parabens), bulking substances or tonicity modifiers (e.g., lactose,
mannitol), covalent attachment of polymers such as polyethylene
glycol to the protein, complexation with metal ions, or
incorporation of the material into or onto particulate preparations
of polymeric compounds such as polylactic acid, polyglycolic acid,
hydrogels, etc, or onto liposomes, microemulsions, micelles,
unilamellar or multilamellar vesicles, erythrocyte ghosts, or
spheroplasts. Such compositions will influence the physical state,
solubility, stability, rate of in vivo release, and rate of in vivo
clearance. Controlled or sustained release compositions include
formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
Also comprehended by the invention are particulate compositions
coated with polymers (e.g., poloxamers or poloxamines). Other
embodiments of the compositions of the invention incorporate
particulate forms protective coatings, protease inhibitors or
permeation enhancers for various routes of administration,
including parenteral, pulmonary, nasal and oral routes. In one
embodiment the pharmaceutical composition is administered
parenterally, paracancerally, transmucosally, transdermally,
intramuscularly, intravenously, intradermally, subcutaneously,
intraperitonealy, intraventricularly, intracranially intratumorally
or more preferably, directly at a central nervous system (CNS)
lesion site or a peripheral nervous system (PNS) lesion site.
[0104] In addition, the term "pharmaceutically effective amount" or
"therapeutically effective amount" refers to an amount (dose)
effective in treating a patient, having, for example, a nerve
injury. It is also to be understood herein that a "pharmaceutically
effective amount" may be interpreted as an amount giving a desired
therapeutic effect, either taken into one dose or in any dosage or
route or taken alone or in combination with other therapeutic
agents. In the case of the present invention, a "pharmaceutically
effective amount" may be understood as an amount of ADP-ribosyl
transferase C3 or ADP-ribosyl transferase C3 analogues (e.g.,
fusion proteins) of the present invention which may for example,
suppress (e.g., totally or partially) the inhibition of neuronal
axon growth, facilitate axon growth, prevent cell apoptosis,
suppress Rho activity, help regenerate injured axon, or which may
help neurons to make new connections with other cells.
[0105] As may be appreciated, a number of modifications may be made
to the polypeptides of the present invention, such as for example
the active agent region (e.g., ADP-ribosyl transferase C3 or
ADP-ribosyl transferase C3 analogue) or the transport agent region
(e.g., a subdomain of HIV Tat protein, or a homeodomain of
antennapedia) and fragments thereof without deleteriously affecting
the biological activity of the polypeptides or fragments.
Polypeptides of the present invention comprises for example, those
containing amino acid sequences modified either by natural
processes, such as posttranslational processing, or by chemical
modification techniques which are known in the art. Modifications
may occur anywhere in a polypeptide including the polypeptide
backbone, the amino acid side-chains and the amino or carboxy
termini. It will be appreciated that the same type of modification
may be present in the same or varying degrees at several sites in a
given polypeptide. Also, a given polypeptide may contain many types
of modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
posttranslational natural processes or may be made by synthetic
methods. Modifications comprise for example, without limitation,
acetylation, acylation, addition of acetomidomethyl (Acm) group,
ADP-ribosylation, amidation, covalent attachment to fiavin,
covalent attachment to a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphatidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation and ubiquitination (for
reference see, Protein-structure and molecular proterties, 2.sup.nd
Ed., T.E. Creighton, W.H. Freeman and Company, New-York, 1993).
[0106] Other type of polypeptide modification may comprises for
example, amino acid insertion (i.e., addition), deletion and
substitution (i.e., replacement), either conservative or
non-conservative (e.g., D-amino acids, desamino acids) in the
polypeptide sequence where such changes do not substantially alter
the overall biological activity of the polypeptide. Polypeptides of
the present invention comprise for example, biologically active
mutants, variants, fragments, chimeras, and analogues; fragments
encompass amino acid sequences having truncations of one or more
amino acids, wherein the truncation may originate from the amino
terminus (N-terminus), carboxy terminus (C-terminus), or from the
interior of the protein. Analogues of the invention involve an
insertion or a substitution of one or more amino acids. Variants,
mutants, fragments, chimeras and analogues may have the biological
properties of polypeptides of the present invention which comprise
for example (without being restricted to the present examples) to
facilitate neuronal axon growth, to suppress the inhibition of
neuronal axon growth, to facilitate neurite growth, to inhibit
apoptosis, to treat nerve injury, to regenerate injured axon and/or
to act as a Rho antagonist.
[0107] As it may be exemplified (Example 13: reverse Tat sequence),
in some instance, the order of the amino acids in a particular
polypeptide is not critical. As for the transport agent region
described herein, the transport function of this region may be
preserved even if the amino acids are not in their original (as it
is found in nature) order (sequence).
[0108] Example of substitutions may be those, which are
conservative (i.e., wherein a residue is replaced by another of the
same general type). As is understood, naturally occurring amino
acids may be sub-classified as acidic, basic, neutral and polar, or
neutral and non-polar. Furthermore, three of the encoded amino
acids are aromatic. It may be of use that encoded polypeptides
differing from the determined polypeptide of the present invention
contain substituted codons for amino acids, which are from the same
group as that of the amino acid being replaced. Thus, in some
cases, the basic amino acids Lys, Arg and His may be
interchangeable; the acidic amino acids Asp and Glu may be
interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln,
and Asn may be interchangeable; the non-polar aliphatic amino acids
Gly, Ala, Val, Ile, and Leu are interchangeable but because of size
Gly and Ala are more closely related and Val, Ile and Leu are more
closely related to each other, and the aromatic amino acids Phe,
Trp and Tyr may be interchangeable.
[0109] It should be further noted that if the polypeptides are made
synthetically, substitutions by amino acids, which are not
naturally encoded by DNA may also be made. For example, alternative
residues include the omega amino acids of the formula NH2(CH2)nCOOH
wherein n is 2-6. These are neutral nonpolar amino acids, as are
sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine,
and norleucine. Phenylglycine may substitute for Trp, Tyr or Phe;
citrulline and methionine sulfoxide are neutral nonpolar, cysteic
acid is acidic, and ornithine is basic. Proline may be substituted
with hydroxyproline and retain the conformation conferring
properties.
[0110] It is known in the art that mutants or variants may be
generated by substitutional mutagenesis and retain the biological
activity of the polypeptides of the present invention. These
variants have at least one amino acid residue in the protein
molecule removed and a different residue inserted in its place (one
or more nucleotide in the DNA sequence is changed for a different
one using known molecular biology techniques, giving a different
amino acid upon translation of the corresponding messenger RNA to a
polypeptide). For example, one site of interest for substitutional
mutagenesis may include but are not restricted to sites identified
as the active site(s), or immunological site(s). Other sites of
interest may be those, for example, in which particular residues
obtained from various species are identical. These positions may be
important for biological activity. Examples of substitutions
identified as "conservative substitutions" are shown in Table 1. If
such substitutions result in a change not desired, then other type
of substitutions, denominated "exemplary substitutions" in Table 1,
or as further described herein in reference to amino acid classes,
are introduced and the products screened.
[0111] In some cases it may be of interest to modify the biological
activity of a polypeptide by amino acid substitution, insertion, or
deletion. For example, modification of a polypeptide may result in
an increase in the polypeptide's biological activity, may modulate
its toxicity, may result in changes in bioavailability or in
stability, or may modulate its immunological activity or
immunological identity. Substantial modifications in function or
immunological identity are accomplished by selecting substitutions
that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation. (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side chain properties:
[0112] (1) hydrophobic: norleucine, methionine (Met), Alanine
(Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile)
[0113] (2) neutral hydrophilic: Cysteine (Cys), Serine (Ser),
Threonine (Thr)
[0114] (3) acidic: Aspartic acid (Asp), Glutamic acid (Glu)
[0115] (4) basic: Asparagine (Asn), Glutamine (Gln), Histidine
(His), Lysine (Lys), Arginine (Arg)
[0116] (5) residues that influence chain orientation: Glycine
(Gly), Proline (Pro); and
[0117] (6) aromatic: Tryptophan (Trp), Tyrosine (Tyr),
Phenylalanine (Phe)
[0118] Non-conservative substitutions will entail exchanging a
member of one of these classes for another.
1TABLE 1 Preferred amino acid substitution Original Exemplary
Conservative residue substitution substitution Ala (A) Val, Leu,
Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln
Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly
(G) Pro Pro His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met,
Ala, Leu Phe, norleucine Leu (L) Norleucine, Ile, Val, Ile Met,
Ala, Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe
(F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T)
Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile,
Leu, Met, Phe, Leu Ala, norleucine
[0119] Amino acids sequence insertions (e.g., additions) include
amino and/or carboxyl-terminal fusions ranging in length from one
residues to polypeptides containing a hundred or more residues, as
well as intrasequence insertions of single or multiple amino acid
residues. Other insertional variants include the fusion of the N-
or C-terminus of the protein to a homologous or heterologous
polypeptide forming a chimera. Chimeric polypeptides (i.e.,
chimeras, polypeptide analogue) comprise sequence of the
polypeptides of the present invention fused to homologous or
heterologous sequence. Said homologous or heterologous sequence
encompass those which, when formed into a chimera with the
polypeptides of the present invention retain one or more biological
or immunological properties.
[0120] Other type of chimera generated by homologous fusion
includes new polypeptides formed by the repetition of two or more
polypeptides of the present invention. The number of repeat may be,
for example, between 2 and 50 units (i.e., repeats). In some
instance, it may be useful to have a new polypeptide with a number
of repeat greater than 50. For example, it may be useful to fuse
(using cross-linking techniques or recombinant DNA technology
techniques) polypeptides such as C3APL, C3APLT, C3APS, C3-TL,
C3-TS, C3Basic1, C3Basic2 and C3Basic3 either to themselves (e.g.,
C3APLT fused to C3APLT) or to another polypeptide of the present
invention (e.g., C3APLT fused to C3APL).
[0121] In addition, a transport agent such as for example, a
subdomain of HIV Tat protein, and a homeodomain of antennapedia may
be repeated more than one time in a polypeptide comprising the
ADP-ribosyl transferase C3 or ADP-ribosyl transferase C3 analogues.
The transport agent region may be either at the amino-terminal
region of an ADP-ribosyl transferase C3 or ADP-ribosyl transferase
C3 analogues or at its carboxy-terminal region or at both regions.
The repetition of a transport agent region may affect (e.g.,
increase) the uptake of the ADP-ribosyl transferase C3 or
ADP-ribosyl transferase C3 analogues by a desired cell.
[0122] Heterologous fusion includes new polypeptides made by the
fusion of polypeptides of the present invention with heterologous
polypeptides. Such polypeptides may include but are not limited to
bacterial polypeptides (e.g., betalactamase,
glutathione-S-transferase, or an enzyme encoded by the E.coli trp
locus), yeast protein, viral proteins, phage proteins, bovine serum
albumin, chemotactic polypeptides, immunoglobulin constant region
(or other immunoglobulin regions), albumin, or ferritin.
[0123] Other type of polypeptide modification includes amino acids
sequence deletions (e.g., truncations). Those generally range from
about 1 to 30 residues, more preferably about 1 to 10 residues and
typically about 1 to 5 residues.
[0124] Mutants, Variants and Analogues Proteins
[0125] Mutant polypeptides will possess one or more mutations,
which are deletions (e.g., truncations), insertions (e.g.,
additions), or substitutions of amino acid residues. Mutants can be
either naturally occurring (that is to say, purified or isolated
from a natural source) or synthetic (for example, by performing
site-directed mutagenesis on the encoding DNA or made by other
synthetic methods such as chemical synthesis). It is thus apparent
that the polypeptides of the invention can be either naturally
occurring or recombinant (that is to say prepared from the
recombinant DNA techniques).
[0126] A protein at least 50% identical, as determined by methods
known to those skilled in the art (for example, the methods
described by Smith, T. F. and Waterman M. S. (1981) Ad. Appl.Math.,
2:482-489, or Needleman, S. B. and Wunsch, C. D. (1970)
J.Mol.Biol., 48: 443-453), to those polypeptides of the present
invention, for example C3APL, C3APLT, C3APS, C3-TL, C3-TS,
C3Basic1, C3Basic2 and C3Basic3 are included in the invention, as
are proteins at least 70% or 80% and more preferably at least 90%
identical to the protein of the present invention. This will
generally be over a region of at least 5, preferably at least 20
contiguous amino acids.
[0127] "Variant" as the term used herein, is a polynucleotide or
polypeptide. that differs from reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusion and truncations in the polypeptide
encoded by the reference sequence, as discussed herein. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequence of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid by one
or more substitutions, additions, deletions, or any combination
therefore. A substituted or inserted amino acid residue may or may
not be one encoded by the genetic code. A variant polynuclotide or
polypeptide may be a naturally occurring such as an allelic
variant, or it may be a variant that is not known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques or by direct
synthesis.
[0128] Amino acid sequence variants may be prepared by introducing
appropriate nucleotide changes into DNA, or by in vitro synthesis
of the desired polypeptide. Such variant include, for example,
deletions, insertions, or substitutions of residues within the
amino acid sequence. A combination of deletion, insertion and
substitution can be made to arrive at the final construct, provided
that the final protein product possesses the desired biological
activity, or characteristics. The amino acid changes also may alter
posttranslational processes such as changing the number or position
of the glycosylation sites, altering the membrane anchoring
characteristics, altering the intra-cellular location by inserting,
deleting or otherwise affecting the transmembrane sequence of the
native protein, or modifying its susceptibility to proteolytic
cleavage.
[0129] Unless otherwise indicated, the recombinant DNA techniques
utilized in the present invention are standard procedures, known to
those skilled in the art. Example of such techniques are explained
in the literature in sources such as J. Perbal, A Practical Guide
to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al
., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989), T. A. Brown (editor), Essential Molecular
Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991),
D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical
Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel
et al. (editors), Current Protocols in Molecular Biology, Greene
Pub. Associates and Wiley-Interscience (1988, including all updates
until present) and are incorporated herein by reference.
[0130] It is to be understood herein, that if a "range" or "group
of substances" is mentioned with respect to a particular
characteristic (e.g. amino acid groups, temperature, pressure, time
and the like) of the present invention, the present invention
relates to and explicitly incorporates herein each and every
specific member and combination of sub-ranges or sub-groups therein
whatsoever. Thus, any specified range or group is to be understood
as a shorthand way of referring to each and every member of a range
or group individually as well as each and every possible sub-ranges
or sub-groups encompassed therein; and similarly with respect to
any sub-ranges or sub-groups therein. Thus, for example,
[0131] with respect to a sequence comprising up to 50 base units it
is to be understood as specifically incorporating herein each and
every individual unit, as well as sub-range of units;
[0132] with respect to reaction time, a time of 1 minute or more is
to be understood as specifically incorporating herein each and
every individual time, as well as sub-range, above 1 minute, such
as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1
to 3 hours, 16 hours, 3 hours to 20 hours etc.;
[0133] with respect to polypeptides, a polypeptide analogue
comprising a particular sequence and having an addition of at least
one amino acid to its amino-terminus or to its carboxy terminus is
to be understood as specifically incorporating each and every
individual possibility, such as for example one, two, three, ten,
eighteen, forty, etc.;
[0134] with respect to polypeptides, a polypeptide analogue having
at least 90% of its amino acid sequence identical to a particular
amino acid sequence is to be understood as specifically
incorporating each and every individual possibility (excluding
100%), such as for example, a polypeptide analogue having 90%,
90.5%, 91%, 93.7%, 97%, 99%, etc., of its amino acid sequence
identical to a particular amino acid sequence.
[0135] with respect to polypeptides, a polypeptide analogue having
at least 70% of its amino acid sequence identical to a particular
amino acid sequence is to be understood as specifically
incorporating each and every individual possibility (excluding
100%), such as for example, a polypeptide analogue having 70%,
72.3%, 73%, 88.6%, 98% etc., of its amino acid sequence identical
to a particular amino acid sequence.
[0136] with respect to polypeptides, a polypeptide analogue having
at least 50% of its amino acid sequence identical to a particular
amino acid sequence is to be understood as specifically
incorporating each and every individual possibility (excluding
100%), such as for example, a polypeptide analogue having 50%, 54%,
66.7%, 70.2%, 84%, 93% etc., of its amino acid sequence identical
to that particular amino acid sequence.
[0137] with respect to polypeptide, a polypeptide comprising at
least one transport agent region is to be understood as
specifically incorporating each and every individual possibility,
such as for example a polypeptide having one, two, five, ten, etc.,
transport agent region.
[0138] and similarly with respect to other parameters such as low
pressures, concentrations, elements, etc. . . .
[0139] It is also to be understood herein that "g" or "gm" is a
reference to the gram weight unit; and that "C", or ".degree. C."
is a reference to the Celsius temperature unit.
2TABLE 2 Abbreviations Abbreviation Full name C3 ADP-ribosyl
transferase C3 NGF Nerve growth factor BDNF Brain-derived
neurotrophic factor C. or .degree.C. Degree Celcius ml milliliter
.mu.l or ul microliter .mu.M or uM micromolar mM millimolar M molar
N normal CNS Central nervous system PNS Peripheral nervous system
HIV Human immunodeficiency virus HIV-1 Human immunodeficiency virus
type-1 kDa kilodalton GST Glutathione S-transferase MTS Membrane
transport sequence SDS-PAGE Sodium dodecyl sulfte polyacrylamide
gel electrophoresis PBS Phosphate buffered saline U unit BBB Basso,
Beattie Breshnahan behavior recovery scale IPTG Isopropyl
.beta.-D-thiogalactopyranoside rpm Rotation per minutes DTT
dithiothreitol PMSF Phenylmethylsulfonyl fluoride NaCl Sodium
chloride MgCl.sub.2 Magnesium chloride HBSS Hank's balanced salt
solution NaOH Sodium hydroxide CSPG chondroitin sulfate
proteoglycan PKN Protein kinase N RSV Rous sarcoma virus MMTV Mouse
mammary tumor virus LTR Long terminal repeat HL Hind limb FL Fore
limb neo neomycin hygro hygromycin IN-1 monoclonal antibody called
IN-1 ADP Adenosine di-phosphate ATP Adenosine tri-phosphate
.sup.32P Isotope 32 of phosphorus DHFR Dihydrofolate reductase PCR
Polymerase chain reaction
[0140] The invention in particular provides C3-like proteins, which
may have additional amino acids added to the carboxy terminal end
of the C3 proteins. Examples of such proteins includes:
[0141] C3APL: (C3 antennapedia -long) created by annealing
sequences from the antennapedia transcription factor to the 3' end
of the sequence encoding C3 cDNA. The long antennapedia sequence of
60 amino acids containing the homeodomain of antennapedia, was
used;
[0142] C3APLT: (C3 antennapedia -truncated) created by annealing
sequences from the antennapedia transcription factor to the 3' end
of the sequence encoding C3 cDNA. This clone with a frameshift
mutation gives a proline-rich transport peptide with good transport
activity. This sequence is truncated i.e. shorter than C3APL.
[0143] C3APS: A short 11 amino acid sequence of antennapedia that
has transmembrane transport properties was fused to the carboxy
terminal of C3 to create C3APS;
[0144] C3-TL: C3 Tat-long created by fusing amino acids 27 to 72 of
Tat to the carboxy terminal of C3 protein;
[0145] C3-TS: C3 Tat-short created by fusing the amino acids
YGRKRRQRRR to the C3 protein;
[0146] C3Basic1 a random basic charge sequence added to the
C-terminal of C3;
[0147] C3Basic2: a random basic charge sequence added to the
C-terminal of C3;
[0148] C3Basic3: C3 Tat-short created by fusing the reverse
sequence of Tat amino acids RRQRRKKR to the C3 protein.
[0149] It has been found that conjugates or fusion proteins
(C3-like proteins) Rho antagonists of the present invention are
effective to stimulate repair in the CNS after spinal cord injury.
The increased cell permeability of new Rho antagonist (new chimeric
C3) would now allow treatment of victims of stroke and
neurodegenerative disease because Rho signaling pathway is
important in repair after stroke (Hitomi, et al. (2000) 67:
1929-39. Trapp et al 2001. Mol.Cell. Neurosci. 17: 883-84).
Treatment with Rho antagonists in the adhesive delivery system
could be used to enhance the rate of axon growth in the PNS. Also,
evidence in the literature now links Rho signaling with formation
of Alzheimer's disease tangles through its ability to activate PKN
which then phosphorylates tau and neurofilaments (Morissette, et
al. (2000) 278: H1769-74., Kawamata, et al. (1998) 18: 7402-10.,
Amano, et al. (1996) 271: 648-50., Watanabe, et al. (1996) 271:
645-8.). Therefore, Rho antagonists are expected to be useful in
the treatment of Alzheimer's disease. The new chimeric C3 drugs
should be able to diffuse readily and therefore can promote repair
for diseases that are neurodegenerative. Examples include, but are
not limited to stroke, traumatic brain injury, Parkinson's disease,
Alzheimer's disease and ALS. Moreover, it is now well established
that Rho signaling antagonists are effective in the treatment of
other diseases. These include, but are not limited to eye diseases
such as glaucoma (Honjo, et al. (2001) 42: 137-44., Rao, et al.
(2001) 42: 1029-1037.), cancer cell migration and metastasis
(Sahai, et al. (1999) 9: 136-45., Takamura, et al. (2001) 33:
577-81., Imamura, et al. (2000) 91: 811-6.). The effect of the Rho
signaling pathway on smooth muscle relaxation are well established.
This has led to the identification of Rho signaling antagonists as
effective in treatment of hypertension (Chitaley, et al. (2001) 3:
139-144., Somlyo (1997) 389: 908-911, Uehata, et al. (1997) 389:
990-994), asthma (Nakahara, et al. (2000) 389: 103-6., Ishizaki, et
al. (2000) 57: 976-83), and vascular disease (Miyata, et al. (2000)
20: 2351-8., Robertson, et al. (2000) 131: 5-9.) as well as penile
erectile dysfunction (Chitaley, et al. (2001) 7: 119-22.). Rho is
also important as a cardioprotective protein (Lee et al. 2001.
FASEB J. 15:1886-1894).
[0150] Rho GTPases include members of the Rho, Rac and Cdc42 family
of proteins. Our invention concerns Rho family members of the Rho
class. Rho proteins consist of different variants encoded by
different genes. For example, PC-12 cells express RhoA, RhoB and
RhoC (Lehmann et al 1999 supra); PC-12 cells: Pheochromocytom cell
line (Greene A and Tischler, A S PNAS 73:2424 (1976). To inactivate
Rho proteins inside cells, Rho antagonists of the C3 family type
are effective because they inactivate all forms of Rho (e.g. RhoA,
Rho B etc). In contrast, gene therapy techniques, such as
introduction of a dominant negative RhoA family member into a
diseased cell, will only inactivate that specific RhoA family
member.
[0151] Recombinant C3 proteins, or C3 proteins that retain the
ribosylation activity are also effective in our delivery system and
are covered by this invention. In addition, Rho kinase is a
well-known target for active Rho, and inactivating Rho kinase has
the same effect as inactivating Rho, at least in terms of neurite
or axon growth (Kimura and Schubert (1992) Journal of Cell
Biology.116:777-783, Keino-Masu, et al. (1996 )Cell.87:175-185,
Matsui, et al. (1996) EMBO J.15:2208-2216, Matsui, et al. (1998) J.
Cell Biol.140:647-657, Ishizaki (1997)FEBS Lett.404: 118-124), the
biological activity that concerns this invention.
[0152] The C3 polypeptides of the present invention include
biologically active fragments and analogues of C3; fragments
encompass amino acid sequences having truncations of one or more
amino acids , wherein the truncation may originate from the amino
terminus, carboxy terminus, or from the interior of the protein.
Fragments containing Glu(173) of C3 are included in this invention
(Saito et al. 1995. FEBS Lett. 371-105). Analogues of the invention
involve an insertion or a substitution of one or more amino acids.
Fragments and analogues will have the biological property of C3
that is capable of inactivating Rho GTPase on Asn(41) on Rho. Also
encompassed by the invention are chimeric polypeptides comprising
C3 amino acid sequences fused to heterologous amino acid sequences.
Said heterologous sequences encompass those which, when formed into
a chimera with C3 retain one or more biological or immunological
properties of C3. A host cell transformed or transfected with
nucleic acids encoding C3 protein or C3 chimeric protein are also
encompassed by the invention. Any host cell which produces a
polypeptide having at least one of the biological properties of C3
may be used. Specific examples include bacterial, yeast, plant,
insect or mammalian cells. In addition, C3 protein may be produced
in transgenic animals. Transformed or transfected host cells and
transgenic animals are obtained using materials and methods that
are routinely available to one skilled in the art. Host cells may
contain nucleic acid sequences having the full-length gene for C3
protein including a leader sequence and a C-terminal membrane
anchor sequence (see below) or, alternatively, may contain nucleic
acid sequences lacking one or both of the leader sequence and the
C-terminal membrane anchor sequence. In addition, nucleic acid
fragments, variants and analogues which encode a polypeptide
capable of retaining the biological activity of C3 may also be
resident in host expression systems.
[0153] C3 is produced as a 26 kDa protein. The full length C3
protein inactivates Rho by ADP-ribosylating asparagine 41 of Rho A
(Han et al. (2001) J. Mol. Biol. 305: 95). Truncated, elongated or
altered C3 proteins or C3-derived peptides that retain the ability
to ribosylate Rho are included in this invention and can be used to
make fusion proteins. The crystal structure of C3 has been
determined giving insight to elements of the C3 protein that could
be changed without affecting ribosylating activity (Han et al.
(2001) J. Mol. Biol. 305: 95).
[0154] The Rho antagonist that is a recombinant proteins can be
made according to methods present in the art. The proteins of the
present invention may be prepared from bacterial cell extracts, or
through the use of recombinant techniques. In general, C3 proteins
according to the invention can be produced by transformation
(transfection, transduction, or infection) of a host cell with all
or part of a C3-encoding DNA fragment in a suitable expression
vehicle. Suitable expression vehicles include: plasmids, viral
particles, and phages. For insect cells, baculovirus expression
vectors are suitable. The entire expression vehicle, or a part
thereof, can be integrated into the host cell genome. In some
circumstances, it is desirable to employ an inducible expression
vector.
[0155] Those skilled in the field of molecular biology will
understand that any of a wide variety of expression systems can be
used to provide the recombinant protein. The precise host cell used
is not critical to the invention. The C3 and C3-like proteins may
be produced in a prokaryotic host (e.g., E. coli or B. subtilis) or
in a eukaryotic host (e.g., Saccharomyces or Pichia; mammalian
cells, e.g., COS, NIH 3T3, CHO, BHK, 293, or HeLa cells; or insect
cells).
[0156] Proteins and polypeptides may also be produced by plant
cells. For plant cells viral expression vectors (e.g., cauliflower
mosaic virus and tobacco mosaic virus) and plasmid expression
vectors (e.g., Ti plasmid) are suitable. Such cells are available
from a wide range of sources (e.g., the American Type Culture
Collection, Rockland, Md.). The methods of transformation or
transfection and the choice of expression vehicle will depend on
the host system selected.
[0157] The host cells harboring the expression vehicle can be
cultured in conventional nutrient media adapted as need for
activation of a chosen gene, repression of a chosen gene, selection
of transformants, or amplification of a chosen gene. One expression
system is the mouse 3T3 fibroblast host cell transfected with a
pMAMneo expression vector (Clontech, Palo Alto, Calif.). pMAMneo
provides an RSV-LTR enhancer linked to a dexamethasone-inducible
MMTV-LTR promotor, an SV40 origin of replication which allows
replication in mammalian systems, a selectable neomycin gene, and
SV40 splicing and polyadenylation sites. DNA encoding a C3 or
C3-like protein would be inserted into the pMAMneo vector in an
orientation designed to allow expression. The recombinant C3 or
C3-like protein would be isolated as described below. Other
preferable host cells that can be used in conjunction with the
pMAMneo expression vehicle include COS cells and CHO cells (ATCC
Accession Nos. CRL 1650 and CCL 61, respectively).
[0158] C3 polypeptides can be produced as fusion proteins. For
example, expression vectors may be used to create lacz fusion
proteins. The pGEX vectors can be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can be
easily purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety. Another strategy to
make fusion proteins is to use the His tag system.
[0159] In an insect cell expression system, Autographa californica
nuclear polyhedrosis virus AcNPV), which grows in Spodoptera
frugiperda cells, is used as a vector to express foreign genes. A
C3 coding sequence can be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter, e.g., the polyhedrin promoter.
Successful insertion of a gene encoding a C3 or C3-like protein
(polypeptide) will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus
lacking the proteinaceous coat encoded by the polyhedrin gene).
These recombinant viruses are then used to infect Spodoptera
frugiperda cells in which the inserted gene is expressed (see,
Lehmann et al for an example of making recombinant MAG
protein).
[0160] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the C3 nucleic acid sequence can be ligated to
an adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
can then be inserted into the adenovirus genome by in vitro or in
vivo recombination. Insertion into a non-essential region of the
viral genome (e.g., region El or E3) will result in a recombinant
virus that is viable and capable of expressing a C3 gene product in
infected hosts.
[0161] Specific initiation signals may also be required for
efficient translation of inserted nucleic acid sequences. These
signals include the ATG initiation codon and adjacent sequences. In
cases where an entire native C3 gene or cDNA, including its own
initiation codon and adjacent sequences, is inserted into the
appropriate expression vector, no additional translational control
signals may be needed. In other cases, exogenous translational
control signals, including, perhaps, the ATG initiation codon, must
be provided. Furthermore, the initiation codon must be in phase
with the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational
control signals and initiation codons can be of a variety of
origins, both natural and synthetic. The efficiency of expression
may be enhanced by the inclusion of appropriate transcription
enhancer elements, transcription terminators.
[0162] In addition, a host cell may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the
gene product in a specific, desired fashion. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of protein
products may be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells that
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited
to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and in
particular, choroid plexus cell lines.
[0163] Alternatively, a C3 protein can be produced by a
stably-transfected mammalian cell line. A number of vectors
suitable for stable transfection of mammalian cells are available
to the public; methods for constructing such cell lines are also
publicly available. In one example, cDNA encoding the C3 protein
may be cloned into an expression vector that includes the
dihydrofolate reductase (DHFR) gene. Integration of the plasmid
and, therefore, the C3 or C3-like protein-encoding gene into the
host cell chromosome is selected for by including 0.01-300 .mu.M
methotrexate in the cell culture medium (as described in Ausubel et
al., supra). This dominant selection can be accomplished in most
cell types. Recombinant protein expression may be increased by
DHFR-mediated amplification of the transfected gene. Methods for
selecting cell lines bearing gene amplifications are known in the
art; such methods generally involve extended culture in medium
containing gradually increasing levels of methotrexate.
DHFR-containing expression vectors commonly used for this purpose
include pCVSEII-DHFR and pAdD26SV(A). Any of the host cells
described above or, preferably, a DHFR-deficient CHO cell line
(e.g., CHO DHFR cells, ATCC Accession No. CRL 9096) are among the
host cells preferred for DHFR selection of a stably-transfected
cell line or DHFR-mediated gene amplification.
[0164] A number of other selection systems may be used, including
but not limited to the herpes simplex virus thymidine kinase,
hypoxanthine-guanine phosphoribosyltransferase, and adenine
phosphoribosyltransferase genes can be employed in tk, hgprt, or
aprt cells, respectively. In addition, gpt, which confer resistance
to mycophenolic acid; neo, which confers resistance to the
aminoglycoside G-418; and hygro, which confers resistance to
hygromycin may be used.
[0165] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described in Janknecht et al.
(1981) Proc. Natl. Acad. Sci. USA 88, 8972, allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines. In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni2+nitriloacetic
acid-agarose columns, and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
[0166] Alternatively, C3, C3-like protein or a portion (fragment)
thereof, can be fused to an immunoglobulin Fc domain. Such a fusion
protein can be readily purified using a protein A column.
[0167] To test Rho antagonists for activity, a tissue culture
bioassay system was used. This bioassay is used to define activity
of Rho antagonists that will be effective in promoting axon
regeneration in spinal cord injury, stroke or neurodegenerative
disease.
[0168] Neurons do not grow neurites on inhibitory myelin
substrates. When neurons are placed on inhibitory substrates in
tissue culture, they remain rounded. When an effective Rho
antagonist is added, the neurons are able to grow neurites on
myelin substrates. The time that it takes for neurons to growth
neurites upon the addition of a Rho antagonist is the same as if
neurons had been plated on growth permissive substrate such as
laminin or polylysine, typically 1 to 2 days in cell culture. The
results can be scored visually. If needed, a quantitative
assessment of neurite growth can be performed. This involved
measuring the neurite length in a) control cultures where neurons
are plated on myelin substrates and left untreated b) in positive
control cultures, such as neurons plated on polylysine c) or
treating cultures with different concentrations of the test
antagonist.
[0169] To test C3 in tissue culture, it has been found that the
best concentration is 25-50 ug/ml. (Lehmann et al, 1999.
J.Neurosci. 19: 7537-7547; Jin & Strittmatter, 1997. J.
Neurosci. 17: 6256-6263). Thus, high concentrations of this Rho
antagonist are needed as compared to the growth factors used to
stimulate neurite outgrowth. Growth factors, such as nerve growth
factor (NGF) are used at concentrations of 1-100 ng/ml in tissue
culture. However, growth factors are not able to overcome growth
inhibition by myelin. Our tissue culture experiments are all
performed in the presence of the growth factor BDNF for retinal
ganglion cells, or NGF for PC-12 cells. When growth factors have
been tested in vivo, typically the highest concentrations possible
are used, in the ug/ml range. Also they are often added to the CNS
with the use of pumps for prolonged delivery (e.g. Ramer et al,
supra). For in vivo experiments the highest concentrations possible
was used when working with C3 stored as a frozen 1 mg/ml
solution.
[0170] The Rho antagonist C3 is stable at 37.degree. C. for at
least 24 hours. The stability of C3 was tested in tissue culture
with the following experiment. The C3 was diluted in tissue culture
medium, left in the incubator at 37.degree. C. for 24 hours, then
added to the bioassay system described above, using retinal
ganglion cells as the test cell type. These cells were able to
extend neurites on inhibitory substrates when treated with C3
stored for 24 hours at 37.degree. C. Therefore, the minimum
stability is 24 hours. This is in keeping with the stability
projection based on amino acid composition (see sequence data,
below).
[0171] A compound can be confirmed as a Rho antagonist in one of
the following ways:
[0172] a) Cells are cultured on a growth inhibitory substrate as
above, and exposed to the candidate Rho antagonist;
[0173] b) Cells of step a) are homogenized and a pull-down assay is
performed. This assay is based on the capability of GST-Rhotektin
to bind to GTP-bound Rho. Recombinant GST-Rhotektin or GST
rhotektin binding domain (GST-RBD) is added to the cell homogenate
made from cells cultured as in a). It has been found that
inhibitory substrates activate Rho, and that this activated Rho is
pulled down by GST-RBD. Rho antagonists will block activation of
Rho, and therefore, an effective Rho antagonist will block the
detection of Rho when cell are cultured as described by a)
above;
[0174] c) An alternate method for this pull-down assay would be to
use the GTPase activating protein, Rho-GAP as bait in the assay to
pull down activated Rho, as described (Diekmann and Hall, 1995. In
Methods in Enzymology Vol. 256 part B 207-215).
[0175] Another method to confirm that a compound is a Rho
antagonist is as follows: When added to living cells antagonists
that inactivate Rho by ADP-ribosylation of the effector domain can
be identified by detecting a molecular weight shift in Rho (Lehmann
et al, 1999 supra). The molecular weight shift can be detected
after treatment of cells with Rho antagonist by homogenizing the
cells, separating the proteins in the cellular homogenate by SDS
polyacrylamide gel electrophoresis. The proteins are transferred to
nitrocellulose paper, then Rho is detected with Rho-specific
antibodies by a Western blotting technique.
[0176] Another method to confirm that compound is a Rho-kinase
antagonist is as follows:
[0177] a) Recombinant Rho kinase tagged with myc epitope tag, or a
GST tag or any suitable tag is expressed in Hela cells or another
suitable cell type by transfection;
[0178] b) The kinase is purified from cell homogenates by
immunoprecipitation using antibodies directed against the specific
tag (e.g., myc tag or the GST tag);
[0179] c) The recovered immunoprecipitates from b) are incubated
with [.sup.32P] ATP and histone type 2 as a substrate in the
presence or absence of the Rho kinase inhibitor. In the absence of
Rho kinase inhibitor activity, the Rho kinase phosphorylated
histone. In the presence of Rho kinase inhibitor the
phosphorylation activity of Rho kinase (i.e. phosphorylation of
histone) is blocked, and as such identified the compound as a Rho
kinase antagonist.
[0180] Turning now to the transport side of the conjugates of the
present invention, known methods are available to add transport
sequences that allow proteins to penetrate into the cell; examples
include membrane translocating sequence (Rojas (1998) 16: 370-375),
Tat-mediated protein delivery (Vives (1997) 272: 16010-16017),
polyargine sequences (Wender et al. 2000, PNAS 24: 13003-13008) and
antennapedia (Derossi (1996) 271: 18188-18193). Examples of known
transport agents, moities, subdomains and the like are also shown
for example in Canadian patent document no. 2,301,157 (conjugates
containing homeodomain of antennapedia) as well as in U.S. Pat.
Nos. 5,652,122, 5,670,617, 5,674,980, 5,747,641, and 5,804,604
(conjugates containing amino acids of Tat HIV protein (hereinafter
Tat HIV protein is sometimes simply referred to as Tat); the entire
contents of each of these patent documents is incorporated herein
by reference.
[0181] A 16 amino acid region of the third alpha-helix of
antennapedia homeodomain has been shown to enable proteins (made as
fusion proteins) to cross cellular membranes (PCT international
publication number WO 99/11809 and Canadian application No.:
2,301,157 (Crisanti et al,) incorporated herein as references).
Here we have generated fusion-proteins comprising C3 and having an
antennapedia homeodomain sequence located at the carboxy-terminal
end of the fusion-protein. The biological activity (e.g., promoting
axon growth) of these fusion proteins was demonstrated on primary
mammalian cells such as neurons. Similarly, HIV Tat protein was
shown to be able to cross cellular membranes (Frankel A. D. et al.,
Cell, 55: 1189). We have shown here using a sequence spanning amino
acid 27 to 72 of HIV Tat, that Tat-mediated delivery of
biologically active C3 protein is possible in neuronal cells and
more specifically, in primary neuronal cells.
[0182] In addition to HIV Tat and antennapedia -mediated transport
of C3 proteins and analogs, new transport sequences (i.e.,
transport polypeptide moiety, transport agent region, etc.) are
presented herein.
[0183] Several receptor-mediated transport strategies have been
used to try and improve function of ADP ribosylases: these methods
include fusing C2 and C3 sequences (Wilde, et al. (2001) 276:
9537-9542.) and use of receptor-mediated transport with the
diphtheria toxin receptor (Aullo, et al. (1993) 12: 921-31; Boquet,
P. et al. (1995) Meth. Enzymol. 256: 297-306).). These methods have
not been demonstrated to dramatically increase the potency of C3.
Moreover, these proteins require receptor-mediated transport. This
means that the cells must express the receptor, and must express
sufficient quantities of the receptor to significantly improve
transport. Moreover, when C3 enters the cell by endocytosis, it
will be locked within a membrane compartment, and therefore most of
it will not be available to inactivate Rho. In the case of
diphtheria toxin, not all cells express the appropriate receptor,
limiting its potential use. The clinical importance for any of
these has not been tested or shown. A C2/C3 fusion protein has also
been made to try and improve the effectiveness of C3. In this case,
the addition of a C2II binding protein to the tissue culture medium
is needed, along with the C2-C3 fusion toxin to allow uptake of C3
by receptor-mediated endocytosis (Barthe et al. (1998) Infection
and Immunity 66:1364). The disadvantage of this system is that much
of the C3 in the cell will be restrained within a membrane
compartment. More importantly, two different proteins must be added
separately for transport to occur (Wahl et al. 2000. J. Cell Biol.
149:263), which make this system difficult to apply to in vivo for
treatment of disease. Moreover, none of the methods to inactivate
Rho with C3 or C3 analogues (C3-like protein) have been
demonstrated to be sufficient to overcome growth inhibition in
tissue culture, or to promote recovery after CNS damage in
vivo.
[0184] One strategy which may be used in accordance with the
present invention is to exploit the antennapedia homeodomain that
is able to transport proteins across the plasma membrane by a
receptor-independent mechanism (Derossi (1996) 271: 18188-18193);
an alternate strategy is to exploit Tat-mediated delivery (Vives
(1997) 272: 16010-16017, Fawell (1994) 91: 664-668, Frankel (1988)
55: 1189-1193).
[0185] The Antennapedia strategy has been used for protein
translocation into neurons (Derossi (1996) 271: 18188-18193).
Antennapedia has, for example, been used to transport
biotin-labeled peptides in order to demonstrate the efficacy of the
technique; see U.S. Pat. No. 6,080,724 (the entire contents of this
patent are incorporated herein by reference). Antennapedia enhances
growth and branching of neurons in vitro (Bloch-Gallego (1993) 120:
485-492). Homeoproteins are transcription factors that regulate
development of body organization, and antennapedia is a Drosophila
homeoprotein. Tat on the other hand is a regulatory protein from
human immunodeficiency virus (HIV). It is a highly basic protein
that is found in the nucleus and can transport reporter genes into
cell. Moreover, Tat-linked proteins can penetrate cells after
intraperitoneal injection, and it can even cross the blood brain
barrier to enter cells within the brain (Schwarze, et al. (1999)
285: 1569-72).
[0186] Other transport sequences that have been tested in other
contexts, (i.e., to show that they work through the use of reporter
sequences), are known. One transport peptide, a 12 mer,
AAVLLPVLLAAP, is rich in proline. It was made as a GST-MTS fusion
protein and is derived from the h region of the Kaposi FGF signal
sequence (Royas et al. 1998 Nature Biotech. 16: 370-375. Another
example is the sperm fertiline alpha peptide,
HPIQIAAFLARIPPISSIGTCILK (This is reviewed in Pecheur, J.
Sainte-Marie, A. Bienvenuie, D. Hoekstra. 1999. J. Membrane Biol.
167: 1-17). It must be noted however that the alpha helix-breaking
propensity of proline (Pro) residues is not a general rule, since
the putative fusion peptide of sperm fertilin alpha displays a high
alpha helical content in the presence of liposomes. However, the
Pro-Pro sequence is required for efficient fusion properties of
fertilin. The C3APLT fusion protein that we tested fits the
requirement of having a two prolines for making an effective
transport peptide. Therefore, proline-rich sequences and random
sequences that have helix-breaking propensity that act as effective
transporters would also be effective if fused to C3.
[0187] In the context of axon growth on inhibitory substrates, axon
regeneration after injury, or axon regeneration in the brain or
spinal cord, no method using these transport sequences has been
devised. In particular, it should be noted that the ability of
antennapedia to enhance growth was tested with neurons placed on
laminin-coated coverslips. Laminin supports axon growth and
overrides growth inhibition (David, et al. (1995) 42: 594-602)
thus, it is not a suitable substrate to test the potential for
regeneration. There is an enormous wealth of literature over the
last 20 years on substances that promote axon growth under such
favorable tissue culture conditions, but none of these has lead to
clinical advances in the treatment of spinal cord injury. The
effect of antennapedia was shown to act as similar to a growth
factors. Growth factors do not overcome growth inhibition by CNS
growth inhibitory substrates (Lehmann, et al. (1999) 19: 7537-7547,
Cai, et al. (1999) 22: 89-101). Growth factors applied in vivo do
not support regeneration, only sprouting (Schnell, et al. (1994)
367: 170-173).
[0188] The transport sequence may be added to the N-terminal
(amino-terminal) sequence of the C3 protein. Alternatively, the
transport sequence may be added on the C-terminal
(carboxy-terminal) end of the C3 protein; because the C-terminal is
already quite basic, this should enhance further the transport
properties. This is likely one of the reasons that C3APLT shows
activity in addition to its basic charge and the proline-rich
sequences.
[0189] The new chimeric C3 may be used to treat spinal cord injury
to promote functional repair. We have demonstrated that both C3APLT
and C3APS can overcome growth inhibition on complex inhibitory
substrates that include myelin and mixed chondroitin sulfate
proteoglycans. Further, we demonstrate that C3APLT can promote
functional recovery after application to injured spinal cord in
adult mice. The new chimeric protein may be used to promote axon
regeneration and reduce scarring after CNS injury. Scarring is a
barrier to nerve regeneration.
[0190] The advantage of the new chimeric C3 is the ability to treat
the injured axons after a significant delay between the injury and
the treatment. Also, the new recombinant protein may be useful in
the treatment of chronic injury. The chimeric C3 can also be used
to treat neurodegenerative diseases such as Alzheimer's disease and
Parkinson's disease where penetration of the Rho antagonist to the
affected neuronal population is required for effective treatment.
The chimeric C3 (fusion proteins) will also be of benefit for the
treatment of stroke and traumatic brain injury. Moreover, much
evidence suggests efficacy in the treatment of cancer cell
migration. Rho antagonists are also useful in the treatment of
disease involving smooth muscle, such as vascular disease,
hypertension, asthma, and penile dysfunction.
[0191] For treatment of spinal cord injury, the conjugate Rho
antagonists of the present invention may be used in conjunction
with cell transplantation. Many different cell transplants have
been extensively tested for their potential to promote regeneration
and repair, including, but not restricted to, Schwann cells,
fibroblasts modified to express growth factors, fetal spinal cord
transplants, macrophages, embryonic or adult stem cells, and
olfactory ensheathing glia. C3 fusion proteins may be used in
conjunction with neurotrophins, apoptosis inhibitors, or other
agents that prevent cell death. They may be used in conjunction
with cell adhesion molecules such as L1, laminin, and artificial
growth matrices that promote axon growth. The chimeric C3
constructs of the present invention may also be used in conjunction
with the use of antibodies that block growth inhibitory protein
substrates to promote axon growth. Examples of such antibody
methods are the use of IN-1 or related antibodies (Schnell and
Schwab (1990) 343: 269-272) or through the use of therapeutic
vaccine approaches (Huang (1999) 24: 639-647).
BRIEF DESCRIPTION OF THE FIGURES
[0192] In drawings which illustrate example embodiments of the
present invention:
[0193] FIG. 1 illustrates the dose response of normal C3 with and
without trituration;
[0194] FIG. 2 illustrates ADP ribosylation by C3APLT and C3APS, but
not C3 after passively adding the compounds to PC-12 cells;
[0195] FIG. 3A illustrates that C3APLT penetrates cells;
[0196] FIG. 3B illustrates a lower level of cell penetration by C3
as compared to FIG. 3A;
[0197] FIG. 4 illustrates the effectiveness of C3APLT and C3APS at
low doses;
[0198] FIG. 5 illustrates the effectiveness of C3APLT and C3APS at
low doses;
[0199] FIG. 6 illustrates the effectiveness of C3APLT to stimulate
axon regeneration of primary neurons;
[0200] FIG. 7 illustrates the effectiveness of C3APLT to promote
functional recovery after spinal cord injury;
[0201] FIG. 8 illustrates effectiveness of Tat transport sequences
to enhance growth as C3-Tat (C3-TL and C3-TS) chimeras;
[0202] FIGS. 9A and 9B illustrate axon regeneration after spinal
cord injury and treatment with C3APLT;
[0203] FIG. 10 illustrates effectiveness of C3APLT to prevent cell
death after spinal cord injury, thereby showing that it is
neuroprotective,
[0204] FIG. 11 illustrates a comparison of C3APLT and C3Basic3 to
promote neurite outgrowth, and;
[0205] FIG. 12 illustrates that C3APLT promotes neurite outgrowth
from retinal neurons plated on inhibitory myelin or CSPG
substrates. Retinal neurons plated on myelin (dark bars) or CSPG
(dotted bars) substrates and treated with C3-05. FIG. 12 A
illustrates the percentage of cells with neurites neurites longer
than 1 cell body diameter (neurite outgrowth); FIG. 12 B
illustrates the length of the longest neurite per cell (neurite
length).
[0206] Referring to FIG. 1, PC-12 cells were plated on inhibitory
myelin substrates (0). Unmodified C3 added to the tissue culture
medium at concentration from 0.00025-50 ug/ml did not significantly
improve neurite outgrowth over the untreated control (grey bars).
C3 was only effective in stimulating neurite outgrowth for cells
plated on myelin substrates after scrape-loading (black bars). This
Figure demonstrates the limited or no penetration in cells when
passively added to the tissue culture medium. Please see Example 4
below for techniques.
[0207] Referring to FIG. 2, this Figure provides a demonstration
that C3APLT and C3APS, ADP ribosylate Rho. Western blot showing
RhoA in untreated cells (lane 1), and cells treated with C3APLT
(lane 2) or C3APS (lane 3). When Rho is ADP ribosylated by C3 it
undergoes a molecular weight shift (Lehmann et al supra), as
observed for lanes 2 and 3. Please see Example 4 below for
techniques.
[0208] Referring to FIG. 3, this Figure shows intracellular
activity after treatment with C3APLT. Detection that the new fusion
C3 penetrates into the cells. Immunocytochemistry with anti-C3
antibody of PC-12 cells plated on myelin and treated with C3 (A) or
C3APLT (13). Cells in A (FIG. 3A) are not immunoreactive because C3
has not penetrated into the cells. Cells in B (FIG. 3B) are
immunoreactive and they are able to extend neurites on myelin
substrates. Please see Example 4 below for techniques.
[0209] Turning to FIG. 4, this Figure shows that C3-antennapedia
fusion proteins promote growth on inhibitory substrates. The
percent of neurons that grow neurites was counted for each
treatment. The dose response experiment shows that C3APLT and C3APS
promote more neurite growth per cell than control PC-12 cells
plated on myelin (0). PC-12 cells were plated on myelin and either
scrape loaded with unmodified C3 (C3 50) left untreated (0) or
treated with various concentrations of C3APLT. Compared to C3 used
at 25 ug/ml, C3APS is effective at stimulating more cells to grow
neurites at 0.0025 ug/ml, a dose 10,000 X less. Please see Example
4 below for techniques.
[0210] FIG. 5 shows a dose-response experiment showing that C3APLT
and C3APS elicit long neurites to grow when cells are plated on
inhibitory substrates. The length of neurites was measured for each
treatment. PC-12 cells were plated on myelin and either scrape
loaded with unmodified C3 (C3 50) left untreated (0) or treated
with various concentrations of C3APLT. Compared to C3 used at 25
ug/ml, C3APS is effective at stimulating more cells to longer
neurite growth at 0.0025 ug/ml, a dose 10,000.times. less. Please
see Example 4 below for techniques.
[0211] As may be seen FIG. 6 shows primary neurons growing on
inhibitory substrates after treatment with C3APLT. Rat retinal
ganglion cells were plated on myelin substrates and treated with
different concentrations of C3APLT. Concentrations of 0.025 and
above promoted significantly longer neurites. This dose is
1000.times. lower than that of C3 needed to promote growth on
myelin.
[0212] Referring to FIG. 7, this Figure shows behavioral recovery
after treatment of adult mice with C3APLT in a dose-response
experiment. Mice received a dorsal hemisection of the spinal cord
and were left untreated (transection), were treated with fibrin
alone (fibrin) or were treated with fibrin plus C3APLT at the
indicated concentrations given in ug/mouse. Each point represents
one animal. The BBB score (see Example 6 for details) was assessed
24 hours after treatment. Animals treated with C3APLT exhibited a
significant improvement in behavioral recovery compared to
untreated animals. The effective dose of 0.5 .mu.g is 100.times.
less than unmodified C3 used (see previous experiment shown in
Canadian patent application 2,325,842). Please see Example 6.
[0213] Referring to FIG. 8, this Figure shows promotion of axon
growth by C3-Tat chimeric proteins. The dose-response experiment
shows that C3-TS and C3-TL promote more neurite growth per cell
than control PC-12 cells plated on myelin. PC-12 cells were plated
on myelin and either scrape loaded with unmodified C3 (scrape load)
left untreated (myelin) or treated with various concentrations of
C3-TS (grey bars) or C3-TL (black bars). Compared to C3 used at 25
ug/ml, C3-TL is effective at stimulating more cells to grow
neurites at 0.0025 ug/ml, a dose 10,000.times. less than C3.
[0214] Referring to FIGS. 9A and 9B, these Figures show axon
regeneration in injured spinal cord, i.e. anatomical regeneration
after treatment with C3APLT. Section of the spinal cord after
anterograde labeling with horseradish peroxidase conjugated to
wheat germ agglutinin (WGA-HRP). A) Sprouting of cut axons into the
dorsal white matter. Arrows show regenerating axons distal to the
lesion. B) Same section 3 mm from the lesion site. Arrows show
regenerating axons.
[0215] Referring to FIG. 10, this Figure shows that C3-APLT
protected neurons from cell death following spinal cord injury.
Apoptotic (dying) cells were counted following TUNEL labeling (see
Example 16) 2 mm rostral to the lesion (Rostral) at the lesion site
(lesion) and 2 mm caudal to the lesion site (caudal). Bars show
average counts of Tunel positive cells from 4 animals treated with
fibrin only after spinal cord injury as control (white bars), or
with C3APLT in fibrin at 1 .mu.g (black bars). Treatment with
C3APLT show significantly reduced numbers of Tunel-labeled cells
(dying cells). Non-injured spinal cord samples were also processed
and these spinal cords did not show Tunel labeling, as
expected.
[0216] Referring to FIG. 11, this Figure shows that C3APLT and
C3Basic3 promote rapid neurite outgrowth compared to untreated
cells when cells are plated on plastic as part of a rapid bioassay
(see Example 4).
[0217] Referring to FIGS. 12A and 12B, to further support the
ability of C3-like chimeric proteins to promote neurite outgrowth
on inhibitory substrates, we examined the response of primary
cultures plated on inhibitory substrates to C3APLT treatment.
Purified retinal ganglion cells (RGCs) were plated on myelin, or
CSPG substrates and treated with varying concentrations of C3APLT.
During the RGC dissection great care was taken in order to try to
limit the amount of mechanical manipulation of the cells, however,
the isolation protocol requires that some triturating take place in
order to dissociate and separate the cells. When RGCs are plated on
inhibitory substrates, they maintained a similar round appearance
to PC-12 cells plated on myelin. Treatment of RGCs with C3APLT
promoted neurite outgrowth and increased neurite length on both
myelin and CSPG substrates. In contrast to the wide range of
concentrations shown to be effective in other PC-12 experiments a
narrower range of C3APLT treatment, 0.025 ug/ml to 50 ug/ml
promoted neurite outgrowth and increased neurite length on myelin.
In the case of RGCs plated on CSPG substrates, effective
concentration ranges of 0.0025 ug/ml to 50 ug/ml were observed.
DETAILED DESCRIPTION
Method for Making the C3APL, C3APLT, and C3APS
[0218] C3APL is the name given to the protein made by ligating a
cDNA encoding C3 (Dillon and Feig (1995) 256: 174-184) with cDNA
encoding the antennapedia homeodomain (Bloch-Gallego (1993) 120:
485-492). The stop codon at the 3' end of the DNA was replaced with
an EcoR I site by polymerase chain reaction (PCR) using the primers
(oligonucleotides) 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3' (SEQ ID
NO: 1) and 5'GGT GGC GAC CAT CCT CCA AAA 3' (SEQ ID NO: 2). The PCR
product was sub-cloned into a pSTBlue-1 vector (Novagen, city),
then cloned into a pGEX-4T vector using BamH I and Not I
restriction site. This vector was called pGEX-4T/C3. The
antennapedia sequence used to add to the 3' end of C3 in pGEX-4T/C3
was created by PCR from the pET-3a vector (Bloch-Gallego (1993)
120: 485-492, Derossi (1994) 269: 10444-10450), subcloned into a
pSTBlue-1 blunt vector, then cloned into the pGEX-4T/C3, using the
restriction sites EcoR I and Sal I, creating pGEX-4T/C3APL. Another
clone (C3APLT) with a frameshift mutation was selected, and the
protein made and tested. When the cultures tested positive despite
the mutation, the clone was resequenced by another company to
confirm the mutation, and this clone was called C3APLT. To confirm
the sequence of C3APLT, the coding sequence from both strands was
sequenced. The sequence for this clone is given in Examples 16 and
17 (nucleotide sequence of C3APLT; SEQ ID NO: 42, amino acid
sequence of C3APLT; SEQ ID NO: 43).
[0219] A shorter version of the Antennapedia (pGEX-4T/C3APS) was
also made. This chimeric sequence was made by ligating
oligonucleotides encoding the short antennapedia peptide (Maizel
(1999) 126: 3183-3190) into the pGEX-4T/C3 vector cut with EcoR I
and Sal I. The recombinant C3APLT and C3APS cDNAs were separately
transformed into bacteria, and after the recombinant proteins were
produced, a bacterial homogenate was obtained by sonication, and
the homogenate cleared by centrifugation. Glutathione-agarose beads
(Sigma) were added to the cleared lysate and placed on a rotating
plate for 2-3 hours, then washed extensively. To remove the
glutathione S transferase sequence from the recombinant protein, 20
U (unit) of Thrombin was added, the beads were left on a rotator
overnight at 4.degree. C. After cleavage with thrombin, the beads
were loaded into an empty 20 ml column, and the proteins eluted
with PBS (phosphate buffered saline). Aliquots containing
recombinant protein were pooled and 100 .mu.l p-aminobenzamidine
agarose beads (Sigma) were added and left mixing for 45 minutes at
4.degree. C. to remove thrombin, then recombinant protein was
isolated from the beads by centrifugation. Purity of the sample was
determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), and bioactivity bioassay with PC-12
cells was performed (See Lehmann et al supra).
[0220] Other possible methods for making bioactive chimeric
proteins include anion exchange chromatography. For this, the GST
tag is not required and can be removed. The cDNA can then be cloned
into a high expression bacterial vector, such as pET, as given in
Example 16.
[0221] The Rho antagonist is a recombinant protein and can be made
according to methods present in the art. The proteins of the
present invention may be prepared from bacterial cell extracts, or
through the use of recombinant techniques by transformation,
transfection, or infection of a host cell with all or part of a
C3-encoding DNA fragment with an antennapedia-derived transport
sequence in a suitable expression vehicle. Those skilled in the
field of molecular biology will understand that any of a wide
variety of expression systems can be used to provide the
recombinant protein. The precise host cell used is not critical to
the invention.
[0222] Any fusion protein can be readily purified by utilizing
either affinity purification techniques or more traditional column
chromatography. Affinity techniques include, but are not restricted
to GST (gluathionie-S-transferase), or the use of an antibody
specific for the fusion protein being expressed, or the use of a
histidine tag. Alternatively, recombinant protein can be fused to
an immunoglobulin Fc domain. Such a fusion protein can be readily
purified using a protein A column. It is envisioned that small
molecule mimetics of the above-described antagonists are also
encompassed by the invention.
[0223] Testing the Bioactivity of C3APLT, C3APS, C3-TL and
C3-TS
[0224] To test the efficacy of C3APLT, C3APS, C3-TL and C3-TS a
number of experiments were performed with PC-12 cells, a neural
cell line, grown on growth inhibitory substrates (see Lehmann et al
supra). PC-12 cells were plated on myelin substrates as described
(Lehmann et al, supra). C3, C3APLT, C3APS, C3-TL or C3-TS were
added at different concentrations without trituration (please refer
to FIGS. 4, 5 and 8 for concentrations used). C3 added passively to
the culture medium in this way was not able to promote neurite
growth in the growth inhibitory substrates because cells must be
triturated for C3 to enter the cells and be active (FIG. 1). Both
C3APLT and C3APS were able to ADP ribosylate Rho to cause a shift
in the molecular weight of RhoA (FIG. 2). Both C3APLT and C3APS
were able to promote neurite growth and enter neurons after being
added passively to the culture medium (FIG. 3, FIGS. 4 and 5).
Dose-response experiment where concentrations of 0.25 ng/ml, 2.5
ng/ml, 25 ng/ml, 250 ng/ml and 2.5 .mu.g/ml and 25 .mu.g/ml were
tested and showed that C3APLT and C3APS helped more neurons
differentiate neurites at doses 10,000 fold less than C3 (FIG. 4).
Dose response experiments where concentrations of 0.25 ng/ml, 2.5
ng/ml, 25 ng/ml, 250 ng/ml and 2.5 .mu.g/ml and 25 .mu.g/ml were
tested and showed that C3APLT was able to promote long neurite
growth when added at a minimum concentration of 0.0025 ug/ml (FIG.
5). These concentrations of 2.5 ng/ml and 25 ng/ml for C3APLT and
C3APS, represent 10,000 and 1,000 times less than the dose needed
with C3, respectively. Moreover, at the highest concentration
tested, 50 ug/ml, these two new Rho antagonists did not exhibit
toxic effects on PC-12 cells, and were able to stimulate neurite
outgrowth on growth inhibitory substrates.
[0225] C3-TL and C3-TS also were tested at concentrations of 0.25
ng/ml, 2.5 ng/ml, 25 ng/ml, 250 ng/ml and 2.5 .mu.g/ml and 25
.mu.g/ml and were found to be able to promote neurite growth on
myelin substrates at doses significantly less than C3 (FIG. 8).
C3Basic3 was tested at 50 ug/ml in a fast growth assay (FIG.
11).
[0226] To verify the ability of C3APLT and C3APS to promote growth
from primary neurons, primary retinal cultures were prepared, and
the neurons were plated on myelin substrates as described with
respect to Example 5. In the absence of treatment with C3APLT or
C3APS, the cells remained round and were not able to grow neurites.
When treated with C3APLT or C3APS, retinal neurons were able to
extend long neurites on inhibitory myelin substrates (FIG. 6).
[0227] Next, was tested the ability of C3APLT and C3APS to promote
growth on a different type of growth inhibitory substrate relevant
to the type of growth inhibitory proteins found at glial scars.
Chamber slides were coated with a mixture of chondroitin sulfate
proteoglycans (Chemicon), and then plated with retinal neurons
(results presented in FIG. 12). The neurons were not able to extend
neurites on the proteoglycan substrates, but when treated with
C3APLT or C3APS, they extended long neurites. These studies
demonstrate that C3APLT and C3APS can be used to promote neurite
growth on myelin and on proteoglycans, the major classes of
inhibitory substrates that prevent repair after injury in the
CNS.
[0228] Testing Ability of C3APLT to Promote Regeneration and
Functional Recovery after Spinal Cord Injury
[0229] To test if C3APLT could promote repair after spinal cord
injury, fully adult mice were used (as described with respect to
Example 6). A dorsal hemisection was made at T8 (thoracic spinal
level 8), and mice were treated with different amounts (FIG. 7) of
C3APLT in a fibrin glue as described (McKerracher, US patent
pending (delivery patent)). In previous known experiments with C3,
it was found that 40-50 .mu.g was needed to promote anatomical
regeneration in optic nerve (Lehmann et all supra). We tested
different doses (see FIG. 7) of C3APLT ranging from 1 .mu.g to 50
.mu.g and assessed animals for behavioral recovery according the
BBB scale (Basso (1995) 12: 1-21).
[0230] The day following surgery and application of C3APLT,
behavioral testing began. The animals were placed in an open field
environment that consisted of a rubber mat approximately
4'.times.3' in size. The animals were left to move randomly, the
movement of the animals were videotaped. For each test two
observers scored the animals for ability to move ankle, knee and
hip joints in the early phase of recovery. Previously C3 treatment
of mice was seen to lead to functional recovery observable 24 hours
after treatment. In mice treated with C3APLT, functional recovery
could be observed as early as 24 hours after spinal cord injury
(FIG. 7). Untreated mice exhibit a function recovery score
according to the BBB scale averaging 0, whereas mice treated with
C3 are able to walk and have a BBB score averaging 8 (FIG. 7). At
higher concentrations of 50 ug, about 50% of the mice treated with
C3APLT died within 24 hours. However, of the mice that survived,
they exhibited good long-term functional recovery. These results
demonstrate that C3APLT effectively promotes functional recovery
early after spinal cord injury, and that it is effective at much
lower doses than C3. However, at high concentrations, C3APLT
appears to exhibit toxicity, and therefore careful doing will be
required for clinical use.
[0231] Qualitative observations of the videotapes showed that only
animals that received C3APLT reached the late phase of recovery
after 30 days of treatment. Untreated control animals did not
typically pass beyond the early phase of recovery. These results
indicate that the application of C3APLT improved long-term
functional recovery after spinal cord injury compared to no
treatment, injury alone, or fibrin adhesive alone.
[0232] To test if the early recovery was due to neuroprotection,
spinal cord sections were examined for apoptosis by Tunel labeling
following manufacturer's instruction (Roche Diagnostic). C3APLT was
able to reduce the number of dying cells observed at the lesion
site. Therefore, C3APLT should be an effective neuroprotective
agent for treatment of ischemia, such as follows stroke.
EXAMPLE 1
Dna and Protein Sequence Details of C3APL
[0233] Nucleotide Sequence of C3APL
[0234] It has been reported that the long version of antennapedia
transport sequence can enhance neurite growth
(Bloch-Gallego,E.,LeRoux,I.- , Joliot,A. H., Volovitch,M.,
Henderson,C. E., Prochiantz,A. 1993. J. Cell Biol. 120:485).
Therefore, this sequence is expected to enhance neurite growth. For
the sequence given below, the start site, is in the GST sequence of
the plasmid (not shown). The vector with the GST sequence is
commercially available and thus the entire GST sequence including
the start was not sequenced. It was desired to determine only the
sequence located 3' to the thrombin cleavage site which releases C3
conjugate from the GST sequence. The GST sequence is cleaved with
thrombin.
[0235] The APL transport sequence (SEQ ID NO.: 44) is as
follows:
[0236]
VMESRKRARQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN
[0237] Nucleotide Sequence of C3APL (SEQ ID NO: 3)
3 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTC TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
CAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC GTG ATG GAA TCC CGC
AAA CGC GCA AGG CAG ACA TAC ACC CGG TAC CAG ACT CTA GAG CTA GAG AAG
GAG TTT CAC TTC AAT CGC TAC TTG ACC CGT CGG CGA AGG ATC GAG ATC GCC
CAC GCC CTG TGC CTC ACG GAG CGC CAG ATA AAG ATT TGG TTC CAG AAT CGG
CGC ATG AAG TGG AAG AAG GAG AAC TGA 3'
[0238] Amino Acid Sequence of C3APL (SEQ ID NO: 4)
4 GSSRVDLQACNAYSINQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KEAIVSYTKSASEINGKLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPE
NIMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGY
ISTSLMNVSQFAGRPIITQFKVAKGSKAGYIDPISAFQGQLEMLLPRHST
YHIDDMRLSSDGKQIIITATMMGTAINPKEFVMESRKRARQTYTRYQTLE
LEKEFHFNRYLTRRRRIEIAHALCLTERQIKIWFQNRRMKWKKEN
[0239] Physical Characteristics of C3APL
[0240] Molecular Weight 34098.03 Daltons
[0241] 295 Amino Acids
[0242] 48 Strongly Basic(+) Amino Acids (K,R)
[0243] 28 Strongly Acidic(-) Amino Acids (D,E)
[0244] 89 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0245] 94 Polar Amino Acids (N,C,Q,S,T,Y)
[0246] 9.847 Isolectric Point
[0247] 20.524 Charge at PH 7.0
[0248] Davis,Botstein,Roth Melting Temp C. 79.48
EXAMPLE 2
DNA and Protein Sequence Details of C3APS
[0249] Nucleotide sequence of C3APS (SEQ ID NO: 5). The start site,
is in the GST sequence of the plasmid, not shown here.
5 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTC TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
CAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC CGC CAG ATC AAG ATT
TGG TTC CAG AAT CGT CGC ATG AAG TGG AAG AAG GTC GAC TCG AGC GGC CGC
ATC GTG ACT GAC TGA 3'
[0250] The APS transport sequence (SEQ ID NO.: 45) is as
follows:
[0251] RQIKIWFQNRRMKWKKVDS
[0252] Amino Acid Sequence for C3APS (SEQ ID NO: 6)
6 GSSRVDLQACNAYSINQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KEAIVSYTKSASEINGKLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPE
NIMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGY
ISTSLMNVSQFAGRPIITQFKVAKGSKAGYIDPISAFQGQLEMLLPRHST
YHIDDMRLSSDGKQIIITATMMGTAINPKEFRQIKIWFQNRRMKWKKVDS SGRIVTD
[0253] Physical Characteristics of C3APS
[0254] Molecular Weight 29088.22 Daltons
[0255] 257 Amino Acids
[0256] 38 Strongly Basic(+) Amino Acids (K,R)
[0257] 23 Strongly Acidic(-) Amino Acids (D,E)
[0258] 79 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0259] 83 Polar Amino Acids (N,C,Q,S,T,Y)
[0260] 9.745 Isolectric Point
[0261] 15.211 Charge at PH 7.0
[0262] Davis,Botstein,Roth Melting Temp C. 78.34
EXAMPLE 3
Method for Making the C3APLT and C3APS Proteins
[0263] C3APL (amino acid sequence: SEQ ID NO.: 4) and C3APLT (amino
acid sequence; SEQ ID NO: 37) are the names given to the proteins
encoded by cDNAs made by ligating the functional domain of C3
transferase and the homeobox region of the transcription factor
called antennapedia (Bloch-Gallego (1993) 120: 485-492) in the
following way. A cDNA encoding C3 (Dillon and Feig (1995) 256:
174-184) cloned in the plasmid vector pGEX-2T was used for the C3
portion of the chimeric protein. The stop codon at the 3' end of
the DNA was replaced with an EcoR I site by polymerase chain
reaction using the primers 5'GAA TTC TTT AGG ATT GAT AGC TGT GCC 3'
(SEQ ID NO: 1) and 5'GGT GGC GAC CAT CCT CCA AAA 3' (SEQ ID NO: 2).
The PCR product was sub-cloned into a pSTBlue-1 vector (Novagen,
city), then cloned into a pGEX-4T vector using BamH I and Not I
restriction site. This vector was called pGEX-4T/C3. The pGEX-4T
vector has a 5' glutathione S transferase (GST) sequence for use in
affinity purification. The antennapedia sequence used to add to the
3' end of C3 in pGEX-4T/C3 was created by PCR from the pET-3a
vector (Bloch-Gallego (1993) 120: 485-492, Derossi (1994) 269:
10444-10450). The primers used were 5'GAA TCC CGC AAA CGC GCA AGG
CAG 3' (SEQ ID NO: 7) and 5'TCA GTT CTC CTT CTT CCA CTT CAT GCG 3'
(SEQ ID NO: 8). The PCR product obtained from the reaction was
subcloned into a pSTBlue-1 blunt vector, then cloned into the
pGEX-4T/C3, using the restriction sites EcoR I and Sal I, creating
pGEX-4T/C3APL and C3APLT. C3APLT was selected for the presence of a
frameshift mutation giving a transport region moiety rich in
prolines.
[0264] A shorter version of the antennapedia (pGEX-4T/C3AP-short)
(amino acid sequence of C3APS; SEQ ID NO.: 6) was also made. This
chimeric sequence was made by ligating oligonucleotides encoding
the short antennapedia peptide (Maizel (1999) 126: 3183-3190) into
the pGEX-4T/C3 vector cut with EcoR I and Sal I. For
pGEX-4T/C3AP-short the sequences of the oligos made were 5'AAT TCC
GCC AGA TCA AGA TTT GGT TCC AGA ATC GTC GCA TGA AGT GGA AGA AGG 3'
(SEQ ID NO: 9) and 5'GGC GGT CTA GTT CTA AAC CAA GCT CTT AGC AGC
GTA GTT CAC CTT CTT CCA GCT 3' (SEQ ID NO: 10). The two strands
were annealed together by mixing equal amounts of the
oligonucleotides, heating at 72.degree. C. for 5 minutes and then
leaving them at room temperature for 15 minutes. The
oligonucleotides were ligated into the pGEX4T/C3 vector and clones
were picked and analyzed.
[0265] To prepare recombinant C3APLT (SEQ ID NO.: 37) and C3APS
(SEQ ID NO.: 6) proteins, the plasmids containing the corresponding
cDNAs (pGEX-4T/C3APLT and pGEX-4T/C3AP-short) were transformed into
bacteria, strain XL-1 blue competent E. coli. The bacteria were
grown in L-broth (10 g/L Bacto-Tryptone, 5 g/L Yeast Extract, 10
g/L NaCl) with ampicillin at 50 ug/ml (BMC-Roche), in a shaking
incubator for 1 hr at 37.degree. C. and 300 rpm. Isopropyl
.beta.-D-thiogalactopyranoside (IPTG), (Gibco) was added to a final
concentration of 0.5 mM to induce the production of recombinant
protein and the culture was grown for a further 6 hours at
37.degree. C. and 250 rpm. Bacteria pellets were obtained by
centrifugation in 250 ml centrifige bottles at 7000 rpm for 6
minutes at 4.degree. C. Each pellet was re-suspended in 10 ml of
Buffer A (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM
DTT) plus 1 mM PMSF. All re-suspended pellets were pooled and
transferred to a 100 ml plastic beaker on ice. The remaining Buffer
A with PMSF was added to the pooled sample. The bacteria sample was
sonicated 6.times.20 seconds using a Branson Sonifier 450 probe
sonicator. Both the bacteria and probe were cooled on ice 1 minute
between sonications. The sonicate was centrifuged in a Sorvall
SS-34 rotor at 16,000 rpm for 12 minutes at 4.degree. C. to clarify
the supernatant. The supernatant was transferred into fresh SS-34
tubes and re-spun at 12,000 rpm for 12 minutes at 4.degree. C. Up
to 20 ml of Glutathione-agarose beads (Sigma) were added to the
cleared lysate and placed on a rotating plate for 2-3 hours. The
beads were washed 4 times with buffer B, (Buffer A, NaCl is 150 mM,
no PSMF) then 2 times with Buffer C (Buffer B+2.5 mM CaCl.sub.2).
The final wash was poured out till the beads created a thick
slurry. To remove the glutathione S transferase sequence from the
recombinant protein, 20 U of Thrombin (Bovine, Plasminogen-free,
Calbiochem) was added, the beads were left on a rotator overnight
at 4.degree. C. After cleavage with thrombin the beads were loaded
into an empty 20 ml column. Approximately 20 aliquots of 1 ml were
collected by elution with PBS. Samples of each aliquot of 0.5 ul
were spotted on nitrocellulose and stained with Amido Black to
determine the protein peak. Aliquots containing fusion proteins
were pooled and 100 .mu.l p-aminobenzamidine agarose beads (Sigma)
were added and left mixing for 45 minutes at 4.degree. C. This last
step removed the thrombin from the recombinant protein sample. The
recombinant protein was centrifuged to remove the beads and then
concentrated using a centriprep-10 concentrator (Amicon). The
concentrated recombinant protein was desalted with a PD-10 column
(Pharmacia, containing Sephadex G-25M) and ten 0.5 ml aliquots were
collected. A dot-blot was done on these samples to determine the
protein peak, and the appropriate aliquots pooled,
filter-sterilized, and stored at -80.degree. C. A protein assay (DC
assay, Biorad) was used to determine the concentration of
recombinant protein. Purity of the sample was determined by
SDS-PAGE, and bioactivity bioassay with PC-12 cells.
EXAMPLE 4
Testing of Efficacy of C3APLT and C3APS in Tissue Culture
[0266] To test the ability of C3APLT and C3APS to overcome growth
inhibition, PC-12 cells were plated on myelin, a growth inhibitory
substrate. The myelin was purified from bovine brain (Norton and
Poduslo (1973) 21: 749-757). In some other experiments chondroitin
sulfate proteoglycan (CSPG) substrates were made from a purchased
protein composition (Chemicon). Before coating coverslips or wells
of a 96 well plate, they were coated with poly-L-lysine (0.025
.mu.g/ml) (Sigma, St. Louis, Mo.), washed with water and allowed to
dry. Myelin stored as a 1 mg/ml solution at -80.degree. C. was
thawed at 37.degree. C., and vortexed. The myelin was plated at 8
ug/well in a 8 well chamber Lab-Tek slides (Nuc, Naperville, Ill.).
The myelin solution was left to dry overnight in a sterile tissue
culture hood. The next morning the substrate was washed gently with
phosphate buffered saline, and then cells in media were added to
the substrate. PC-12 cells (Lehmann et al., 1999) were grown in
DMEM with 10% horse serum (HS) and 5% fetal bovine serum (FBS). Two
days prior to use the PC-12 cells were differentiated by 50 ng/ml
of nerve growth factor (NGF). After the cells were primed, 5 ml of
trypsin was added to the culture dish to detach the cells, the
cells were pelleted and re-suspended in 2 ml of DMEM with 1% HS and
50 ng/ml of nerve growth factor. Approximately, 5000 to 7000 cells
were then plated on 8 well chamber Lab-Tek slides (Nuc, Naperville,
Ill.) coated myelin. The cells were placed on the test substrates
at 37.degree. C. for 3-4 hours to allow the cells to settle. The
original media was carefully removed by aspiration, taking care not
to disrupt the cells and replaced with DMEM with 1% HS, 50 ng/ml of
NGF and varying amounts of the C3, C3APLT, or C3APS, depending on
the dose desired. After two days, the cells were fixed (4%
paraformaldehyde and 0.5% glutaraldehyde). For control experiments
with unmodified C3, NGF primed PC-12 cells were trypsinized to
detach them from the culture dish, the cells were washed once with
scrape loading buffer (114 mM KCL, 15 mM NaCl, 5.5 mM MgCl.sub.2,
and 10 mM Tris-HCL) and then the cells were scraped with a rubber
policeman into 0.5 ml of scraping buffer in the presence of 25 or
50 .mu.g/ml of C3. The cells were pelleted and resuspended in 2 ml
of DMEM, 1% HS and 50 ng/ml nerve growth factor before plating. At
least four experiments were analyzed for each treatment. For each
well, twelve images were collected with a 20.times. objective using
a Zeiss Axiovert microscope. For each image, the numbers of cells
with and without neurites were counted and the lengths of the
neurites were determined. Since myelin is phase dense, cells plated
on myelin . substrates were immuno-stained with anti-.beta.III
tubulin antibody before analysis. Quantitative analysis of neurite
outgrowth was with the aid of Northern Eclipse software (Empix
Imaging, Mississauga, Ontario, Canada). Data analysis and
statistics were with Microsoft Excel.
[0267] For a fast bioassay, the compounds were tested in tissue
culture as described above, except that the cells were plated on
the tissue culture plastic rather than on inhibitory substrates.
For these experiments the plates were fixed and the neurites
counted five hours after plating the cells. The test compounds
(C3APLT and C3Basic3) were able to promote faster growth on tissue
culture plastic than cells plated without treatment (FIG. 11).
[0268] To examine ADP ribosylation by C3, C3APLT, and C3APS, the
compounds were added to PC-12 cell cultures, as described above.
The cells were harvested by centrifugation, cell homogenates
prepared and the proteins separated by SDS polyacrylamide gel
electrophoresis. The proteins were then transferred to
nitrocellulose and the Western blots probed with anti-RhoA antibody
(Santa Cruz).
EXAMPLE 5
Testing Ability of C3APLT and C3APS to Override Inhibition of
Multiple Growth Inhibitory Proteins
[0269] Myelin substrates were made as described in Example 4 and
plated on tissue culture chamber slides. P1 to P3 rat pups were
decapitated, the heads washed in ethanol and the eye removed and
placed in a petri dish with Hanks buffered saline solution (HBSS,
from Gibco). A hole was cut in the cornea, the lens removed, and
the retina squeezed out. Typically, four retinas per preparation
were used. The retinas were removed to a 15 ml tube and the volume
brought to 7 ml. A further 7 ml of dissociation enzymes and papain
were added. The dissociation enzyme solution was made as follows:
30 mg DL cysteine was added to a 15 ml tube (Sigma DL cystein
hydrochloride), and 70 ml HBSS, 280 ul of 10 mg.ml bovine serum
albumin were added and the solution mixed and pH adjusted to 7 with
0.3 N NaOH. The dissociate solution was filter-sterilized and kept
frozen in 7 ml aliquots, and before use 12.5 units papain per ml
(Worthington) was added. After adding the dissociation solution to
the retina, the tube was incubated for 30 minutes on a rocking tray
at 37.degree. C. The retinas were then gently triturated,
centrifuged and washed with HBSS. The HBSS was replaced with growth
medium (DMEM (Gibco), 10% fetal bovine serum, and 50 ng/ml brain
derived neurotrophic factor (BDNF) vitamins,
penicillin-streptomycin, in the presence or absence of C3APLT or
C3APS. Cells were plated on test substrates of myelin or CSPG in
chamber slides prepared as described in Example 4, above. A
quantitative analysis was completed as described for Example 4
above. Neurons were visualized by fluorescent microscopy with
anti-.beta.III tubulin antibody, which detects growing retinal
ganglion cells (RGCs). Results are presented in FIG. 6.
EXAMPLE 6
Treatment of Injured Mouse Spinal Cord with C3APLT and Measurement
of Recovery of Motor Function in Treated Mice
[0270] Adult Balb-c mice were anaesthetized with 0.6 ml/kg hypnorm,
2.5 mg/kg diazepam and 35 mg/kg ketamine. This does gives about 30
minutes of anaesthetic, which is sufficient for the entire
operation. A segment of the thoracic spinal column was exposed by
removing the vertebrae and spinus process with microrongeurs (Fine
Science Tools). A spinal cord lesion was then made dorsally,
extending past the central canal with fine scissors, and the lesion
was recut with a fine knife. This lesion renders all of the control
animals paraplegic. The paravertebral muscle were closed with
reabsorbable sutures, and the skin was closed with 2.0 silk
sutures. After surgery, the bladder was manually voided every 8-10
hours until the animals regained control, typically 2-3 days. Food
was placed in the cage for easy access, and sponge-water used for
easy accessibility of water after surgery. Also, animals received
subcutaneous injection Buprenorphine (0.05 to 0.1 mg/kg) every 8-12
hours for the first 3 days. Any animals that lost 15-20% of body
weight were killed.
[0271] Rho antagonists (C3 or C3-like proteins) were delivered
locally to the site of the lesion by a fibrin-based tissue adhesive
delivery system (McKerracher, Canadian patent application No.
2,325,842). Recombinant C3APLT was mixed with fibrinogen and
thrombin in the presence of CaCl.sub.2. Fibrinogen is cleaved by
thrombin, and the resulting fibrin monomers polymerize into a
three-dimensional matrix. We added C3APLT as part of a fibrin
adhesive, which polymerized within about 10 seconds after being
placed in the injured spinal cord. We tested C3APLT applied to the
spinal cord lesion site after the lesion was made. For control we
injected fibrin adhesive alone, or transected the cord without
further treatment. For behavioral testing, the BBB scoring method
was used to examine locomotion in an open field environment (Basso
(1995) 12: 1-21). Results are presented in FIG. 7. The environment
was a rubber mat approximately 4'.times.3' in size, and animals
were placed on the mat and videotaped for about 4 minutes. Care was
taken not to stimulate the peroneal region or touch the animals
excessively during the taping session. The video tapes were
digitized and observed by two observers to assign BBB scores. The
BBB score, modified for mice, was as follows:
7 Score Description 1 No observable hindlimb (HL) movement. 2
Slight movement of one or two joints. 3 Extensive movement of one
joint and/or slight movement of one other joint. 4 Extensive
movement of two joints. 5 Slight movement of all three joints of
the HL. 6 Slight movement of two joints and extensive movement of
the third. 7 Extensive movement of two joints and slight movement
of the third. 8 Extensive movement of all three joints of the HL
walking with no weight support. 9 Extensive movement of all three
joints, walking with weight support. 10 Frequent to consistent
dorsal stepping with weight support. 11 Frequent plantar stepping
with weight support. 12 Consistent plantar stepping with weight
support, no coordination. 13 Consistent plantar stepping with
consistent weight support, occasional FL-HL coordination. 14
Consistent plantar stepping with consistent weight support,
frequent FL-HL coordination. 15 Consistent plantar stepping with
consistent weight support, consistent FL-HL coordination;
predominant paw position during locomotion is rotated internally or
externally, or consistent FL-HL coordination with occasional dorsal
stepping. 16 Consistent plantar stepping with consistent weight
support, consistent FL-HL coordination; predominant paw position is
parallel to the body; frequent to consistent toe drag, or curled
toes, trunk instability. 17 Consistent plantar stepping with
consistent weight support, consistent FL-HL coordination;
predominant paw position is parallel to the body, no toe drag, some
trunk instability. 18 Consistent plantar stepping with consistent
weight support, consistent FL-HL coordination; predominant paw
position is parallel to the body, no toe drag and consistent
stability in the locomotion.
EXAMPLE 7
Treatment of Injured Mouse Spinal Cord with C3APLT and Assessment
of Anatomical Recovery
[0272] Mice that received a spinal cord injury and treated as
controls or with C3APLT, as described for Example 6 were assessed
for morphological changes to the scar and for axon regeneration. To
study axon regeneration, the corticospinal axons were identified by
anterograde labeling. For anterograde labeling studies, the animals
were anaesthetized as above, and the cranium over the motor cortex
was removed. With the fine glass micropipetter (about 100 um in
diameter) the cerebral cortex was injected with 2-4 ul of horse
radish peroxidase conjugated to wheat germ agglutinin (2%), a
marker that is taken up by nerve cells and transported
anterogradely into the axon that extends into the spinal cord.
After injection of the anterograde tracer, the cranium was
replaced, and the skin closed with 5-0 silk sutures. The animals
were sacrificed with chloral hydrate (4.9 mg/10 g) after 48 hours,
and perfused with 4% paraformaldehyde in phosphate buffer as a
fixative. The spinal cord was removed, cryoprotected with sucrose
and cryostat sections placed on slides for histological
examination.
EXAMPLE 8
DNA and Protein Sequence Details of C3-TL
[0273] The Tat coding sequence was obtained by polymerase chain
reaction of the plasmid SVCMV-TAT (obtained form Dr. Eric Cohen,
Universite de Montreal) that contains the entire HIV-1 Tat coding
sequence. To isolate the transport sequence of the Tat protein, PCR
was used. The first primer (5'GAATCCAAGCACCAGGAAGTCAGCC 3' (SEQ ID
NO.: 11)) and the second primer (5' ACC AGCCACCACCTTCTGATA 3' (SEQ
ID NO.: 12)) used corresponded to amino acids 27 to 72 of the HIV
Tat protein. Upon verification and purification, the PCR product
was sub cloned into a pSTBlue-1 blunt vector. This transport
segment of the Tat protein was then cloned into pGEX-4T/C3 at the
3' end of C3, using the restriction sites EcoR I and Sac I. The new
C3-Tat fusion protein was called C3-TL. Recombinant protein was
made as described in Example 3.
[0274] DNA Sequence of C3-TL (SEQ ID NO.: 13)
8 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTC TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
CAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC AAG CAT CCA GGA AGT
CAG CCT AAA ACT GCT TGT ACC AAT TGC TAT TGT AAA AAG TGT TGC TTT CAT
TGC CAA GTT TGT TTC ATA ACA AAA GCC TTA GGC ATC TCC TAT GGC AGG AAG
CGG AGA CAG CGA CGA AGA GCT CAT CAG AAC AGT CAG ACT CAT CAA GCT TCT
CTA TCA AAG CAG TAA 3'
[0275] The TL transport peptide sequence by itself is as
follows:(SEQ ID NO.: 46)
[0276]
KHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKRRQRRAHQNSQTHQASLSKQ.
[0277] The Protein Sequence of C3-TL (SEQ ID NO.: 14)
9 GSSRVDLQACNAYSINQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KEAIVSYTKSASEINGKLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPE
NIMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGY
ISTSLMNVSQFAGRPIITQFKVAKGSKAGYIDPISAFQGQLEMLLPRHST
YHIDDMRLSSDGKQIIITATMMGTAINPKEFKHPGSQPKTACTNCYCKKC
CFHCQVCFITKALGISYGRKRRQRRRAHQNSQTHQASLSKQ.
[0278] Molecular Weight 32721.40 Daltons
[0279] 291 Amino Acids
[0280] 43 Strongly Basic(+) Amino Acids (K,R)
[0281] 21 Strongly Acidic(-) Amino Acids (D,E)
[0282] 82 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0283] 104 Polar Amino Acids (N,C,Q,S,T,Y)
[0284] 9.688 Isolectric Point
[0285] 22.655 Charge at PH 7.0
[0286] Total Number of Bases Translated is 876
10 % A = 37.44 [328] % G = 17.58 [154] % T = 28.31 [248] % C =
16.67 [146]
EXAMPLE 9
DNA and Protein Sequence Details of C3-TS
[0287] A shorter Tat construct was also made (C3-TS). To make the
shorter C3 Tat fusion protein the following oligonucleotides were
5'AAT TCT ATG GTC GTA AAA AAC GTC GTC AAC GTC GTC GTG 3' (SEQ ID
NO.: 15) and 5' GAT ACC AGC ATT TTT TGC AGC AGT TGC AGC AGC ACA GCT
3' (SEQ ID NO.: 16). The two oligonucleotide strands were annealed
together by combining equal amounts of the oligonucleotides,
heating at 72.degree. C. for 5 minutes and then letting the
oligonucleotide solution cool at room temperature for 15 minutes.
The oligonucleotides were ligated into the pGEX4T/C3 vector at the
3' end of C3. The construct was sequenced. All plasmids were
transformed into XL-1 blue competent cells. Recombinant protein was
made as described in Example 3.
[0288] Nucleotide Sequence of C3-TS (SEQ ID NO.: 17)
11 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTC TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
CAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC TAT GGT GCT AAA AAA
CGT CGT CAA CGT CGT CGT GTC GAC TCG AGC GGC CCG CAT CGT GAC TGA
3'
[0289] The TS transport peptide sequence by itself is as follows:
(SEQ ID NO.: 47)
[0290] YGAKKRRQRRRVDSSGPHRD
[0291] The Protein Sequence of C3-TS (SEQ ID NO.: 18)
12 GSSRVDLQACNAYSINQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KEAIVSYIKSASETNGKLRQNKGVINGFPSNLIKQVELLDKSFNKMKTPE
NIMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGY
ISTSLMNVSQFAGRPIITQFKVAKGSKAGYIDPISAFQGQLEMLLPRHST
YHIDDMRLSSDGKQIIITATMMGTAINPKEFYGAKKRRQRRRVDSSGPHR D
[0292] Molecular Weight 26866.62 Daltons
[0293] 238 Amino Acids
[0294] 36 Strongly Basic(+) Amino Acids (K,R)
[0295] 21 Strongly Acidic(-) Amino Acids (D,E)
[0296] 71 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0297] 78 Polar Amino Acids (N,C,Q,S,T,Y)
[0298] 9.802 Isolectric Point
[0299] 15.212 Charge at PH 7.0
[0300] Total Number of Bases Translated is 717
13 % A = 38.91 [279] % G = 17.43 [125] % T = 28.45 [204] % C =
15.20 [109]
EXAMPLE 10
[0301] The following example illustrates how a coding sequence can
be modified without affecting the efficacy of the translated
protein. The example shows modifications to C3Basic3 that would not
affect the activity. Sequences may include the entire GST sequence,
as shown here that includes the start site, which would not be
removed enzymatically. Also, the transport sequence shown in this
example has changes in amino acid composition surrounding the
active sequence due to a difference in the cloning strategy, and
the His tag has been omitted. However, the active region is : R R K
Q R R K R R. This sequence is contained in the C3Basic3, and is the
active transport sequence in the sequence below. Also note that the
C-terminal region of the protein after this active region differs
from C3Basic3. That is because the cloning strategy was changed,
the restriction sites differ, and therefore non-essential amino
acids 3' terminal to the transport sequence are transplanted and
included in the protein.
[0302] Nucleic Acid Sequence: (SEQ ID NO.: 19)
[0303] 1413 base pairs
[0304] single strand
[0305] linear sequence
14 5' ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA
CCC ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG
TAT GAG CGC GAT GAA GGT GAT AAA TGG CGA AAC AAA AAG TTT GAA TTG GGT
TTG GAG TTT CCC AAT CTT CCT TAT TAT ATT GAT GGT GAT GTT AAA TTA ACA
CAG TCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC ATG TTG GGT
GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA ATG CTT GAA GGA GCG GTT TTG
GAT ATT AGA TAC GGT GTT TCG AGA ATT GCA TAT AGT AAA GAC TTT GAA ACT
CTC AAA GTT GAT TTT CTT AGC AAG CTA CCT GAA ATG CTG AAA ATG TTC GAA
GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT GGT GAT CAT GTA ACC CAT CCT
GAC TTC ATG TTG TAT GAC GCT CTT GAT GTT GTT TTA TAC ATG GAC CCA ATG
TGC CTG GAT GCG TTC CCA AAA TTA GTT TGT TTT AAA AAA CGT ATT GAA GCT
ATC CCA CAA ATT GAT AAG TAC TTG AAA TCC AGC AAG TAT ATA GCA TGG CCT
TTG CAG GGC TGG CAA GCC ACG TTT GGT GGT GGC GAC CAT CCT CCA AAA TCG
GAT CTG GTT CCG CGT GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT
TAT TCC ATT AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT
ATT GAT CAA GCA AAA GCT TGG GGT AAT GCT GAG TAT AAA AAG TAT GGA CTA
AGC AAA TCA GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA
ATA AAT GGA AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA
AAT TTA ATA AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG
ACC CCT GAA AAT ATT ATG TTA TTT AGA GGC GAG GAG CCT GCT TAT TTA GGA
ACA GAA TTT CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG
GCT TTT GAA AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT
GGA TAT ATT AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCA GGA AGA CCA
ATT ATT ACA AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAG
CCT ATT AGT GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT
ACT TAT CAT ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA
ATT ACA GCA ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC AGA AGG
AAA CAA AGA AGA AAA AGA AGA CTG CAG GCG GCC GCA TCG TGA 3'
[0306] Amino Acid Sequence (SEQ ID NO: 20)
[0307] 479 amino acids
[0308] linear, single strand
15 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL
EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVL
DIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTH
PDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIA
WPLQGWQATFGGGDHPPKSDLVPRGSSRVDLQACNAYSINQKAYSNTYQE
FTNTDQAKAWGNAQYKKYGLSKSEKEAIVSYTKSASETNGKLRQNKGVIN
GFPSNLIKQVELLDKSFNKMKTPENIMLFRGDDPAYLGTEFQNTLLNSNG
TINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPIITRFKVAKGS
KAGYIDPISAFQGQLEMLLPRHSTYHDDMRLSSDGKQIIITATMMGTAIN
PKEFRRKQRRKRRLQAAAS.
[0309] Molecular Weight 53813.02 Daltons
[0310] 470 Amino Acids
[0311] 68 Strongly Basic(+) Amino Acids (K,R)
[0312] 55 Strongly Acidic(-) Amino Acids (D,E)
[0313] 149 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0314] 121 Polar Amino Acids (N,C,Q,S,T,Y)
[0315] 9.137 Isolectric Point
[0316] 14.106 Charge at PH 7.0
[0317] Total Number of Bases Translated is 1413
16 % A = 34.61 [489] % G = 19.75 [279] % T = 29.51 [417] % C =
15.99 [226] % Ambiguous = 0.14 [2] % A + T = 64.12 [906] % C + G =
35.74 [505]
[0318] Davis,Botstein,Roth Melting Temp C. 79.20
EXAMPLE 11
Additional Chimeric C3 Proteins that would be Effective to
Stimulate Repair in the CNS
[0319] The following sequences could be added to the amino terminal
or carboxy terminal of C3 or a truncated C3 that retains its
enzymatic activity.
[0320] 1) Sequences of polyarginine as described (Wender, et al.
(2000) 97: 13003-8.). These could be from 6 to 9 or more
arginines.
[0321] 2) Sequences of poly-lysine
[0322] 3) Sequences of poly-histidine
[0323] 4) Sequences of arginine and lysine mixed.
[0324] 5) Basic stretches of amino acids containing non-basic amino
acids stretch where the sequence added retains transport
characteristics.
[0325] 6) Sequences of 5-15 amino acids containing at least 50%
basic amino acids
[0326] 7) Sequences longer than 15-30 amino acids containing at
least 30% basic amino acids.
[0327] 8) Sequences longer than 50 amino acids containing at least
18% basic amino acids.
[0328] 9) Any of the above where the amino acids are chemically
modified, such as by addition of cyclohexyl side chains, other side
chains, different alkyl spacers.
[0329] 10) Sequences that have proline residues with helix-breaking
propensity to act as effective transporters.
EXAMPLE 12
Additional Chimeric C3 Proteins that would be Effective to
Stimulate Repair in the CNS
[0330] C3Basic1: C3 fused to a randomly designed basic tail
[0331] C3Basic2: C3 fused to a randomly designed basic tail
[0332] C3 Basic3: C3 fused to the reverse Tat sequence
[0333] We have designed the following DNA encoding a chimeric C3
with membrane transport properties. The protein is designated
C3Basic1. This sequence was designed with C3 fused to a random
basic sequence. The construct was made to encode the peptide given
below.
[0334] K R R R R R P K K R R R A K R R (SEQ ID NO.: 21)
[0335] The construct was made by synthesizing the two
oligonucleotides given below, annealing them together, and ligating
them into the pGEX-4T/C3 vector with an added histidine tag.
[0336] 5' AAG AGA AGG CGA AGA AGA CCT AAG AAG AGA CGA AGG GCG AAG
AGG AGA 3' (SEQ ID NO.: 22)
[0337] 5'TTC TCT TCC GCT TCT TCT GGA TTC TTC TCT GCT TCC CGC TTC
TCC TCT 3' (SEQ ID NO.: 23)
[0338] DNA Sequence of C3Basic1 (SEQ ID NO: 24)
17 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC AAG AGA AGG CGA AGA
AGA CCT AAG AAG AGA CGA AGG GCG AAG AGG AGA CAC CAC CAC CAC CAC CAC
GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA 3'
[0339] Protein Sequence of C3Basic1 (SEQ ID NO: 25)
18 GSSRVDLQACNAYSTNQKAYSNTYQEFTNDQAKAWGNAQYKKYGLSKSEK
EAIVSYTKSASEINGKLRQNKGVINGFPSNUKQVELLDKSFNKMKTPENI
MLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYIS
TSLMNVSQFAGRPIITKFKVAKGSKAGYDPISAFQGQLEMLLPRHSTYHD
DMRLSSDGKIIITATMMGTAINPKEFKRRRRRPKKRRRAKRRHHHHHHVD SSGRIVTD.
[0340] Molecular Weight 29897.03 Daltons
[0341] 263 Amino Acids
[0342] 44 Strongly Basic(+) Amino Acids (K,R)
[0343] 23 Strongly Acidic(-) Amino Acids (D,E)
[0344] 75 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0345] 79 Polar Amino Acids (N,C,Q,S,T,Y)
[0346] 10.024 Isolectric Point
[0347] 22.209 Charge at PH 7.0
[0348] Davis,Botstein,Roth Melting Temp C. 78.56
EXAMPLE 13
Additional Chimeric C3 Protein that would be Effective to Stimulate
Repair in the CNS
[0349] We have designed the following DNA encoding a chimeric C3
with membrane transport properties. The protein is designated
C3Basic2. This sequence was designed with C3 fused to a random
basic sequence. The construct was made to encode the peptide given
below.
[0350] K R R R R K K R R Q R R R(SEQIDNO.:26)
[0351] The construct was made by synthesizing the two
oligonucleotides given below, annealing them together, and ligating
them into the pGEX4T/C3 vector with an added histidine tag.
[0352] 5' AAG CGT CGA CGT AGA AAG AAA CGT AGA CAG CGT AGA CGT 3'
(SEQ ID NO.: 27)
[0353] 5'TTC GCA GCT GCA TCT TTC TTT GCA TCT GTC GCA TCT GCA 3'
(SEQ ID NO.: 28)
[0354] DNA Sequence of C3Basic2 (SEQ ID NO.: 29)
19 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GPA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT PAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GPA TTT
CPA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC AAG CGT CGA CGT AGA
AAG AAA CGT AGA CAG CGT AGA CGT CAC CAC CAC CAC CAC CAC GTC GAC TCG
AGC GGC CGC ATC GTG ACT GAC TGA 3'
[0355] Protein Sequence of C3Basic2 (SEQ ID NO.: 30)
20 GSSRVDLQACNAYSINQKAYSNTYQEFTNDQAKAWGNAQYKKYGLSKSEK
EAIVSYTKSASETNGKLRQNKGVTNGFPSNLIKQVELLDKSFNXMKTPEN
TMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYI
STSLMNVSQFAGRPIITKFKVAKGSKAGYIDPISAFQGQLEMLLPRHSTY
HTDDMRLSSDGKQIIITATMMGTATNPKEFKRRRRKKRRQRRRHHHHHHV DSSGRIVTD.
[0356] Molecular Weight 29572.61 Daltons
[0357] 260 Amino Acids
[0358] 42 Strongly Basic(+) Amino Acids (K,R)
[0359] 23 Strongly Acidic(-) Amino Acids (D,E)
[0360] 74 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0361] 80 Polar Amino Acids (N,C,Q,S,T,Y)
[0362] 9.956 Isolectric Point
[0363] 20.210 Charge at PH 7.0
[0364] Davis,Botstein,Roth Melting Temp C. 78.45
EXAMPLE 14
Additional Chimeric C3 Protein that would be Effective to Stimulate
Repair in the CNS
[0365] We have designed the following DNA encoding a chimeric C3
with membrane transport properties. The protein is designated
C3Basic3. This sequence was designed with C3 fused to a reverse Tat
sequence. The construct was made to encode the peptide given
below
[0366] R R K Q R R K R R (SEQIDNO.: 31)
[0367] The construct was made by synthesizing the two
oligonucleotides given below, annealing them together, and ligating
them into the pGEX4T/C3 vector with an added histidine tag, then
subcloning in pGEX-4T/C3.
[0368] 5'AGA AGG AAA CAA AGA AGA AAA AGA AGA 3' (SEQ ID NO.:
32)
[0369] 5' TCT TCC TTT GTT TCT TCT TTT TCT TCT 3' (SEQ ID NO.:
33)
[0370] DNA Sequence of C3Basic3 (SEQ ID NO.: 34)
21 5' GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAT GCT TAT TCC ATT
AAT CAA AAG GCT TAT TCA AAT ACT TAC CAG GAG TTT ACT AAT ATT GAT CAA
GCA AAA GCT TGG GGT AAT GCT CAG TAT AAA AAG TAT GGA CTA AGC AAA TCA
GAA AAA GAA GCT ATA GTA TCA TAT ACT AAA AGC GCT AGT GAA ATA AAT GGA
AAG CTA AGA CAA AAT AAG GGA GTT ATC AAT GGA TTT CCT TCA AAT TTA ATA
AAA CAA GTT GAA CTT TTA GAT AAA TCT TTT AAT AAA ATG AAG ACC CCT GAA
AAT ATT ATG TTA TTT AGA GGC GAC GAC CCT GCT TAT TTA GGA ACA GAA TTT
CAA AAC ACT CTT CTT AAT TCA AAT GGT ACA ATT AAT AAA ACG GCT TTT GAA
AAG GCT AAA GCT AAG TTT TTA AAT AAA GAT AGA CTT GAA TAT GGA TAT ATT
AGT ACT TCA TTA ATG AAT GTT TCT CAA TTT GCA GGA AGA CCA ATT ATT ACA
AAA TTT AAA GTA GCA AAA GGC TCA AAG GCA GGA TAT ATT GAC CCT ATT AGT
GCT TTT CAG GGA CAA CTT GAA ATG TTG CTT CCT AGA CAT AGT ACT TAT CAT
ATA GAC GAT ATG AGA TTG TCT TCT GAT GGT AAA CAA ATA ATA ATT ACA GCA
ACA ATG ATG GGC ACA GCT ATC AAT CCT AAA GAA TTC AGA AGG AAA CAA AGA
AGA AAA AGA AGA CAC CAC CAC CAC CAC CAC GTC GAC TCG AGC GGC CGC ATC
GTG ACT GAC TGA 3'
[0371] Protein Sequence of C3Basic3 (SEQ ID NO.: 35)
22 GSSRVDLQACNAYSTNQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KIEAIVSYTKSASEINGKLRQNKGVINGFPSNIKQVELLDKSFNKMKTPE
NIMLFRGDDPAYLGTEFQNTLLNSNGTTNKTAFEKAKAKFLNKDRLEYGY
ISTSLMNVSQFAGRLPIITRFKVAKGSKAGYIDPISAFQGQLEMLLARHS
TYHIDDMRLSSDGKQIIITATMMGTAINPKEFRRKQRRKRRHHHHHHVDS SGRIVTD.
[0372] Molecular Weight 29441.47 Daltons
[0373] 260 Amino Acids
[0374] 39 Strongly Basic(+) Amino Acids (K,R)
[0375] 23 Strongly Acidic(-) Amino Acids (D,E)
[0376] 76 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0377] 80 Polar Amino Acids (N,C,Q,S,T,Y)
[0378] 9.833 Isolectric Point
[0379] 17.211 Charge at PH 7.0
[0380] Davis,Botstein,Roth Melting Temp C. 78.29
EXAMPLE 15
Sequences for C3APLT
[0381] One of the clones that was selected from the subcloning of
C3APL into pGEX encoded a protein that was not the expected size
but had good biological activity. This clone that had a frameshift
mutation leading to a truncation, and this clone was called C3APLT.
The clone was resequenced and the chromatograms analyzed to confirm
the sequence. To confirm the sequences of C3APLT, the coding
sequence from both strands of pGEX-4T/C3APLT were sequenced by
double strand sequencing of the full length of the clone
(BioS&T, Montreal, Quebec).
[0382] The DNA Sequence for C3APLT is as follows: (SEQ ID NO.:
36)
23 GGATCCTCTA GAGTCGACCT GCAGGCATGC AATGCTTATT 60 CCATTAATCA
AAAGGCTTAT TCAAATACTT ACCAGGAGTT TACTAATATT GATCAAGCAA 120
AAGCTTGGGG TAATGCTCAG TATAAAAAGT ATGGACTAAG CAAATCAGAA AAAGAAGCTA
180 TAGTATCATA TACTAAAAGC GCTAGTGAAA TAAATGGAAA GCTAAGACAA
AATAAGGGAG 240 TTATCAATGG ATTTCCTTCA AATTTAATAA AACAAGTTGA
ACTTTTAGAT AAATCTTTTA 300 ATAAAATGAA GACCCCTGAA AATATTATGT
TATTTAGAGG CGACGACCCT GCTTATTTAG 360 GAACAGAATT TCAAAACACT
CTTCTTAATT CAAATGGTAC AATTAATAAA ACGGCTTTTG 420 AAAAGGCTAA
AGCTAAGTTT TTAAATAAAG ATAGACTTGA ATATGGATAT ATTAGTACTT 480
CATTAATGAA TGTTTCTCAA TTTGCAGGAA GACCAATTAT TACAAAATTT AAAGTAGCAA
540 AAGGCTCAAA GGCAGGATAT ATTGACCCTA TTAGTGCTTT TGCAGGACAA
CTTGAAATGT 600 TGCTTCCTAG ACATAGTACT TATCATATAG ACGATATGAG
ATTGTCTTCT GATGGTAAAC 660 AAATAATAAT TACAGCAACA ATGATGGGCA
CAGCTATCAA TCCTAAAGAA TTCGTGATGA 720 ATCCCGCAAA CGCGCAAGGC
AGACATACAC CCGGTACCAG ACTCTAGAGC TAGAGAAGGA 780 GTTTCACTTC
AATCGCTACT TGACCCGTCG GCGAAGGATC GAGATCGCCC ACGCCCTGTG 840
CCTCACGGAG CGCCAGATAA AGATTTGGTT CCAGAATCGG CGCATGAAGT GGAAGAAGGA
887 GAACTGA
[0383] The APLT transport peptide sequence by itself is as follows
(SEQ ID NO.: 48):
[0384] VMNPANAQGRHTPGTRL
[0385] The Protein Sequence for C3APLT is as follows: (SEQ ID NO.:
37)
24 GSSRVDLQACNAYSINQKAYSNTYQEFTNIDQAKAWGNAQYKKYGLSKSE
KIEAIVSYTKSASEINGKLRQNKGVINGFPSNLIKQVELLDKSFNKMKTP
ENIMLFRGDDPAYLGTEFQNTLLNSNGTINKTAFEKAKAKFLNIKDRLEY
GYISTSLMNVSQFAGRPIITKFKVAKGSKAGYIDPISAFAGQLEMLLPRH
STYHIDDMRLSSDGKQIIITATMMGTATNPKEFVMNPANAQGRHTPGTRL
[0386] Molecular Weight 27574.42 Daltons
[0387] 248 Amino Acids
[0388] 33 Strongly Basic(+) Amino Acids (K,R)
[0389] 21 Strongly Acidic(-) Amino Acids (D,E)
[0390] 76 Hydrophobic Amino Acids (A,I,L,F,W,V)
[0391] 80 Polar Amino Acids (N,C,Q,S,T,Y)
[0392] 9.636 Isoelectric Point
[0393] 12.379 Charge at PH 7.0
EXAMPLE 16
Subcloning and Sequences for C3APLT in pET
[0394] C3 has been reported to be stably expressed in E. coli by
both pGEX-series and pET-series vectors (e.g., Dillon and Feig,
1995 Meth. Enzymol. 256: 174-184. Small GTPases and Their
Regulators. Part B. Rho Family. W. E. Balch, C. J. Der, and A.
Hall, eds.; Lehmann et al., 1999 supra; Han et al., 2001. J. Mol.
Biol. 395: 95-107). The fusion proteins were expressed well in the
pGEX vector, for synthesis and testing. However, for large-scale
production it is more efficient to synthesize recombinant proteins
without an affinity tag that increases the size of the protein
produced. Also, it is more economical to synthesize proteins in
large scale by affinity chromatography using automated FPLC
systems. The polymerase chain reaction was used to transfer
recombinant construct C3APLT into the pET T7 polymerase based
system E. coli expression system (reviewed by Studier et al., 1990.
Meth. Enzymol. 185: 60-89. Gene Expression Technology. D. V.
Goeddel, ed.). A similar PCR approach is suitable for others in the
fusion protein series of C3-based constructs with transport
sequences. The pET3a vector DNA was obtained from Dr. Jerry
Pelletier, McGill University. PCR primers were obtained from
Invitrogen. The upper (5') primer was 5'-GGA TCT GGT TCC GCG TCA
TAT GTC TAG AGT CGA CCT G-3 (37 b) (SEQ ID NO.:38). Underlined is
the Nde I site that was introduced into the primer to replace the
BamHI site in pGEX4T-C3APLT. The lower primer was 5'-CGC GGA TCC
ATT AGT TCT CCT TCT TCC ACT TC-3' (32 b) (SEQ ID NO.:39). This
primer introduced two changes in the coding strand DNA of
pGEX4T-C3APLT, replacing the EcoRI site from pGEX4T-C3APLT with a
BamiH I site (underlined) and replacing a TGA stop codon with the
strong stop sequence TAAT (the italicized ATTA sequence in the
complementary primer). Compared to pGEX4T-C3APLT, the predicted
N-terminal sequence of pET3a-C3APLT is Met-Ser rather than
Gly-Ser-Ser, a loss of one serine and a substitution of Met for
Gly. There were no changes in amino acid sequence at the C-terminus
of C3APLT.
[0395] The target C3APLT gene was amplified using Pfu polymerase
(Invitrogen/Canadian Life Technologies) with buffer, DNA and
deoxyribonucleotide concentrations recommended by the manufacturer.
The PCR was carried out as follows : 95.degree. C. for 5 minutes,
10 cycles of 94.degree. C. for 2 minutes followed by 56.degree. C.
for 2 minutes then extension at 70.degree. C. for 2 minutes, then
30 cycles of 94.degree. C. for 2 minutes followed by 70.degree. C.
Completed reactions were stored at 4.degree. C. The QIAEXII kit
(Qiagen) was used to purify the agarose gel slice containing DNA
band. The purified PCR product DNA and the vector were digested
with BamH I and Nde I (both obtained from New England BioLabs)
following the instructions of the manufacturer. The digestion
products were separated from extraneous DNA by agarose gel
electrophoresis and purified with the QIAEXII kit. . The insert and
vector DNA were incubated together overnight at 16.degree. C. with
T4 DNA ligase according to directions provided by the manufacturer
(New England BioLabs). Competent E. coli (DH5.alpha., obtained from
Invitrogen/Canadian Life Technologies) were transformed with the
ligation mixture.
[0396] DNA was prepared from purified colonies using the Qiagen
plasmid midi kit, and the entire insert and junction sequences were
verified by double strand sequencing of the full length of the
clone (BioS&T, Montreal, Quebec) with forward primer 5' AAA TTA
ATA CGA CTC ACT ATA GGG 3' (24 bases) (SEQ ID NO.: 40)and reverse
T7 terminator sequencing primer 5' GCT AGT TAT TGC TCAGCG G 3' (19
bases) (SEQ ID NO.: 41). The sequence of the C3APLT cDNA in pET is
given in SEQ ID NO.: 42. The amino acid sequence is given in SEQ.
ID NO.: 43.
EXAMPLE 17
Modifications of Sequences
[0397] Any of sequences given in Examples 1, 2, 8, 9, 10, 11, 12
and 13, 15 and 16 could be modified to retain C3 enzymatic activity
and effective transport sequences. For example amino acids encoded
from DNA at the 3' end of the sequence that represents the
translation of the restriction sites used in cloning may be removed
without affecting activity. Some of the amino terminal amino acids
may also be removed without affecting activity. The minimal amount
of sequence needed for biological activity of the C3 portion of the
fusion protein is not known but could be easily determined by known
techniques. For example, increasingly more of the 5' end of the
cDNA encoding C3 could be removed, and the resulting proteins made
and tested for biological activity. Similarly, increasing amounts
of the 3' end could be removed and the fragments tested for
biological activity. Next, fragments testing the central region
could be tested for retention of C3 activity. Therefore, the C3
portion of the protein could be truncated to include just the amino
acids needed for activity. Alternatively mutations could be made in
the coding regions of C3, and the resulting proteins tested for
activity. The transport sequences could be modified to add or
remove one or more amino acids or to completely change the
transport peptide, but retain the transport characteristics in
terms of effective dose compared to C3 in our tissue culture
bioassay (Example 4). New transport sequences could be tested for
biological activity to improve the efficiency of C3 activity by
plating neurons and testing them on inhibitory substrates, as
described in Example 4.
[0398] As discussed previously, it has been determined in tissue
culture studies, that the minimum amount of C3 that can be used to
induce growth on inhibitory substrates is 25 ug/ml (Lehmann, et al.
(1999) J. Neurosci. 19: 7537-7547; Morii, N and Narumiya, S. (1995)
Methods in Enzymology, Vol 256 part B, pg.196-206. If the cells are
not triturated, even this dose is ineffective (FIG. 1). In the
context of the present invention it has been determined, for
example, that at least 40 .mu.g of C3/20 g mouse needs to be
applied to injured mouse spinal cord or rat optic nerve
(McKerracher, Canadian patent application No.: 2,325,842).
Calculating doses that would be required to treat an adult human on
an equivalent dose per weight scale up used for rat and mice
experiments, it would be necessary to apply 120 mg/kg of C3 (i.e.
alone) to the injured human spinal cord. This large amount of
recombinant C3 protein needed, creates significant problems for
manufacturing, due to the large-scale protein purification and
cost. It also limits the dose ranging that can be tested because of
the large amount of protein needed for minimal effective doses.
[0399] Fusion proteins of the present invention are much more
effective than C3 (i.e., alone) in promoting neurite outgrowth on
myelin substrate. For example, concentrations of C3APLT and C3APS,
10,000 and 1,000 times less than the concentration needed for C3
may be used with comparable (similar) effects without exhibiting
toxic effects (e.g., on PC-12 cells). C3-TL and C3-TS are also able
to promote neurite growth on myelin substrates at doses
significantly less than C3. In vivo results also indicate that
lower dose of the fusion proteins may be required to promote
regeneration and functional recovery after spinal cord injury in
mice. Thus, fusion proteins of the present invention represent a
significant improvement and advantage over C3 in both manufacture
cost and doses required for treatment.
Sequence CWU 1
1
48 1 27 DNA Artificial Sequence Oligonucleotide used to remove the
stop codon from ADP-ribosyl transferase C3 (Clostridium botulinum)
cDNA. 1 gaattcttta ggattgatag ctgtgcc 27 2 21 DNA Artificial
Sequence Oligonucleotide used to remove the stop codon from
ADP-ribosyl transferase C3 (Clostridium botulinum) cDNA. 2
ggtggcgacc atcctccaaa a 21 3 888 DNA Artificial Sequence Sequence
of C3APL includes ADP-ribosyl transferase C3 (Clostridium
botulinum) and Antennapedia sequence. 3 gga tcc tct aga gtc gac ctg
cag gca tgc aat gct tat tcc att aat 48 Gly Ser Ser Arg Val Asp Leu
Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5 10 15 caa aag gct tat tca
aat act tac cag gag ttt act aat att gat caa 96 Gln Lys Ala Tyr Ser
Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 gca aaa gct
tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144 Ala Lys Ala
Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45 tca
gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata 192 Ser
Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55
60 aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca
240 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser
65 70 75 80 aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt aat
aaa atg 288 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn
Lys Met 85 90 95 aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 336 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 100 105 110 tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 384 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 115 120 125 aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 432 Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140 aga ctt gaa tat gga tat
att agt act tca tta atg aat gtc tct caa 480 Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln 145 150 155 160 ttt gca gga
aga cca att att aca caa ttt aaa gta gca aaa ggc tca 528 Phe Ala Gly
Arg Pro Ile Ile Thr Gln Phe Lys Val Ala Lys Gly Ser 165 170 175 aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 576 Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185
190 atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
624 Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
195 200 205 tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 672 Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 210 215 220 gct atc aat cct aaa gaa ttc gtg atg gaa tcc cgc
aaa cgc gca agg 720 Ala Ile Asn Pro Lys Glu Phe Val Met Glu Ser Arg
Lys Arg Ala Arg 225 230 235 240 cag aca tac acc cgg tac cag act cta
gag cta gag aag gag ttt cac 768 Gln Thr Tyr Thr Arg Tyr Gln Thr Leu
Glu Leu Glu Lys Glu Phe His 245 250 255 ttc aat cgc tac ttg acc cgt
cgg cga agg atc gag atc gcc cac gcc 816 Phe Asn Arg Tyr Leu Thr Arg
Arg Arg Arg Ile Glu Ile Ala His Ala 260 265 270 ctg tgc ctc acg gag
cgc cag ata aag att tgg ttc cag aat cgg cgc 864 Leu Cys Leu Thr Glu
Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg 275 280 285 atg aag tgg
aag aag gag aac tga 888 Met Lys Trp Lys Lys Glu Asn 290 295 4 295
PRT Artificial Sequence Sequence of C3APL includes ADP-ribosyl
transferase C3 (Clostridium botulinum) and Antennapedia sequence. 4
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala
Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile Asn Pro Lys
Glu Phe Val Met Glu Ser Arg Lys Arg Ala Arg 225 230 235 240 Gln Thr
Tyr Thr Arg Tyr Gln Thr Leu Glu Leu Glu Lys Glu Phe His 245 250 255
Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg Ile Glu Ile Ala His Ala 260
265 270 Leu Cys Leu Thr Glu Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg 275 280 285 Met Lys Trp Lys Lys Glu Asn 290 295 5 774 DNA
Artificial Sequence Sequence of C3APS Includes ADP-ribosyl
transferase C3 (Clostridium botulinum) and Antennapedia sequence. 5
gga tcc tct aga gtc gac ctg cag gca tgc aat gct tat tcc att aat 48
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 caa aag gct tat tca aat act tac cag gag ttt act aat att gat
caa 96 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 gca aaa gct tgg ggt aat gct cag tat aaa aag tat gga
cta agc aaa 144 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 tca gaa aaa gaa gct ata gta tca tat act aaa
agc gct agt gaa ata 192 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 aat gga aag cta aga caa aat aag gga
gtt atc aat gga ttt cct tca 240 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 aat tta ata aaa caa gtt gaa
ctt tta gat aaa tct ttt aat aaa atg 288 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 aag acc cct gaa aat
att atg tta ttt aga ggc gac gac cct gct tat 336 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 tta gga aca
gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 aat
aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 aga ctt gaa tat gga tat att agt act tca tta atg aat gtc tct caa
480 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 ttt gca gga aga cca att att aca caa ttt aaa gta gca
aaa ggc tca 528 Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala
Lys Gly Ser 165 170 175 aag gca gga tat att gac cct att agt gct ttt
cag gga caa ctt gaa 576 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 atg ttg ctt cct aga cat agt act tat
cat ata gac gat atg aga ttg 624 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 tct tct gat ggt aaa caa ata
ata att aca gca aca atg atg ggc aca 672 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 gct atc aat cct aaa
gaa ttc cgc cag atc aag att tgg ttc cag aat 720 Ala Ile Asn Pro Lys
Glu Phe Arg Gln Ile Lys Ile Trp Phe Gln Asn 225 230 235 240 cgt cgc
atg aag tgg aag aag gtc gac tcg agc ggc cgc atc gtg act 768 Arg Arg
Met Lys Trp Lys Lys Val Asp Ser Ser Gly Arg Ile Val Thr 245 250 255
gac tga 774 Asp 6 257 PRT Artificial Sequence Sequence of C3APS
Includes ADP-ribosyl transferase C3 (Clostridium botulinum) and
Antennapedia sequence. 6 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys
Asn Ala Tyr Ser Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn
Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn Gly
Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80
Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85
90 95 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala
Tyr 100 105 110 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn
Gly Thr Ile 115 120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys
Phe Leu Asn Lys Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr
Ser Leu Met Asn Val Ser Gln 145 150 155 160 Phe Ala Gly Arg Pro Ile
Ile Thr Gln Phe Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly Tyr
Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu
Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205
Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210
215 220 Ala Ile Asn Pro Lys Glu Phe Arg Gln Ile Lys Ile Trp Phe Gln
Asn 225 230 235 240 Arg Arg Met Lys Trp Lys Lys Val Asp Ser Ser Gly
Arg Ile Val Thr 245 250 255 Asp 7 24 DNA Artificial Sequence
Oligonucleotide used in the amplification of Antennapedia sequence
7 gaatcccgca aacgcgcaag gcag 24 8 27 DNA Artificial Sequence
Oligonucleotide used in the amplification of Antennapedia sequence
8 tcagttctcc ttcttccact tcatgcg 27 9 54 DNA Artificial Sequence
Oligonucleotide used in the cloning of sequences from Antennapedia
9 aattccgcca gatcaagatt tggttccaga atcgtcgcat gaagtggaag aagg 54 10
54 DNA Artificial Sequence Oligonucleotide used in the cloning of
sequences from Antennapedia 10 ggcggtctag ttctaaacca agctcttagc
agcgtagttc accttcttcc agct 54 11 26 DNA Artificial Sequence
Oligonucleotide used inthe amplification of a sequence
corresponding to amino acid 27-72 of HIV-1 Tat 11 gaatccaagc
atccaggaag tcagcc 26 12 21 DNA Artificial Sequence Oligonucleotide
used inthe amplification of a sequence corresponding to amino acid
27-72 of HIV-1 Tat 12 accagccacc accttctgat a 21 13 876 DNA
Artificial Sequence Sequence of C3-TL Includes ADP-ribosyl
transferase C3 (Clostridium botulinum) and HIV-1 Tat sequence. 13
gga tcc tct aga gtc gac ctg cag gca tgc aat gct tat tcc att aat 48
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 caa aag gct tat tca aat act tac cag gag ttt act aat att gat
caa 96 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 gca aaa gct tgg ggt aat gct cag tat aaa aag tat gga
cta agc aaa 144 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 tca gaa aaa gaa gct ata gta tca tat act aaa
agc gct agt gaa ata 192 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 aat gga aag cta aga caa aat aag gga
gtt atc aat gga ttt cct tca 240 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 aat tta ata aaa caa gtt gaa
ctt tta gat aaa tct ttt aat aaa atg 288 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 aag acc cct gaa aat
att atg tta ttt aga ggc gac gac cct gct tat 336 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 tta gga aca
gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 aat
aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 aga ctt gaa tat gga tat att agt act tca tta atg aat gtc tct caa
480 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 ttt gca gga aga cca att att aca caa ttt aaa gta gca
aaa ggc tca 528 Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala
Lys Gly Ser 165 170 175 aag gca gga tat att gac cct att agt gct ttt
cag gga caa ctt gaa 576 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 atg ttg ctt cct aga cat agt act tat
cat ata gac gat atg aga ttg 624 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 tct tct gat ggt aaa caa ata
ata att aca gca aca atg atg ggc aca 672 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 gct atc aat cct aaa
gaa ttc aag cat cca gga agt cag cct aaa act 720 Ala Ile Asn Pro Lys
Glu Phe Lys His Pro Gly Ser Gln Pro Lys Thr 225 230 235 240 gct tgt
acc aat tgc tat tgt aaa aag tgt tgc ttt cat tgc caa gtt 768 Ala Cys
Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln Val 245 250 255
tgt ttc ata aca aaa gcc tta ggc atc tcc tat ggc agg aag cgg aga 816
Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg Lys Arg Arg 260
265 270 cag cga cga aga gct cat cag aac agt cag act cat caa gct tct
cta 864 Gln Arg Arg Arg Ala His Gln Asn Ser Gln Thr His Gln Ala Ser
Leu 275 280 285 tca aag cag taa 876 Ser Lys Gln 290 14 291 PRT
Artificial Sequence Sequence of C3-TL Includes ADP-ribosyl
transferase C3 (Clostridium botulinum) and HIV-1 Tat sequence. 14
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala
Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro
Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp
Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala
Ile Asn Pro Lys Glu Phe Lys His Pro Gly Ser Gln Pro Lys Thr 225 230
235 240 Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gln
Val 245 250 255 Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly Arg
Lys Arg Arg 260 265 270 Gln Arg Arg Arg Ala His Gln Asn Ser Gln Thr
His Gln Ala Ser Leu 275 280 285 Ser Lys Gln 290 15 39 DNA
Artificial Sequence Oligonucleotide used in the cloning of
sequences from HIV-1 Tat 15 aattctatgg tcgtaaaaaa cgtcgtcaac
gtcgtcgtg 39 16 39 DNA Artificial Sequence Oligonucleotide used in
the cloning of sequences from HIV-1 Tat 16 gataccagca ttttttgcag
cagttgcagc agcacagct 39 17 756 DNA Artificial Sequence Sequence of
C3-TS Includes ADP-ribosyl transferase C3 (Clostridium botulinum)
and HIV-1 Tat sequence. 17 gga tcc tct aga gtc gac ctg cag gca tgc
aat gct tat tcc att aat 48 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys
Asn Ala Tyr Ser Ile Asn 1 5 10 15 caa aag gct tat tca aat act tac
cag gag ttt act aat att gat caa 96 Gln Lys Ala Tyr Ser Asn Thr Tyr
Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 gca aaa gct tgg ggt aat
gct cag tat aaa aag tat gga cta agc aaa 144 Ala Lys Ala Trp Gly Asn
Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45 tca gaa aaa gaa
gct ata gta tca tat act aaa agc gct agt gaa ata 192 Ser Glu Lys Glu
Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 aat gga
aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca 240 Asn Gly
Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80
aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt aat aaa atg 288
Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85
90 95 aag acc cct gaa aat att atg tta ttt aga ggc gac gac cct gct
tat 336 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala
Tyr 100 105 110 tta gga aca gaa ttt caa aac act ctt ctt aat tca aat
ggt aca att 384 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn
Gly Thr Ile 115 120 125 aat aaa acg gct ttt gaa aag gct aaa gct aag
ttt tta aat aaa gat 432 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys
Phe Leu Asn Lys Asp 130 135 140 aga ctt gaa tat gga tat att agt act
tca tta atg aat gtc tct caa 480 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr
Ser Leu Met Asn Val Ser Gln 145 150 155 160 ttt gca gga aga cca att
att aca caa ttt aaa gta gca aaa ggc tca 528 Phe Ala Gly Arg Pro Ile
Ile Thr Gln Phe Lys Val Ala Lys Gly Ser 165 170 175 aag gca gga tat
att gac cct att agt gct ttt cag gga caa ctt gaa 576 Lys Ala Gly Tyr
Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185 190 atg ttg
ctt cct aga cat agt act tat cat ata gac gat atg aga ttg 624 Met Leu
Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205
tct tct gat ggt aaa caa ata ata att aca gca aca atg atg ggc aca 672
Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210
215 220 gct atc aat cct aaa gaa ttc tat ggt gct aaa aaa cgt cgt caa
cgt 720 Ala Ile Asn Pro Lys Glu Phe Tyr Gly Ala Lys Lys Arg Arg Gln
Arg 225 230 235 240 cgt cgt gtc gac tcg agc ggc ccg cat cgt gac tga
756 Arg Arg Val Asp Ser Ser Gly Pro His Arg Asp 245 250 18 251 PRT
Artificial Sequence Sequence of C3-TS Includes ADP-ribosyl
transferase C3 (Clostridium botulinum) and HIV-1 Tat sequence. 18
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Gln Phe Lys Val Ala
Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile Asn Pro Lys
Glu Phe Tyr Gly Ala Lys Lys Arg Arg Gln Arg 225 230 235 240 Arg Arg
Val Asp Ser Ser Gly Pro His Arg Asp 245 250 19 1413 DNA Artificial
Sequence Includes GST sequences, ADP-ribosyl transferase C3 (C.
botulinum) sequence and a random basic amino acid sequence. 19 atg
tcc cct ata cta ggt tat tgg aaa att aag ggc ctt gtg caa ccc 48 Met
Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10
15 act cga ctt ctt ttg gaa tat ctt gaa gaa aaa tat gaa gag cat ttg
96 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30 tat gag cgc gat gaa ggt gat aaa tgg cga aac aaa aag ttt
gaa ttg 144 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe
Glu Leu 35 40 45 ggt ttg gag ttt ccc aat ctt cct tat tat att gat
ggt gat gtt aaa 192 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
Gly Asp Val Lys 50 55 60 tta aca cag tct atg gcc atc ata cgt tat
ata gct gac aag cac aac 240 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr
Ile Ala Asp Lys His Asn 65 70 75 80 atg ttg ggt ggt tgt cca aaa gag
cgt gca gag att tca atg ctt gaa 288 Met Leu Gly Gly Cys Pro Lys Glu
Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 gga gcg gtt ttg gat att
aga tac ggt gtt tcg aga att gca tat agt 336 Gly Ala Val Leu Asp Ile
Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 aaa gac ttt gaa
act ctc aaa gtt gat ttt ctt agc aag cta cct gaa 384 Lys Asp Phe Glu
Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 atg ctg
aaa atg ttc gaa gat cgt tta tgt cat aaa aca tat tta aat 432 Met Leu
Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140
ggt gat cat gta acc cat cct gac ttc atg ttg tat gac gct ctt gat 480
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145
150 155 160 gtt gtt tta tac atg gac cca atg tgc ctg gat gcg ttc cca
aaa tta 528 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro
Lys Leu 165 170 175 gtt tgt ttt aaa aaa cgt att gaa gct atc cca caa
att gat aag tac 576 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln
Ile Asp Lys Tyr 180 185 190 ttg aaa tcc agc aag tat ata gca tgg cct
ttg cag ggc tgg caa gcc 624 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro
Leu Gln Gly Trp Gln Ala 195 200 205 acg ttt ggt ggt ggc gac cat cct
cca aaa tcg gat ctg gtt ccg cgt 672 Thr Phe Gly Gly Gly Asp His Pro
Pro Lys Ser Asp Leu Val Pro Arg 210 215 220 gga tcc tct aga gtc gac
ctg cag gca tgc aat gct tat tcc att aat 720 Gly Ser Ser Arg Val Asp
Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 225 230 235 240 caa aag gct
tat tca aat act tac cag gag ttt act aat att gat caa 768 Gln Lys Ala
Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 245 250 255 gca
aaa gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 816 Ala
Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 260 265
270 tca gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata
864 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile
275 280 285 aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt
cct tca 912 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe
Pro Ser 290 295 300 aat tta ata aaa caa gtt gaa ctt tta gat aaa tct
ttt aat aaa atg 960 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser
Phe Asn Lys Met 305 310 315 320 aag acc cct gaa aat att atg tta ttt
aga ggc gac gac cct gct tat 1008 Lys Thr Pro Glu Asn Ile Met Leu
Phe Arg Gly Asp Asp Pro Ala Tyr 325 330 335 tta gga aca gaa ttt caa
aac act ctt ctt aat tca aat ggt aca att 1056 Leu Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 340 345 350 aat aaa acg
gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 1104 Asn Lys
Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 355 360 365
aga ctt gaa tat gga tat att agt act tca tta atg aat gtt tct caa
1152 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser
Gln 370 375 380 ttt gca gga aga cca att att aca aaa ttt aaa gta gca
aaa ggc tca 1200 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val
Ala Lys Gly Ser 385 390 395 400 aag gca gga tat att gac cct att agt
gct ttt cag gga caa ctt gaa 1248 Lys Ala Gly Tyr Ile Asp Pro Ile
Ser Ala Phe Gln Gly Gln Leu Glu 405 410 415 atg ttg ctt cct aga cat
agt act tat cat ata gac gat atg aga ttg 1296 Met Leu Leu Pro Arg
His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 420 425 430 tct tct gat
ggt aaa caa ata ata att aca gca aca atg atg ggc aca 1344 Ser Ser
Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 435 440 445
gct atc aat cct aaa gaa ttc aga agg aaa caa aga aga aaa aga aga
1392 Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg Arg Lys Arg
Arg 450 455 460 ctg cag gcg gcc gca tcg tga 1413 Leu Gln Ala Ala
Ala Ser 465 470 20 470 PRT Artificial Sequence Includes GST
sequences, ADP-ribosyl trans- ferase C3 (C. botulinum) sequence and
a random basic amino acid sequence. 20 Met Ser Pro Ile Leu Gly Tyr
Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu
Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg
Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly
Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55
60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met
Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg
Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe
Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg
Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His
Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu
Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val
Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185
190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val
Pro Arg 210 215 220 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala
Tyr Ser Ile Asn 225 230 235 240 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln
Glu Phe Thr Asn Ile Asp Gln 245 250 255 Ala Lys Ala Trp Gly Asn Ala
Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 260 265 270 Ser Glu Lys Glu Ala
Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 275 280 285 Asn Gly Lys
Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 290 295 300 Asn
Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 305 310
315 320 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala
Tyr 325 330 335 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn
Gly Thr Ile 340 345 350 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys
Phe Leu Asn Lys Asp 355 360 365 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr
Ser Leu Met Asn Val Ser Gln 370 375 380 Phe Ala Gly Arg Pro Ile Ile
Thr Lys Phe Lys Val Ala Lys Gly Ser 385 390 395 400 Lys Ala Gly Tyr
Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 405 410 415 Met Leu
Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu 420 425 430
Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 435
440 445 Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg Arg Lys Arg
Arg 450 455 460 Leu Gln Ala Ala Ala Ser 465 470 21 16 PRT
Artificial Sequence Random basic amino acid sequence of C3Basic1 21
Lys Arg Arg Arg Arg Arg Pro Lys Lys Arg Arg Arg Ala Lys Arg Arg 1 5
10 15 22 48 DNA Artificial Sequence Oligonucleotide used in the
cloning of a random basic amino acid sequence in C3Basic1 22
aagagaaggc gaagaagacc taagaagaga cgaagggcga agaggaga 48 23 48 DNA
Artificial Sequence Oligonucleotide used in the cloning of a random
basic amino acid sequence in C3Basic1 23 ttctcttccg cttcttctgg
attcttctct gcttcccgct tctcctct 48 24 792 DNA Artificial Sequence
Sequence of C3Basic1 includes ADP-ribosyl transferase C3
(Clostridium botulinum) sequence and a sequence encoding a random
basic amino acid sequence and a Histidine tag. 24 gga tcc tct aga
gtc gac ctg cag gca tgc aat gct tat tcc att aat 48 Gly Ser Ser Arg
Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5 10 15 caa aag
gct tat tca aat act tac cag gag ttt act aat att gat caa 96 Gln Lys
Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30
gca aaa gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144
Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35
40 45 tca gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa
ata 192 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu
Ile 50 55 60 aat gga aag cta aga caa aat aag gga gtt atc aat gga
ttt cct tca 240 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly
Phe Pro Ser 65 70 75 80 aat tta ata aaa caa gtt gaa ctt tta gat aaa
tct ttt aat aaa atg 288 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys
Ser Phe Asn Lys Met 85 90 95 aag acc cct gaa aat att atg tta ttt
aga ggc gac gac cct gct tat 336 Lys Thr Pro Glu Asn Ile Met Leu Phe
Arg Gly Asp Asp Pro Ala Tyr 100 105 110 tta gga aca gaa ttt caa aac
act ctt ctt aat tca aat ggt aca att 384 Leu Gly Thr Glu Phe Gln Asn
Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 aat aaa acg
gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432 Asn Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140 aga
ctt gaa tat gga tat att agt act tca tta atg aat gtt tct caa 480 Arg
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln 145 150
155 160 ttt gca gga aga cca att att aca aaa ttt aaa gta gca aaa ggc
tca 528 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly
Ser 165 170 175 aag gca gga tat att gac cct att agt gct ttt cag gga
caa ctt gaa 576 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly
Gln Leu Glu 180 185 190 atg ttg ctt cct aga cat agt act tat cat ata
gac gat atg aga ttg 624 Met Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu 195 200 205 tct tct gat ggt aaa caa ata ata att
aca gca aca atg atg ggc aca 672 Ser Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr 210 215 220 gct atc aat cct aaa gaa ttc
aag aga agg cga aga aga cct aag aag 720 Ala Ile Asn Pro Lys Glu Phe
Lys Arg Arg Arg Arg Arg Pro Lys Lys 225 230 235 240 aga cga agg gcg
aag agg aga cac cac cac cac cac cac gtc gac tcg 768 Arg Arg Arg Ala
Lys Arg Arg His His His His His His Val Asp Ser 245 250 255 agc ggc
cgc atc gtg act gac tga 792 Ser Gly Arg Ile Val Thr Asp 260 25 263
PRT Artificial Sequence Sequence of C3Basic1 includes ADP-ribosyl
transferase C3 (Clostridium botulinum) sequence and a sequence
encoding a random basic amino acid sequence and a Histidine tag. 25
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile Asn Pro Lys
Glu Phe Lys Arg Arg Arg Arg Arg Pro Lys Lys 225 230 235 240 Arg Arg
Arg Ala Lys Arg Arg His His His His His His Val Asp Ser 245 250 255
Ser Gly Arg Ile Val Thr Asp 260 26 13 PRT Artificial Sequence
Random amino acid sequence of C3Basic2 26 Lys Arg Arg Arg Arg Lys
Lys Arg Arg Gln Arg Arg Arg 1 5 10 27 39 DNA Artificial Sequence
Oligonucleotide used in the cloning of a random basic amino acid
sequence in C3Basic2 27 aagcgtcgac gtagaaagaa acgtagacag cgtagacgt
39 28 39 DNA Artificial Sequence Oligonucleotide used in the
cloning of a random basic amino acid sequence in C3Basic2 28
ttcgcagctg catctttctt tgcatctgtc gcatctgca 39 29 783 DNA Artificial
Sequence Sequence of C3Basic2 includes sequences from
ADP-ribosyl-transferase C3 (Clostridium botulinum) and a sequence
encoding a random basic amino acid sequence and a histidine tag. 29
gga tcc tct aga gtc gac ctg cag gca tgc aat gct tat tcc att aat 48
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 caa aag gct tat tca aat act tac cag gag ttt act aat att gat
caa 96 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 gca aaa gct tgg ggt aat gct cag tat aaa aag tat gga
cta agc aaa 144 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 tca gaa aaa gaa gct ata gta tca tat act aaa
agc gct agt gaa ata 192 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 aat gga aag cta aga caa aat aag gga
gtt atc aat gga ttt cct tca 240 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 aat tta ata aaa caa gtt gaa
ctt tta gat aaa tct ttt aat aaa atg 288 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 aag acc cct gaa aat
att atg tta ttt aga ggc gac gac cct gct tat 336 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 tta gga aca
gaa ttt caa aac act ctt ctt aat tca aat ggt aca att 384 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 aat
aaa acg gct ttt gaa aag gct aaa gct aag ttt tta aat aaa gat 432 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 aga ctt gaa tat gga tat att agt act tca tta atg aat gtt tct caa
480 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 ttt gca gga aga cca att att aca aaa ttt aaa gta gca
aaa ggc tca 528 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser 165 170 175 aag gca gga tat att gac cct att agt gct ttt
cag gga caa ctt gaa 576 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 atg ttg ctt cct aga cat agt act tat
cat ata gac gat atg aga ttg 624 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 tct tct gat ggt aaa caa ata
ata att aca gca aca atg atg ggc aca 672 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 gct atc aat cct aaa
gaa ttc aag cgt cga cgt aga aag aaa cgt aga 720 Ala Ile Asn Pro Lys
Glu Phe Lys Arg Arg Arg Arg Lys Lys Arg Arg 225 230 235 240 cag cgt
aga cgt cac cac cac cac cac cac gtc gac tcg agc ggc cgc 768 Gln Arg
Arg Arg His His His His His His Val Asp Ser Ser Gly Arg 245 250 255
atc gtg act gac tga 783 Ile Val Thr Asp 260 30 260 PRT Artificial
Sequence Sequence of C3Basic2 includes sequences from
ADP-ribosyl-transferase C3 (Clostridium botulinum) and a sequence
encoding a random basic amino acid sequence and a histidine tag. 30
Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5
10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp
Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly
Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys
Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly
Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu
Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn
Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr
Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn
Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135
140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln
145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala
Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe
Gln Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His Ser Thr Tyr
His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly Lys Gln Ile
Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile Asn Pro Lys
Glu Phe Lys Arg Arg Arg Arg Lys Lys Arg Arg 225 230 235 240 Gln Arg
Arg Arg His His His His His His Val Asp Ser Ser Gly Arg 245 250 255
Ile Val Thr Asp 260 31 9 PRT Artificial Sequence Reverse HIV-1 Tat
amino acid sequence of C3Basic3 31 Arg Arg Lys Gln Arg Arg Lys Arg
Arg 1 5 32 27 DNA Artificial Sequence Oligonucleotide used in the
cloning of a reverse HIV Tat sequence in C3Basic3 32 agaaggaaac
aaagaagaaa aagaaga 27 33 27 DNA Artificial Sequence Oligonucleotide
used in the cloning of a reverse HIV Tat sequence in C3Basic3 33
tcttcctttg tttcttcttt ttcttct 27 34 771 DNA Artificial Sequence
Sequence of C3Basic3 includes sequences from ADP-ribosyl tranferase
C3 (C. botulinum) and a sequence encoding a reverse HIV-1 Tat amino
acid sequence and a Histidine tag 34 gga tcc tct aga gtc gac ctg
cag gca tgc aat gct tat tcc att aat 48 Gly Ser Ser Arg Val Asp Leu
Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5 10 15 caa aag gct tat tca
aat act tac cag gag ttt act aat att gat caa 96 Gln Lys Ala Tyr Ser
Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 gca aaa gct
tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144 Ala Lys Ala
Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45 tca
gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata 192 Ser
Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50 55
60 aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct tca
240 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser
65 70 75 80 aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt aat
aaa atg 288 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn
Lys Met 85 90 95 aag acc cct gaa aat att atg tta ttt aga ggc gac
gac cct gct tat 336 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp
Asp Pro Ala Tyr 100 105 110 tta gga aca gaa ttt caa aac act ctt ctt
aat tca aat ggt aca att 384 Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu
Asn Ser Asn Gly Thr Ile 115 120 125 aat aaa acg gct ttt gaa aag gct
aaa gct aag ttt tta aat aaa gat 432 Asn Lys Thr Ala Phe Glu Lys Ala
Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140 aga ctt gaa tat gga tat
att agt act tca tta atg aat gtt tct caa 480 Arg Leu Glu Tyr Gly Tyr
Ile Ser Thr Ser Leu Met Asn Val Ser Gln 145 150 155 160 ttt gca gga
aga cca att att aca aaa ttt aaa gta gca aaa ggc tca 528 Phe Ala Gly
Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser 165 170 175 aag
gca gga tat att gac cct att agt gct ttt cag gga caa ctt gaa 576 Lys
Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln Gly Gln Leu Glu 180 185
190 atg ttg ctt cct aga cat agt act tat cat ata gac gat atg aga ttg
624 Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu
195 200 205 tct tct gat ggt aaa caa ata ata att aca gca aca atg atg
ggc aca 672 Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met
Gly Thr 210 215 220 gct atc aat cct aaa gaa ttc aga agg aaa caa aga
aga aaa aga aga 720 Ala Ile Asn Pro Lys Glu Phe Arg Arg Lys Gln Arg
Arg Lys Arg Arg 225 230 235 240 cac cac cac cac cac cac gtc gac tcg
agc ggc cgc atc gtg act gac 768 His His His His His His Val Asp Ser
Ser Gly Arg Ile Val Thr Asp 245 250 255 tga 771 35 256 PRT
Artificial Sequence Sequence of C3Basic3 includes sequences from
ADP-ribosyl tranferase C3 (C. botulinum) and a sequence encoding a
reverse HIV-1 Tat amino acid sequence and a Histidine tag 35 Gly
Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5 10
15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln
20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu
Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser
Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val
Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys Gln Val Glu Leu
Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr Pro Glu Asn Ile
Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110 Leu Gly Thr Glu
Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115 120 125 Asn Lys
Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140
Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln 145
150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys
Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Gln
Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His Ser Thr Tyr His
Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly Lys Gln Ile Ile
Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile Asn Pro Lys Glu
Phe Arg Arg Lys Gln Arg Arg Lys Arg Arg 225 230 235 240 His His His
His His His Val Asp Ser Ser Gly Arg Ile Val Thr Asp 245 250 255 36
887 DNA Artificial Sequence Sequence of C3APLT includes sequences
from ADP-ribosyl transferase C3 (Clostridium botulinum) and a
sequence encoding a proline rich region. 36 gga tcc tct aga gtc gac
ctg cag gca tgc aat gct tat tcc att aat 48 Gly Ser Ser Arg Val Asp
Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn 1 5 10 15 caa aag gct tat
tca aat act tac cag gag ttt act aat att gat caa 96 Gln Lys Ala Tyr
Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln 20 25 30 gca aaa
gct tgg ggt aat gct cag tat aaa aag tat gga cta agc aaa 144 Ala Lys
Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser Lys 35 40 45
tca gaa aaa gaa gct ata gta tca tat act aaa agc gct agt gaa ata 192
Ser Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile 50
55 60 aat gga aag cta aga caa aat aag gga gtt atc aat gga ttt cct
tca 240 Asn Gly Lys Leu Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro
Ser 65 70 75 80 aat tta ata aaa caa gtt gaa ctt tta gat aaa tct ttt
aat aaa atg 288 Asn Leu Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe
Asn Lys Met 85 90 95 aag acc cct gaa aat att atg tta ttt aga ggc
gac gac cct gct tat 336 Lys Thr Pro Glu Asn Ile Met Leu Phe Arg Gly
Asp Asp Pro Ala Tyr 100 105 110 tta gga aca gaa ttt caa aac act ctt
ctt aat tca aat ggt aca att 384 Leu Gly Thr Glu Phe Gln Asn Thr Leu
Leu Asn Ser Asn Gly Thr Ile 115 120 125 aat aaa acg gct ttt gaa aag
gct aaa gct aag ttt tta aat aaa gat 432 Asn Lys Thr Ala Phe Glu Lys
Ala Lys Ala Lys Phe Leu Asn Lys Asp 130 135 140 aga ctt gaa tat gga
tat att agt act tca tta atg aat gtt tct caa 480 Arg Leu Glu Tyr Gly
Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln 145 150 155 160 ttt gca
gga aga cca att att aca aaa ttt aaa gta gca aaa ggc tca 528 Phe Ala
Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly Ser
165 170 175 aag gca gga tat att gac cct att agt gct ttt gca gga caa
ctt gaa 576 Lys Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly Gln
Leu Glu 180 185 190 atg ttg ctt cct aga cat agt act tat cat ata gac
gat atg aga ttg 624 Met Leu Leu Pro Arg His Ser Thr Tyr His Ile Asp
Asp Met Arg Leu 195 200 205 tct tct gat ggt aaa caa ata ata att aca
gca aca atg atg ggc aca 672 Ser Ser Asp Gly Lys Gln Ile Ile Ile Thr
Ala Thr Met Met Gly Thr 210 215 220 gct atc aat cct aaa gaa ttc gtg
atg aat ccc gca aac gcg caa ggc 720 Ala Ile Asn Pro Lys Glu Phe Val
Met Asn Pro Ala Asn Ala Gln Gly 225 230 235 240 aga cat aca ccc ggt
acc aga ctc tag agctagagaa ggagtttcac 767 Arg His Thr Pro Gly Thr
Arg Leu 245 ttcaatcgct acttgacccg tcggcgaagg atcgagatcg cccacgccct
gtgcctcacg 827 gagcgccaga taaagatttg gttccagaat cggcgcatga
agtggaagaa ggagaactga 887 37 248 PRT Artificial Sequence Sequence
of C3APLT includes sequences from ADP-ribosyl transferase C3
(Clostridium botulinum) and a sequence encoding a proline rich
region. 37 Gly Ser Ser Arg Val Asp Leu Gln Ala Cys Asn Ala Tyr Ser
Ile Asn 1 5 10 15 Gln Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr
Asn Ile Asp Gln 20 25 30 Ala Lys Ala Trp Gly Asn Ala Gln Tyr Lys
Lys Tyr Gly Leu Ser Lys 35 40 45 Ser Glu Lys Glu Ala Ile Val Ser
Tyr Thr Lys Ser Ala Ser Glu Ile 50 55 60 Asn Gly Lys Leu Arg Gln
Asn Lys Gly Val Ile Asn Gly Phe Pro Ser 65 70 75 80 Asn Leu Ile Lys
Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met 85 90 95 Lys Thr
Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr 100 105 110
Leu Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile 115
120 125 Asn Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys
Asp 130 135 140 Arg Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn
Val Ser Gln 145 150 155 160 Phe Ala Gly Arg Pro Ile Ile Thr Lys Phe
Lys Val Ala Lys Gly Ser 165 170 175 Lys Ala Gly Tyr Ile Asp Pro Ile
Ser Ala Phe Ala Gly Gln Leu Glu 180 185 190 Met Leu Leu Pro Arg His
Ser Thr Tyr His Ile Asp Asp Met Arg Leu 195 200 205 Ser Ser Asp Gly
Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr 210 215 220 Ala Ile
Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly 225 230 235
240 Arg His Thr Pro Gly Thr Arg Leu 245 38 37 DNA Artificial
Sequence Oligonucleotide used in the cloning of C3APLT in pET
vector 38 ggatctggtt ccgcgtcata tgtctagagt cgacctg 37 39 32 DNA
Artificial Sequence Oligonucleotide used in the cloning of C3APLT
in pET vector 39 cgcggatcca ttagttctcc ttcttccact tc 32 40 24 DNA
Artificial Sequence Oligonucleotide used in the sequencing of
C3APLT 40 aaattaatac gactcactat aggg 24 41 19 DNA Artificial
Sequence Oligonucleotide used in the sequencing of C3APLT 41
gctagttatt gctcagcgg 19 42 888 DNA Artificial Sequence Sequence of
C3APLT in a pET vector includes sequences from ADP-ribosyl
transferase C3 (Clostridium botulinum) and a sequence encoding a
proline rich region. 42 atg tct aga gtc gca ctg cag gca tgc aat gct
tat tcc att aat caa 48 Met Ser Arg Val Ala Leu Gln Ala Cys Asn Ala
Tyr Ser Ile Asn Gln 1 5 10 15 aag gct tat tca aat act tac cag gag
ttt act aat att gat caa gca 96 Lys Ala Tyr Ser Asn Thr Tyr Gln Glu
Phe Thr Asn Ile Asp Gln Ala 20 25 30 aaa gct tgg ggt aat gct cag
tat aaa aag tat gga cta agc aaa tca 144 Lys Ala Trp Gly Asn Ala Gln
Tyr Lys Lys Tyr Gly Leu Ser Lys Ser 35 40 45 gaa aaa gaa gct ata
gta tca tat act aaa agc gct agt gaa ata aat 192 Glu Lys Glu Ala Ile
Val Ser Tyr Thr Lys Ser Ala Ser Glu Ile Asn 50 55 60 gga aag cta
aga caa aat aag gga gtt atc aat gga ttt cct tca aat 240 Gly Lys Leu
Arg Gln Asn Lys Gly Val Ile Asn Gly Phe Pro Ser Asn 65 70 75 80 tta
ata aaa caa gtt gaa ctt tta gat aaa tct ttt aat aaa atg aag 288 Leu
Ile Lys Gln Val Glu Leu Leu Asp Lys Ser Phe Asn Lys Met Lys 85 90
95 acc cct gaa aat att atg tta ttt aga ggc gac gac cct gct tat tta
336 Thr Pro Glu Asn Ile Met Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu
100 105 110 gga aca gaa ttt caa aac act ctt ctt aat tca aat ggt aca
att aat 384 Gly Thr Glu Phe Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr
Ile Asn 115 120 125 aaa acg gct ttt gaa aag gct aaa gct aag ttt tta
aat aaa gat aga 432 Lys Thr Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu
Asn Lys Asp Arg 130 135 140 ctt gaa tat gga tat att agt act tca tta
atg aat gtt tct caa ttt 480 Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu
Met Asn Val Ser Gln Phe 145 150 155 160 gca gga aga cca att att aca
aaa ttt aaa gta gca aaa ggc tca aag 528 Ala Gly Arg Pro Ile Ile Thr
Lys Phe Lys Val Ala Lys Gly Ser Lys 165 170 175 gca gga tat att gac
cct att agt gct ttt gca gga caa ctt gaa atg 576 Ala Gly Tyr Ile Asp
Pro Ile Ser Ala Phe Ala Gly Gln Leu Glu Met 180 185 190 ttg ctt cct
aga cat agt act tat cat ata gac gat atg aga ttg tct 624 Leu Leu Pro
Arg His Ser Thr Tyr His Ile Asp Asp Met Arg Leu Ser 195 200 205 tct
gat ggt aaa caa ata ata att aca gca aca atg atg ggc aca gct 672 Ser
Asp Gly Lys Gln Ile Ile Ile Thr Ala Thr Met Met Gly Thr Ala 210 215
220 atc aat cct aaa gaa ttc gtg atg aat ccc gca aac gcg caa ggc aga
720 Ile Asn Pro Lys Glu Phe Val Met Asn Pro Ala Asn Ala Gln Gly Arg
225 230 235 240 cat aca ccc ggt acc aga ctc tag agctagagaa
ggagtttcac ttcaatcgct 774 His Thr Pro Gly Thr Arg Leu 245
acttgacccg tcggcgaagg atcgagatcg cccacgccct gtgcctcacg gagcgccaga
834 taaagatttg gttccagaat cggcgcatga agtggaagaa ggaggactaa ctga 888
43 247 PRT Artificial Sequence Sequence of C3APLT in a pET vector
includes sequences from ADP-ribosyl transferase C3 (Clostridium
botulinum) and a sequence encoding a proline rich region. 43 Met
Ser Arg Val Ala Leu Gln Ala Cys Asn Ala Tyr Ser Ile Asn Gln 1 5 10
15 Lys Ala Tyr Ser Asn Thr Tyr Gln Glu Phe Thr Asn Ile Asp Gln Ala
20 25 30 Lys Ala Trp Gly Asn Ala Gln Tyr Lys Lys Tyr Gly Leu Ser
Lys Ser 35 40 45 Glu Lys Glu Ala Ile Val Ser Tyr Thr Lys Ser Ala
Ser Glu Ile Asn 50 55 60 Gly Lys Leu Arg Gln Asn Lys Gly Val Ile
Asn Gly Phe Pro Ser Asn 65 70 75 80 Leu Ile Lys Gln Val Glu Leu Leu
Asp Lys Ser Phe Asn Lys Met Lys 85 90 95 Thr Pro Glu Asn Ile Met
Leu Phe Arg Gly Asp Asp Pro Ala Tyr Leu 100 105 110 Gly Thr Glu Phe
Gln Asn Thr Leu Leu Asn Ser Asn Gly Thr Ile Asn 115 120 125 Lys Thr
Ala Phe Glu Lys Ala Lys Ala Lys Phe Leu Asn Lys Asp Arg 130 135 140
Leu Glu Tyr Gly Tyr Ile Ser Thr Ser Leu Met Asn Val Ser Gln Phe 145
150 155 160 Ala Gly Arg Pro Ile Ile Thr Lys Phe Lys Val Ala Lys Gly
Ser Lys 165 170 175 Ala Gly Tyr Ile Asp Pro Ile Ser Ala Phe Ala Gly
Gln Leu Glu Met 180 185 190 Leu Leu Pro Arg His Ser Thr Tyr His Ile
Asp Asp Met Arg Leu Ser 195 200 205 Ser Asp Gly Lys Gln Ile Ile Ile
Thr Ala Thr Met Met Gly Thr Ala 210 215 220 Ile Asn Pro Lys Glu Phe
Val Met Asn Pro Ala Asn Ala Gln Gly Arg 225 230 235 240 His Thr Pro
Gly Thr Arg Leu 245 44 64 PRT Artificial Sequence Amino acid
sequence of Antennapedia from C3APL 44 Val Met Glu Ser Arg Lys Arg
Ala Arg Gln Thr Tyr Thr Arg Tyr Gln 1 5 10 15 Thr Leu Glu Leu Glu
Lys Glu Phe His Phe Asn Arg Tyr Leu Thr Arg 20 25 30 Arg Arg Arg
Ile Glu Ile Ala His Ala Leu Cys Leu Thr Glu Arg Gln 35 40 45 Ile
Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Glu Asn 50 55
60 45 19 PRT Artificial Sequence Amino acid sequence of
Antennapedia from C3APS 45 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg Met Lys Trp Lys Lys 1 5 10 15 Val Asp Ser 46 60 PRT Artificial
Sequence Amino acid sequence of HIV-1 Tat from C3-TL 46 Lys His Pro
Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys 1 5 10 15 Lys
Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr Lys Ala Leu 20 25
30 Gly Ile Ser Tyr Gly Arg Lys Arg Arg Gln Arg Arg Arg Ala His Gln
35 40 45 Asn Ser Gln Thr His Gln Ala Ser Leu Ser Lys Gln 50 55 60
47 20 PRT Artificial Sequence Amino acid sequence of HIV-1 Tat from
C3-TS 47 Tyr Gly Ala Lys Lys Arg Arg Gln Arg Arg Arg Val Asp Ser
Ser Gly 1 5 10 15 Pro His Arg Asp 20 48 17 PRT Artificial Sequence
Amino acid sequence of the proline rich region of C3APLT 48 Val Met
Asn Pro Ala Asn Ala Gln Gly Arg His Thr Pro Gly Thr Arg 1 5 10 15
Leu
* * * * *