U.S. patent application number 09/947137 was filed with the patent office on 2002-09-26 for methods and compositions for targeting compounds to muscle.
This patent application is currently assigned to Auburn University. Invention is credited to Samoilova, Tatiana I., Smith, Bruce F..
Application Number | 20020137023 09/947137 |
Document ID | / |
Family ID | 21951436 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137023 |
Kind Code |
A1 |
Smith, Bruce F. ; et
al. |
September 26, 2002 |
Methods and compositions for targeting compounds to muscle
Abstract
Compositions for use in targeting therapies to muscle cells are
provided. The compositions comprise peptides which are capable of
binding muscle cells in vivo.
Inventors: |
Smith, Bruce F.; (Auburn,
AL) ; Samoilova, Tatiana I.; (Auburn, AL) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Auburn University
|
Family ID: |
21951436 |
Appl. No.: |
09/947137 |
Filed: |
September 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09947137 |
Sep 5, 2001 |
|
|
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09084605 |
May 26, 1998 |
|
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60047863 |
May 29, 1997 |
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Current U.S.
Class: |
435/5 ; 435/183;
435/6.16 |
Current CPC
Class: |
G01N 2800/2878 20130101;
C12N 15/87 20130101; A61K 48/00 20130101; A61K 47/62 20170801; C07K
14/4708 20130101; G01N 33/6887 20130101; G01N 2800/2885 20130101;
A61K 38/00 20130101; C07K 7/06 20130101; C07K 14/4716 20130101 |
Class at
Publication: |
435/5 ; 435/6;
435/183 |
International
Class: |
C12Q 001/70; C12Q
001/68; C12N 009/00 |
Claims
That which is claimed:
1. A method for determining muscle-type specific peptides, said
method comprising constructing a bacteriophage library which
expresses random peptides at the amino terminus of a phage protein,
and hybridizing the resulting phage to muscle cells of interest
wherein said peptides are selected based on in vivo binding.
2. The method of claim 1, wherein said peptides are random
peptides.
3. The method of claim 1, wherein said peptides are 7-mer
peptides.
4. The method of claim 1, wherein said muscle-type specific
peptides are species specific.
5. The peptides of claim 1, wherein said muscle-type specific
peptides are species independent.
6. The method of claim 1, wherein said muscle-type specific
peptides are capable of binding to specific muscle cells selected
from the group consisting of smooth, skeletal and cardiac muscle
cells.
7. The method of claim 6, wherein said muscle cells are skeletal
muscle cells.
8. The method of claim 6, wherein said muscle cells are cardiac
muscle cells.
9. The method of claim 1, wherein said muscle-type specific peptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.
10. A method for targeting a pharmaceutical composition to muscle
cells, said method comprising ligating a muscle-type specific
peptide to said pharmaceutical composition.
11. The method of claim 10, wherein said pharmaceutical composition
is selected from drugs, proteins, and nucleic acids.
12. The method of claim 10, wherein said pharmaceutical composition
is useful for treating a myopathy.
13. The method of claim 10, wherein said peptides are random
peptides.
14. The method of claim 10, wherein said peptides are 7-mer
peptides.
15. The method of claim 10, wherein said muscle-type specific
peptides are species specific.
16. The peptides of claim 10, wherein said muscle-type specific
peptides are species independent.
17. The method of claim 10, wherein said muscle-type specific
peptides are capable of binding to specific muscle cells selected
from the group consisting of smooth, skeletal and cardiac muscle
cells.
18. The method of claim 17, wherein said muscle cells are skeletal
muscle cells.
19. The method of claim 17, wherein said muscle cells are cardiac
muscle cells.
20. The method of claim 10, wherein said muscle-type specific
peptide comprises an amino acid sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID
NO:28.
21. A nucleotide sequence encoding the peptide having an amino acid
sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.
22. A method for treating a patient having a muscular disorder,
said method comprising administering to said patient a
pharmaceutically-effect- ive amount of a composition comprising a
muscle-type specific peptide operably linked to a pharmaceutical
composition useful for treating said disorder.
23. The method of claim 22, wherein said peptide is species
specific.
24. The method of claim 22, wherein said peptide is species
independent.
25. The method of claim 22, wherein said peptide is capable of
binding specific muscle cells selected from the group consisting of
cardiac, skeletal, and smooth muscle cells.
26. The method of claim 22, wherein said muscle-type specific
peptide comprises an amino acid sequence selected from the group
consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID
NO:28.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/084,605, filed May 26, 1998 and provisional application
serial No. 60/047,863 filed May 29, 1997, which are hereby
incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] The invention relates to methods for gene therapy,
particularly for targeting genes, proteins, pharmaceuticals, or
other compounds to muscle.
BACKGROUND OF THE INVENTION
[0003] The capabilities to introduce a particular foreign or native
gene sequence into a mammal and to control the expression of that
gene are of substantial value in the fields of medical and
biological research. Such capabilities provide a means for studying
gene regulation and for designing a therapeutic basis for the
treatment of disease.
[0004] In addition to introducing the gene into mammals, providing
expression of the gene at the site of interest remains a challenge.
Methods have been developed to deliver DNA to target cells by
capitalizing on indigenous cellular pathways of macromolecular
transport. In this regard, gene transfer has been accomplished via
the receptor-mediated encytosis pathway employing molecular
conjugate vectors.
[0005] Inherited diseases of muscle pose a therapeutic challenge,
as muscle is the single largest tissue of the body. Pharmacologic
approaches do not significantly alter the course of many of these
diseases as such approaches fail to correct the underlying genetic
deficit. New approaches, relying on the transfer of genetic
material have been advocated. However, current methodologies used
for gene therapy are limited in their usefulness with regard to
treating myopathies. Local administration of gene therapy vectors
or transplantation of donor cells only treat the immediate area of
the injection site. Effective therapy with these methods requires
multiple injections and carry the safety risks associated with
disease transplantation and the use of immunosuppressive drugs.
[0006] Duchenne muscular dystrophy is probably the most common
inherited progressive lethal disorder of mankind. The rate of
occurrence of the disease is attributable to the very high mutation
rate of the gene. The progression of the clinical disease is
characterized by skeletal muscle tissue deterioration and wasting.
Duchenne muscular dystrophy (DMD) follows a degenerative course
which confines sufferers to a wheelchair by the age of about 12
years old and results in death by the third decade due to cardiac
or respiratory failure.
[0007] DMD results from mutations, mainly frame-shift deletions, in
a single, recessively inherited gene. The gene has been cloned and
represents the largest gene so far identified in the human genome,
spanning at least 2.3 megabases in the short arm of the X
chromosome.
[0008] Positional cloning of the X-linked gene has revealed that
defects of the dystrophin gene lead to either Duchenne or Becker
muscular dystrophy (BMD). The 14 kilobase (kb) dystrophin mRNA
encodes a 3685 amino acid protein of 427 kilodaltons (kD) with
overall similarity to the cytoskeletal proteins .beta.-spectrin and
.alpha.-actinin. These proteins perform structural roles in static
and dynamic cellular processes and all cell types. Dystrophin is
associated with the cytoplasmic face of the sarcolemma and is thus
an essential component of the muscle cytoskeleton. Although the
exact function of dystrophin is unknown, it has been postulated to
contribute to stabilization of the sarcolemma.
[0009] The current approaches to the in vivo transfer of dystrophin
cDNA for the treatment of DMD have involved direct intramuscular
injection of naked plasmid DNA or recombinant viral vectors.
However, these techniques suffer from the limitation that skeletal
muscle comprises a large proportion of the cells of the body,
making widespread intramuscular transduction impractical in a
clinical context. Furthermore, some muscles affected in DMD, such
as the heart and diaphragm, are not readily accessible to direct
injection.
[0010] Current approaches to gene therapy in inherited myopathies
are limited in practicality either by the mode of administration or
the capacity of the vector used for gene transfer. Therefore,
methods are needed for targeting specific compositions to muscle
cells.
SUMMARY OF THE INVENTION
[0011] Compositions and methods for targeting genes, proteins,
pharmaceuticals, or other compounds to muscle are provided. The
compositions comprise peptide sequences which bind muscle, in vivo,
with high specificity. Both species-specific and
species-independent sequences have been determined.
[0012] The compositions are useful in gene therapy methods for the
treatment of myopathies, gene therapy of disease where muscles
potentially serve as reservoirs of protein production, and delivery
of a wide variety of drugs to muscles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 provides the structure of phage in the library.
[0014] FIG. 2 provides the scheme for selection of tissue-specific
phage in vivo.
[0015] FIG. 3 provides the experimental scheme and results. The
sequences shown in the Figure are set forth in SEQ ID NO:1
(WDANGKT), SEQ ID NO:2 (ASSLNIA), SEQ ID NO:3 (LAPKQLP), SEQ ID
NO:4 (VSAAPYP), SEQ ID NO:5 (AAANVWH), SEQ ID NO:6 (AYPGFAL), SEQ
ID NO:7 (TATITTK), SEQ ID NO:8 (MSTQSIN), SEQ ID NO:9 (SGLPAYP),
SEQ ID NO:10 (AGMAHIR), SEQ ID NO:11 (STSXITH), SEQ ID NO:12
(SYFSAPP), SEQ ID NO:13 (QLSLLLA), SEQ ID NO:14 (WKPATFY), SEQ ID
NO:15 (APLYPPS), SEQ ID NO:16 (HLSNWPR), SEQ ID NO:17 (SMQTHPF),
SEQ ID NO:18 (SHWHNSE), SEQ ID NO:19 (HPAHVAK) and SEQ ID NO:20
(TTQHMLK).
DETAILED DESCRIPTION OF THE INVENTION
[0016] The invention is drawn to peptide sequences that are capable
of binding muscle with high specificity. Both species-specific and
species-independent peptide sequences have been identified. The
peptide sequences are useful for targeting components to
muscle.
[0017] The peptides of the invention are generally short peptide
ligands. The peptide ligands exhibit at least three-fold,
preferably ten-fold, more preferably greater than ten-fold binding
affinity for muscle cells.
[0018] The peptides of the invention are cell-binding and
cell-entry peptides. For the most part, the peptides will comprise
at least about 5 to about 50 amino acids, preferably at least about
5 to about 30 amino acids, more preferably at least about 7 to
about 20 amino acids. It is recognized that consensus sequences may
be identified among the peptides that are capable of binding to a
target. Such consensus sequences identify key amino acids or
patterns of amino acids that are essential for binding. Consensus
sequences may be determined by an analysis of peptide patterns that
are capable of binding muscle cells. Once recognized the consensus
regions will be used in constructing other peptides for use in
muscular targeting. Such consensus sequences may be tested by
constructing peptides and determining the effect of the consensus
sequence on binding. In this manner, as long as the consensus
sequence is present, the peptide will bind the target. In some
cases, longer peptides will be useful as such peptides may be more
easily bound or more readily enter into the target cell.
[0019] The peptides can be classified into linear, cyclic and
conformational types. While the invention is not bound by any
particular mode of action, it is postulated that the shorter
peptides, generally from about 7 to about 20 amino acids are
involved in linear binding to the target muscle cells. Longer
peptides assume conformational folding and are involved in
conformational binding. Cyclic peptide structures can also be
constructed for use in the invention. In this manner, a core
peptide region such as a consensus peptide binding sequence will be
flanked with identical sequences to form cyclic peptides. Such
libraries are available commercially, for example the Ph.D..TM.
Phage display peptide library kits from New England Biolabs, Inc.
See also, Parmley et al. (1988) Gene 73:305-318; Cortese et al.
(1995) Curr. Opin. Biotechnol 6:73-80; Noren (1996) NEB Transcript
8(1):1-5; and Devlin et al. (1990) Science 249:404-406.
[0020] Peptides of the invention can be determined which are
capable of binding any type of muscle cells including skeletal,
smooth, cardiac, and the like. Based on the selective binding
protocols, peptides which are tissue or muscle-type specific or
alternatively which are capable of binding to different muscle
cells can be determined. In the same manner, the peptides may be
species independent, that is, the peptides will bind to the muscle
type from any mammalian species. Alternatively, the peptides may be
species specific. By species specific is intended that peptides are
specific for muscle cells from a particular species and will not
bind to muscle cells from another species. Therefore, the peptides
may be characterized by tissue specificity or alternatively by
species specificity. Mammalian species of interest include, but are
not limited to human, rat, dog, chimpanzee, etc.
[0021] Multiple muscle targets can be utilized to select for
muscle-binding peptides. Peptides can be selected against
differentiated myotube cell lines from any mammal, as well as
against primary muscle samples from mammals, including quadracep,
tibialis anterior, soleus, cardiac, and the like.
[0022] Methods are available in the art for the determination of
the peptides of the invention. Such methods include selection from
a bacteriophage library which expresses random peptides, mirror
image phage display to isolate naturally-occurring L-enantiomers in
a peptide library, and the like. See, for example, Barry et al.
(1996) Nature Medicine 2:299-305; Schumacher et al. (1996) Science
271:1854-1857; Pasqualini et al. (1996) Nature 380:364-366; and the
references cited therein, herein incorporated by reference.
[0023] Peptides of the invention can be determined from phage
libraries which have been used to select random peptides that bind
single proteins. See, Barry et al. (1996) Nature Medicine
2:299-305; Devlin et al. (1990) 249:404-406; Cwirla et al. (1990)
Proc. Natl. Acad. Sci. USA 87:6378-6382; and the references cited
therein. In this manner, peptide libraries can be constructed
utilizing a number of random amino acids. The random amino acids
are fused to the amino terminus of a phage protein and expressed as
a bacteriophage library. See, Barry et al. (1996) Nature Medicine
2:299-305, herein incorporated by reference. The phage is
hybridized to the cells of interest, at different temperatures,
generally about 4.degree. C. and about 37.degree. C. After repeated
selection, peptides which exhibit a higher affinity for the cells
of interest are isolated. Methods for preparing libraries
containing diverse populations are also disclosed in Gordon et al.
(1994) J. Med. Chem. 37:1385-1401; Ecker and Crooke (1995)
BioTechnology 13:351-360; Goodman and Ro, Peptidomimetics For Drug
Design, in "Burger's Medicinal Chemistry and Drug Discovery", Vol.
1, M. E. Wolff (Ed.) John Wiley & Sons 1995, pages 803-861;
Blondelle et al. (1995) Trends Anal. Chem. 14:83-92; and Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, 1989. Each of these references are herein incorporated by
reference.
[0024] After in vitro screening of the peptides, the peptides of
the invention are selected based on in vivo binding. Such methods
for in vivo binding are known in the art. See, for example,
Pasqualini et al. (1996) Nature 380:364-366, and the references
cited therein. While the invention is discussed in terms of
peptides, it is recognized that other molecules may be identified
in the same manner. Such molecules include organic chemicals,
modified peptides, proteins such as antibodies, antibody fragments,
and the like.
[0025] The screening method of the invention comprises
administering a library of molecules to the muscle cells of
interest and identifying those molecules which are capable of
binding. See, for example, U.S. Pat. No. 5,622,699, herein
incorporated by reference.
[0026] For peptides capable of binding human muscle cells,
chimpanzees can be used as the target mammal. Thus, species
independent peptides can be identified which do not cross react
with other mammalian species. For peptides which cross-react or are
species independent, after screening in a first mammal, the
peptides are screened in at least a second mammal and those which
bind can be determined. As noted above, because the mammal is
typically sacrificed to determine binding, a primate species can be
used for human, generally chimpanzees.
[0027] The in vivo selection method of the invention represents the
first methods where peptides have been determined which bind
differentiated or "adult" muscle. Previous attempts have selected
based on muscle cells in tissue culture. Muscle cells in culture
are not characteristic of adult muscle even if collected from
developed muscle. Once the cells are cultured they convert to an
embryonic phenotype. See Gambke et al. (1984) J. Biol Chem.
259:12092-12100. Thus, the peptides of the invention have different
binding specificities and affinities. It is using the methods of
the invention that tissue and species-independent peptides can be
determined.
[0028] Once peptides have been selected which show an affinity for
the target tissue, they may be modified by methods known in the
art. Such methods include random mutagenesis, as well as synthesis
of the compounds for selected amino acid substitutions. Peptides of
various length can be constructed and tested for the effect on
binding affinity and specificity. In this manner, the binding
affinity may be increased or altered. Thus, peptides may be
identified which exhibit specific binding to muscle cells, as well
as peptides which exhibit specific binding and internalization by
the muscle cells of interest.
[0029] The peptides find use in targeting genes, proteins,
pharmaceuticals, or other compounds to particular muscle tissue. In
this manner, the peptides can be used in any vector systems for
delivery of specific nucleotides or compositions to the target
cells. By nucleotide is intended gene sequences, DNA, RNA, as well
as antisense nucleic acids.
[0030] The nucleotide sequences encoding the muscle-specific
peptides are also encompassed. Where necessary, the nucleotide
sequences can be used in the construction of fusion proteins or
vectors for use in the invention. Such methods are known in the
art. Additionally the construction of expression cassettes are
known as well as promoters, terminators, enhancers, etc., necessary
for expression.
[0031] A number of vector systems are known for the introduction of
foreign or native genes into mammalian cells. These include SV40
virus (See, e.g., Okayama et al. (1985) Mol. Cell Biol.
5:1136-1142); Bovine papilloma virus (See, e.g., DiMaio et al.
(1982) Proc. Natl. Acad. Sci. USA 79:4030-4034); adenovirus (See,
e.g., Morin et al. (1987) Proc. Natl. Acad. Sci. USA 84:4626; Yifan
et al. (1995) Proc. Natl. Acad. Sci. USA 92:1401-1405; Yang et al.
(1996) Gene Ther. 3:137-144; Tripathy et al. (1996) Nat. Med.
2:545-550; Quantin et al. (1992) Proc. Natl. Acad. Sci. USA
89:2581-2584; Rosenfeld et al. (1991) Science 252:431-434; Wagner
(1992) Proc. Natl. Acad. Sci. USA 89:6099-6103; Curiel et al.
(1992) Human Gene Therapy 3:147-154; Curiel (1991) Proc. Natl.
Acad. Sci. USA 88:8850-8854; LeGal LaSalle et al. (1993) Science
259:590-599); Kass-Eisler et al. (1993) Proc. Natl. Acad. Sci. USA
90:11498-11502); adeno-associated virus (See, e.g., Muzyczka et al.
(1994) J. Clin. Invest. 94:1351; Xiao et al. (1996) J. Virol.
70:8098-8108); herpes simplex virus (See, e.g., Geller et al.
(1988) Science 241:1667; Huard et al. (1995) Gene Therapy
2:385-392; U.S. Pat. No. 5,501,979); retrovirus-based vectors (See,
for example, Curran et al. (1982) J. Virol. 44:674-682; Gazit et
al. (1986) J. Virol. 60:19-28; Miller, A. D. (1992) Curr. Top.
Microbiol. Immunol. 158:1-24; Cavanaugh et al. (1994) Proc. Natl.
Acad. Sci. USA 91:7071-7075; Smith et al. (1990) Mol. Cell. Biol.
10:3268-3271); herein incorporated by reference.
[0032] In the same manner, the peptides can be used in any
mammalian expression vector to target the expression system to the
appropriate target muscle cells. See, for example, Wu et al. (1991)
J. Biol. Chem. 266:14338-14342; Wu and Wu (J. Biol Chem. (1988))
263:14621-14624; Wu et al. (1989) J. Biol. Chem. 264:16985-16987;
Zenke et al. (1990) Proc. Natl. Acad. Sci. USA 87:3655-3659; Wagner
et al. (1990) 87:3410-3414.
[0033] Standard techniques for the construction of the vectors of
the present invention are well-known to those of ordinary skill in
the art and can be found in such references as Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring
Harbor, N.Y., 1989). A variety of strategies are available for
ligating fragments of DNA, the choice of which depends on the
nature of the termini of the DNA fragments and which choices can be
readily made by those of skill in the art.
[0034] Where the peptides of the invention are targeting a gene for
expression in muscles, the gene to be expressed will be provided in
an expression cassette with the appropriate regulatory elements
necessary for expression of the gene. Such regulatory elements are
well known in the art and include promoters, terminators,
enhancers, etc.
[0035] The peptides of the invention may also be utilized to target
liposomes, polylysine, or other polycation conjugates, and
synthetic molecules. See, for example, de Haan et al. 91996)
Immunology 89:488-493; Gorlach et al. (1996) DTWDTsch Tierarytl
Wochenschr 103:312-315; Benameur et al. (1995) J. Phar. Pharmacol.
47:812-817; Bonanomi et al. (1987) J. Microencapsul 4:189-200;
Zekom et al. (1995) Transplant Proc. 27:3362-3363.
[0036] In this manner, the peptides of the invention can be used to
provide therapies for neuromuscular and muscular diseases,
including Duchenne muscular dystrophy, Becker muscular dystrophy,
myotic dystrophy, myotiona congenita, dermatomyositis,
polymyositis, polyneuropathy, and other myopathies. That is, genes,
proteins, or pharmaceuticals can be directed to muscles in those
patients suffering from the particular disease.
[0037] Peptides of the invention also find use in treating cardiac
diseases and in targeting drugs to the heart. See, for example,
Onodera et al. (1997) Transplant Proc. 29:1907-1908; Langtry (1997)
Drugs 53:867-884; Donahue (1997) Proc. Natl. Acad. Sci. USA
94:4664-4668; Kass-Eisler et al. (1993) Proc. Natl. Acad. Sci. USA
90:11498-11502; Guzman et al. (1993) Circ. Res. 73:1202-1207; Barr
et al. (1994) Gene Ther. 1:51-58; Gojo et al. (1997) J. Ther.
Cardiovas. Surg. 113:10-18; March et al. (1995) Hum Gene. Ther.
6:41-53; Villarreal et al. (1996) J. Mol. Cell Cardiol. 28:735-742;
Wang et al. (1996 Transplantation 61:1726-1729; Lee et al. (1996)
J. Thorac Cardiovasc. Surg. 111:246-252; and the references cited
therein.
[0038] In this manner, cardiac peptide directed therapies are
useful for the treatment of a number of acquired and inherited
cardiovascular diseases. Previous gene transfer approaches have
been limited by relatively low efficiencies of gene transduction.
Thus, the present approach provides a means to increase recombinant
gene expression and pharmaceutical concentration in the muscular
layers of the coronary arteries as well as the myocardium.
[0039] The compositions of the invention may be provided as
pharmaceutical formulations suitable for parenteral (e.g.,
subcutaneous, intradermal, intramuscular, intravenous and
intraarticular), oral, or inhalation administration. Alternatively,
pharmaceutical formulations of the present invention may be
suitable for administration to the mucous membranes of the subject
(e.g., intranasal administration). The formulations may be
conveniently prepared in unit dosage form and may be prepared by
any of the methods well-known in the art.
[0040] Any inert pharmaceutically-acceptable carrier may be used,
such as saline, or phosphate-buffered saline, or any such carrier
in which the compositions of the present invention have suitable
solubility properties for use in the methods of the present
invention. Reference is made to Remington's Pharmaceutical
Sciences, Merck Publishing Company Easton, Pa., Osol (ed.) (1980)
for methods of formulating pharmaceutical compositions.
[0041] The peptides of the invention may also be utilized for gene
therapy of disease where muscles may serve as a reservoir of
protein production, such as diabetes, hemophilia, and the like.
Proteins which can be delivered include hGH (Dhawan et al. (1991)
Science 254:1509-1512); factor 1.times. protein (Dai et al. (1992)
Proc. Natl. Acad. Sci. USA 89:10892-10895; Yas et al. (1992) Proc.
Natl. Acad. Sci. USA 89:3357-3361; and Roman et al. (1992) Somatic
Cell and Molecular Genetics 18:247-258) insulin; and the like. See,
generally, Barr et al. (1991) Science 245:1507-1509.
[0042] In the same manner, the peptides of the invention may be
utilized to target pharmaceuticals and chemotherapeutic agents to
treat muscle disease such as cancers or tumors of muscle origin.
See, for example, Velez-Yanguas et al. (1996) Orthop. Clin. North
Am. 27:545-549; Douglas et al. (1995) Tumor Targeting 1:67-84; and
LeBricon (1995) Metabolism 44:1340-1348.
[0043] In one example of methods of the invention, the peptides are
utilized in a vector system as the ligand-binding domain for the
treatment of muscular disease, particularly DMD. The method
provides intravenous injection of an expression vector comprising
the targeting peptides of the invention operably linked with an
expression cassette comprising the Duchenne dystrophy gene to
deliver the dystrophin product to the muscles.
[0044] Because of the insert size limitations of most retroviral
and adenoviral vectors, they may be incapable of accommodating the
14 kb fall-length dystrophin cDNA. In these cases, a 6.3 kb
dystrophin cDNA may be utilized and expressed in the vector. See,
England et al. (1990) Nature 343:180-182.
[0045] Alternatively, adenovirus vectors are known in the art which
are capable of expressing the full-length DMD gene. See, for
example, Haeker et al. (1996) Human Gene Therapy 7:1907-1914, which
discloses the expression of full-length human dystrophin from an
adenoviral vector which has all viral genes deleted. The peptides
of the invention could be used to direct such a vector system to
skeletal muscle.
[0046] The following experiments are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
[0047] Phage Presentation Libraries May be Utilized to Identify
Novel Ligands for Gene Transfer to Muscle
[0048] A key factor in any targeting schema is the availability of
appropriate specific molecules on the target cells which can be
exploited. Of note in this regard, the available ligands for
targeting muscle were until recently limited in number and
functional applicability. Many markers of skeletal muscle were
described during studies of differentiation and therefore are only
expressed in myoblasts, mononuclear myocytes, and immature
myotubes. However, if skeletal muscle is to be rescued from
degeneration due to the absence of dystrophin, mature adult
myotubes must be targeted. Thus, the identification of cell surface
markers specific to mature myotubes is of critical importance to
the implementation of the gene delivery strategies for the
treatment of DMD. Systems which, while utilizing a variety of
different approaches, fundamentally share the similarity of
examining libraries of peptides for their ability to bind to
specific cell types, both in vitro and in vivo are useful. See,
FIG. 1 which sets forth the structure of phage in the library.
EXAMPLE 2
[0049] Selection of Peptides against Myoblasts and Myotubes
[0050] For MD gene therapy, muscle cells will need to be targeted.
Candidate muscle-binding peptides are selected by panning against
C.sub.2C.sub.12 myotubes (Barry et al. (1996) Mature Medicine
2:299-305).
[0051] In round one of selection, the phage library is injected
intravenously and multiple tissues may be recovered. In subsequent
rounds, the phage purified from specific tissues recovered in round
one is injected intravenously, and thus, only that tissue is
recovered. In rounds 2-4, separate animals are needed for each
tissue being examined. After the last round, the phages are plated
on a lawn of bacteria and a number of phage plaques are isolated
and the DNA sequenced. Of these a certain percentage will represent
the dominant selected clone. FIG. 2 provides a scheme for selection
of tissue specific phage.
[0052] Therefore, muscle-specific peptides can be identified which
will bind to, and be internalized by, the target cells. Specific
peptides sequences include:
1 WDA NGK T (Trp-Asp-Ala-Asn-Gly-Lys-Thr) ASS LNI A
(Ala-Ser-Ser-Leu-Asn-Ile-Ala) MST QSI N
(Met-Ser-Thr-Gln-Ser-Ile-Asn) SGL PAY P
(Ser-Gly-Leu-Pro-Ala-Tyr-Pro) GET RAP L
(Gly-Glu-Thr-Arg-Ala-Pro-Leu) CGH HPV YAC
(Cys-Gly-His-His-Pro-Val-Tyr-Ala-Cys) QWH GPL H
(Gln-Trp-His-Gly-Pro-Leu-His) HVH TLP T
(His-Val-His-Thr-Leu-Pro-Thr) MHH TRF Y
(Met-His-His-Thr-Arg-Phe-Tyr); the sequences set forth in SEQ ID
NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ
ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26,
respectively.
[0053] NO:1, SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:29,
SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26,
respectively.
[0054] See, also Table 1 for further in vivo selection results.
2TABLE 1 NUMBER OF PHAGE CLONES REMARKS SEQUENCE TISSUE ANIMAL
SEQUENCED FREQUENCY (Selection details) ASSLNIA skeletal mouse 10
40 3 in vitro rounds on C.sub.2C.sub.12 myotubes (SEQ ID NO:2)
muscle (differentiated myoblasts) followed by 2 in vivo rounds on
skeletal muscle WDANGKT skeletal mouse 27 19 4 in vivo round sin
skeletal muscle (SEQ ID NO:1) muscle WDANGKT skeletal mouse + 10 10
2 in vitro on C.sub.2C.sub.12 myotubes followed (SEQ ID NO:1)
muscle dog by 2 in vivo rounds on skeletal muscle GETRAPL skeletal
mouse 27 37 4 in vivo rounds in skeletal muscle (SEQ ID NO:29)
muscle GETRAPL neuro mouse 34 12 4 in vivo rounds in neural
muscular (SEQ ID NO:29) muscular tissue CGHHPVYAC skeletal dog 20
100 3 in vivo rounds in cardiac muscle. (SEQ ID NO:21) muscle This
sequence represents a cyclic peptide where the C residues join to
form a ring structure CGHHPVYAC cardiac dog 40 20 3 in vivo rounds
in cardiac muscle (SEQ ID NO:21) muscle SGTLFAN cardiac dog 40 28 3
in vivo rounds in cardiac muscle (SEQ ID NO:22) muscle KAPLTPV
cardiac dog 40 8 3 in vivo rounds in cardiac muscle (SEQ ID NO:23)
muscle QWHGPLH cardiac mouse 36 20 3 in vivo rounds in cardiac
muscle (SEQ ID NO:24) muscle HVHTLPT Cardiac mouse 36 11 4 in vivo
rounds in cardiac muscle (SEQ ID NO:25) muscle MHHTRFY Cardiac
mouse 36 8 4 in vivo rounds in cardiac muscle (SEQ ID NO:26)
muscle
[0055] In the Table, "Four rounds of selection . . . ," means that
in round 1, the phage library was injected intravenously, and a
specific tissue was recovered. In round 1, multiple tissues are
recovered from one animal (skeletal muscle, cardiac muscle, etc.).
In any subsequent round, the phage purified from a specific tissue
is injected intravenously, and only that tissue is recovered. Thus,
in rounds 2-4, separate animals are needed for each tissue being
examined. After the last round, the phage are plated on a lawn of
bacteria and a number of phage plaques are isolated and the DNA
sequenced. This is represented in the Table by "number of phage
clones sequenced." Of these, a certain percentage will represent
the dominant selected clone. For example, 4 of 10 plaques in the
first experiment revealed the ASSLNIA (SEQ ID NO:2) peptide, giving
a frequency of 40%.
[0056] The experiments disclosed herein, represent the first in
vivo selection in any species other than mouse. Generally, phage
was left to circulate for one hour.
[0057] Peptide APVHRPL (SEQ ID NO:27) was selected via two rounds
in vivo, in canine cardiac muscle. The clone was found in the
washed tissue fraction and may represent a very high affinity
ligand.
[0058] HAIYPRH (SEQ ID NO:28) was selected via four rounds in vivo,
in murine muscle, at a low frequency (4%). This clone may represent
a significant muscle binding peptide.
[0059] The frequency of appearance of each amino acid within the
library is known. Thus, the frequency of amino acids within the
pool of selected peptides can be compared. A number of amino acids
have been observed to be over-represented. These amino acids, (H,
A, G, V, S, Y, T, N, R) may be significant for muscle binding. Such
amino acids may play a role in consensus sequences that are
important for binding. One of skill in the art may identify
consensus sequences by analysis as well as construction of
synthetic sequences and testing binding.
EXAMPLE 3
[0060] Selection of A Panel of Muscle-binding Peptides
[0061] The peptides identified in Example 2 will be further
characterized as described below in Section iii. In addition, a
larger panel of muscle-binding peptides will be isolated to
increase the likelihood that the most specific and highest affinity
muscle-targeting peptides will be identified. Peptides of longer
amino acid sequences will also be tested. See FIG. 3 for an overall
scheme for peptide selection.
[0062] i. Selection of Muscle-binding Peptides
[0063] Using phage libraries (such as those presented on pIII, 15-
and 20-mers presented on pVIII), a variety of myotube-binding
peptides will be selected. Multiple muscle targets will be selected
against to identify common muscle-binding peptides and increase the
likelihood of identifying a wide panel of peptides. Peptides will
be selected against differentiated mouse C.sub.2C.sub.12 and So18
myotube cell lines and against primary muscle samples from mouse,
rat and dog quadricep, tibialis anterior, and soleus muscles. A
phage selection strategy will be employed to identify peptides
which target endocytosing receptors. Selection will be carried
through sufficient cycles until increased binding by the population
is observed or sequencing of phage clones indicates that the
population has collapsed down to a finite number of peptide clones.
Clones from this round (round N) will be screened as described
below in section iii. Clones from the round N-1 will also be
tested, since it is conceivable that truly cell-specific peptides
may only transiently appear in the selection process and may be
out-competed by less specific, higher affinity/avidity peptides in
later rounds of selection. If selection against myotubes does not
yield any useful peptides, then selection and screening will be
carried out against myoblast cell lines or fresh myoblast
isolates.
[0064] ii. Selection of Muscle-binding Peptides with New
Libraries
[0065] While useful peptides have already been isolated from a
20-mer library and a 7-mer library, it should be noted that any one
library vastly under-represents the total number of possible random
20-mer peptides. There is a "space" of .about.10.sup.26 possible
20-mer peptides. Our one library presents .about.10.sup.8 peptides
and therefore represents only 1 in 10.sup.18 of these possible
20-mers. The number of phage clones is the limitation to how many
peptides a library can contain. Therefore, production of more
10.sup.8 transformant peptide will produce entirely independent
peptides to be screened (up to 10.sup.18 could in theory be
created). It is not known that 20 amino acids are required for
cell-binding, however, the 20-mer libraries out complete 12-mer
libraries for selection against a variety of cell targets (Barry et
al. (1996) Mature Medicine 2:299-305; and not shown). This suggests
large peptides are better for cell binding/entry and that peptides
even larger than 20 amino acids may be useful. An additional
benefit is derived by using large peptide libraries, since any one
20-mer peptide also represents 14 6-mer peptides, 12 8-mer peptides
etc. Given these features, new 20-mer and larger libraries will be
created on pIII and pVIII to increase the probability that novel,
useful peptides can be identified. New 20-mer libraries will be
created using randomly synthesized oligonucleotides (Barry et al.
(1996) Mature Medicine 2:299-305). To create even larger peptide
libraries, genomic DNA from E. coli or mammalian sources will be
randomly sheared to small fragments (.about.200 base pairs),
blunted, and cloned into pIII or pVIII. Once created, these
libraries will be selected as in section i and screened as in
section iii.
[0066] iii. Rapid Screening of Peptide Ligands with a Heptapeptide
Phage Display Library
[0067] a. Ph.D..TM. Phage Display Peptide Library (New England
Biolabs) is a ligand screening system which is based on a
combinatorial library of random peptide 7-mers fused to a minor
coat protein (piii) of the filamentius coliphage M13.
[0068] b. The heptapeptide library consists of 2.times.10.sup.9
electroporated sequences amplified once to yield .apprxeq.100
copies of each sequence in 10 .mu.l of the supplied phage.
[0069] c. Phage display creates a physical linkage between a
selectable function (the displayed peptide sequence) and the DNA
encoding that function.
[0070] d. This allows rapid identification of peptide ligands for a
variety of receptor target molecules by an in vitro selection
process called biopanning.
[0071] e. Biopanning is carried out by simply passing the pool of
phage displayed peptides over a plate coated with the target,
washing away the unbound phage, and eluting the bound phage.
[0072] Advantages
[0073] a. Unlike the use of known ligands, the process requires no
prior knowledge of the biology of the target cells.
[0074] b. Molecular recognition and selection are not influenced by
the immunogenicity of candidate targets.
[0075] c. As a result, peptide ligands should be more easily
isolated and incorporated into biological vectors by cloning or by
chemical conjugation to synthetic vectors.
[0076] iv. Screening Peptides for Specificity
[0077] Candidate peptides generated in sections ii and iii will be
screened for specific binding to myotubes. Initial screens will
compare the binding of these peptides on phage against the myotube
lines C.sub.2C.sub.12 and So18 and against heterologous cell lines
including fibroblasts, macrophages, dendritic cells, and
hepatocytes. Peptides will be screened not only for binding muscle
cells, but also for low-level binding to hepatocytes (cell lines
and primary isolates), since the liver is a tremendous sink for
adenovirus in vivo. The liver would therefore be a tissue to avoid
targeting with new peptides. Likewise, peptides will be screened
for low binding to macrophage and dendritic cells. It is likely
that delivery of potentially immunogenic gene products and vector
antigens to these potent antigen-presenting cells would increase
the problem of immune-dependent destruction of
adenovirus-transfected cells (Yang et al. (1996) Gene Therapy
3:412-420).
[0078] Some of the current immunogenicity of adenovirus vectors
could be due to low-level infection of similar antigen-presenting
cells. If so, selected peptides that give lower binding than
wild-type fiber protein to these immune cells might actually
attenuate the existing immune responses against
adenovirus-transfected cells.
[0079] v. Optimization of Peptides by Mutagenesis
[0080] As described in section ii, the phage libraries vastly
under-represent all possible peptides within a particular library.
That any peptides can be selected to bind cells would seem
remarkable with only this information. Conversely, this poor
representation implies that any peptide selected to bind a cell has
only a 10.sup.-18 chance of being "the" optimal peptide for binding
its target. Therefore, it is likely that any of these lead peptides
can be improved by random mutagenesis. Mutagenic libraries will be
constructed by insertion of randomly mutated oligonucleotides based
on the candidate peptide's sequence into the pIII or pVIII gene in
phagemid vector fAFF1 or p8V2 (Barry et al. (1996) Mature Medicine
2:299-305). Random insertion of mutant bases in positions 1 and 2
of each codon will be constrained at a ratio of 70:10:10:10 such
that 70% of each will contain the normal base and 30% will contain
another base. The third position of each codon will be restricted
to G or T as described previously (Barry et al. (1996) Mature
Medicine 2:299-305). Once generated, these libraries will be panned
as previously performed (Barry et al. (1996) Mature Medicine
2:299-305) and consensus amino acids will be identified by
sequencing a pool of selected mutant peptide-presenting phage. In
addition, the optimal sequence will be determined as described in
sections i and iv.
EXAMPLE 4
[0081] Vertebrate Animals
[0082] Inbred Balb C mice will be used to screen phage presentation
libraries. Additional mice will be used following a similar
protocol to assay the ability of novel peptide ligands cloned into
fiber knob, and into recombinant adenoviruses to bind to muscle.
The phage library will be injected intravenously under deep
halothane anesthesia. At periods of time ranging from 5 to 30
minutes, the mice will be euthanized by cervical dislocation and
immediately immersed in liquid nitrogen. Approximately 20 mice will
be allowed to recover from anesthesia following intravenous
injection, and euthanized at two days to one week following
injection to assess the localization of recombinant adenovirus gene
expression.
[0083] The initial use of the mouse allows a larger number of
potential muscle-specific binding motifs to be screened than would
a larger animal model. Initial screens will utilize myoblast and
myotube cultures; however, the fetal/neonatal gene expression
pattern of these cells while in culture may not permit the
isolation of binding moieties appropriate for adult skeletal
muscle. Thus, the use of in vivo screening of phage libraries will
significantly shorten the interval involved in discovery of these
novel binding moieties. Subsequently, the ability of modified
fiber, and then of modified adenovirus to specifically target
muscle after intravenous injection can only be tested in vivo. The
use of the M-PFK deficient dog model provides a readily assessable
animal model of a myopathy in which the ability of tropism modified
adenovirus to deliver a therapeutic gene can be investigated. (See
Example 5).
EXAMPLE 5
[0084] Therapy in a Canine Model
[0085] i. Pre-treatment Studies
[0086] Neonatal dogs (1 day to 1 week old) from an M-PFK colony
will be tested for M-PFK deficiency using the PCR-based
allele-specific test that we devised. Five juvenile (4-9 months)
dogs, which have been determined to be homozygous mutant for M-PFK
deficiency, will be used in this study. Each dog will have skeletal
muscle biopsies taken from the gastrocnemius and triceps muscles.
Portions of these biopsies will be frozen and evaluated
histochemically. Tissue samples will also be homogenized and
assayed for M-PFK activity. Detailed medical records are kept on
all colony animals, and any episodes of anemia or pigmenturia will
be recorded. Pre-treatment magnetic resonance spectroscopy (MRS)
studies will be performed on each animal to establish parameters
for these investigations. Whole blood and muscle will be frozen at
-70.degree. C. to provide pre-treatment controls for Southern
hybridizations, PCR amplification of vector sequences, PFK activity
studies, and M-PFK-specific immunoblots. Serum samples will be
frozen as controls for the antigenicity studies described
below.
[0087] ii. In vivo Gene Delivery to M-PFK Deficient Dogs
[0088] After completion of the pre-treatment studies, each dog will
be sedated, and a 21 gauge intravenous catheter placed in the
cephalic vein. Tropism-modified vectors comprising the peptides and
expression cassettes containing M-PFK will be delivered as a slow
injection. During injection, the dogs will be monitored for signs
associated with adverse reactions such as anaphylaxis, and should
such reactions occur, administration of the vector will be
suspended and appropriate treatments initiated.
[0089] iii. Studies to Detect the Transferred Canine M-PFK cDNA
[0090] Skeletal muscle biopsies will be taken from the
contralateral gastrocnemius and triceps muscle at one, two, three,
four, six and eight weeks following the injection of the vector,
and monthly thereafter. DNA will be recovered from this tissue
sample and examined by PCR for the transferred virus.
[0091] iv. Immunological Detection of Tissue Specific Expression of
M-PFK
[0092] To determine if the vector which has been transferred to the
muscle is expressing M-PFK, M-PFK specific immunoblots will be
performed. One hundred to one hundred fifty micrograms of protein
are loaded into each well of a 7.5% polyacrylamide gel. Following
electrophoresis the gel is stained with coomassie brilliant blue to
show protein content, and the electrophoretically transferred to
0.45 micron nitrocellulose membranes. The membranes are blocked
with 5% milk powder for 1 hr, followed by 0.05% Tween 20 for 30
min. The membranes are then incubated with the primary antibody,
guinea pig anti-rabbit M-PFK, for 18 hr at room temperature, with
gentle agitation. Three washes with Tris-buffered saline (0.2 M
Tris, 5 M NaCl, pH 7.5; TBS), 30 seconds to one minute each, are
followed by a 3 hr incubation with peroxidase-conjugated rabbit
anti-guinea pig IgG (Sigma), at room temperature. A final three
rinses in TBS are followed by the addition of 4 chloro-naphthol to
allow for color development.
[0093] v. Biochemical Detection of Enzyme Activity from Transferred
M-PFK
[0094] To determine if the expressed M-PFK possesses enzymatic
activity, the muscle biopsy samples will be evaluated by
biochemical assay. Phosphofructokinase from muscle biopsies will be
prepared for activity assay by homogenization for 15 seconds in 5
volumes of PFK extraction buffer [50 mM Tris-phosphate, pH 8.0, 25
mM NaF, 0.1 mM ethylenediamine-tetraacetic acid (EDTA), 25 mM
(NH.sub.4).sub.2SO.sub.4, 1 mM adenosine triphosphate (ATP), 10 mM
dithiothreitol (DTT), 0.5 mM phenylmethyl-sulfonyl fluoride (PMSF),
1 mM p-aminobenzamindine, 250 .mu.g/liter Leupeptin, 0.1 mM
Na-p-tosyl-L-lysine chloromethyl ketone]. The homogenate is then
sonicated and centrifuged at 20,000.times.g for 1 hour. The
supernatant is saved and stored at -20.degree. C. Enzyme activity
will be determined by a calorimetric reaction in 50 mM
tris(hydroxymethyl)methylglycine, pH 8.4, 5mM MgCl.sub.2, 0.15 mM
NADH, 0.1 mM DTT, 0.25 mM ATP, 2.4 mM Fructose-6-phosphate, 0.18
units aldolase, 0.6 units triose phosphate isomerase, and 0.1 unit
glycerophosphate dehydrogenase. The decrease in absorbance at a
wavelength of 340 nm is monitored for 10 min at 26.degree. C. and
one unit of PFK activity is defined as the amount of enzyme needed
to convert 1 .mu.mole of fructose-6-phosphate to fructose-1,
6-diphosphate in one minute. All studies will be compared to
pretreatment values as well as values from ag-matched normal and
M-PFK deficient dogs, and dogs treated with a control vector.
[0095] vi. Histochemical Localization of Tissue-specific Expression
of M-PFK
[0096] Determination of the number and type of muscle cells
expressing M-PFK activity will be performed by histochemical
staining for M-PFK. Briefly, small pieces of muscle, frozen in
liquid nitrogen-cooled isopentane, will be mounted in a cryostat
and sectioned at 6 microns. These sections will be placed on
microscope slides, dried, and incubated in a solution containing 10
mM Na.sub.2HAsO.sub.4, 2 mM fructose-6 phosphate, 1 mM
diphosphopyridine nucleotide (DPNH) 1 mM ATP, 1 mM MgSO.sub.4, and
4 mg/ml NBT. The present of PFK activity is seen as a dark blue
stain within the cells.
[0097] vii. Evaluation of the Efficacy of Gene Transfer in Treating
M-PFK Deficiency
[0098] To evaluate the suitability of the molecular conjugate
vectors for treating inherited myopathies, the effects of treatment
on the clinical signs of M-PFK deficiency will be evaluated. The
dogs will be observed closely for signs of myopathy, including
fatigue, exercise intolerance, and muscle cramping. Light levels of
long duration exercise such as walking will be used to attempt to
induce signs of myopathy. If stable expression is seen in muscle
biopsy specimens exceeding one month post injection, MRS studies
will be performed to determine the effect of M-PFK expression on
the metabolic block. With the dog placed under general anesthesia,
the muscle will be tetanically stimulated and the level of
inorganic phosphorus determined. The results of these studies will
be compared to the pretreatment values to determine the extent of
improvement in muscle function. Additionally, the dogs will be
closely observed during this procedure for episodes of cramping or
muscle contracture.
[0099] viii. Detection of Immune Response to the Adenoviral
Vector
[0100] To determine if the vectors induce an immune response in the
animal being treated, serum samples taken prior to treatment and
each biopsy will be hybridized with nitrocellulose blots of
adenoviral proteins run on SDS polyacrylamide gels. A secondary,
labeled anti canine IgG will be utilized to visualize any canine
anti-adenoviral antibodies. Antibodies to the transferred M-PFK
gene are not expected as the canine M-PFK will be used and the
deficiency is due to a point mutation.
[0101] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0102] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
29 1 7 PRT Artificial Sequence Phage display library peptides 1 Trp
Asp Ala Asn Gly Lys Thr 1 5 2 7 PRT Artificial Sequence Phage
display library peptides 2 Ala Ser Ser Leu Asn Ile Ala 1 5 3 7 PRT
Artificial Sequence Phage display library peptides 3 Leu Ala Pro
Gln Lys Leu Pro 1 5 4 7 PRT Artificial Sequence Phage display
library peptides 4 Val Ser Ala Ala Pro Tyr Pro 1 5 5 7 PRT
Artificial Sequence Phage display library peptides 5 Ala Ala Ala
Asn Val Trp His 1 5 6 7 PRT Artificial Sequence Phage display
library peptides 6 Ala Tyr Pro Gly Phe Ala Leu 1 5 7 7 PRT
Artificial Sequence Phage display library peptides 7 Thr Ala Thr
Ile Thr Thr Lys 1 5 8 7 PRT Artificial Sequence Phage display
library peptides 8 Met Ser Thr Gln Ser Ile Asn 1 5 9 7 PRT
Artificial Sequence Phage display library peptides 9 Ser Gly Leu
Pro Ala Tyr Pro 1 5 10 7 PRT Artificial Sequence Phage display
library peptides 10 Ala Gly Met Ala His Ile Arg 1 5 11 7 PRT
Artificial Sequence Phage display library peptides 11 Ser Thr Ser
Xaa Ile Thr His 1 5 12 7 PRT Artificial Sequence Phage display
library peptides 12 Ser Tyr Phe Ser Ala Pro Pro 1 5 13 7 PRT
Artificial Sequence Phage display library peptides 13 Gln Leu Ser
Leu Leu Leu Ala 1 5 14 7 PRT Artificial Sequence Phage display
library peptides 14 Trp Lys Pro Ala Thr Phe Tyr 1 5 15 7 PRT
Artificial Sequence Phage display library peptides 15 Ala Pro Leu
Tyr Pro Pro Ser 1 5 16 7 PRT Artificial Sequence Phage display
library peptides 16 His Leu Ser Asn Trp Pro Arg 1 5 17 7 PRT
Artificial Sequence Phage display library peptides 17 Ser Met Gln
Thr His Pro Phe 1 5 18 7 PRT Artificial Sequence Phage display
library peptides 18 Ser His Trp His Asn Ser Glu 1 5 19 7 PRT
Artificial Sequence Phage display library peptides 19 His Pro Ala
His Val Ala Lys 1 5 20 7 PRT Artificial Sequence Phage display
library peptides 20 Thr Thr Gln His Met Leu Lys 1 5 21 9 PRT
Artificial Sequence Phage display library peptides 21 Cys Gly His
His Pro Val Tyr Ala Cys 1 5 22 7 PRT Artificial Sequence Phage
display library peptides 22 Ser Gly Thr Leu Phe Ala Asn 1 5 23 7
PRT Artificial Sequence Phage display library peptides 23 Lys Ala
Pro Leu Thr Pro Val 1 5 24 7 PRT Artificial Sequence Phage display
library peptides 24 Gln Trp His Gly Pro Leu His 1 5 25 7 PRT
Artificial Sequence Phage display library peptides 25 His Val His
Thr Leu Pro Thr 1 5 26 7 PRT Artificial Sequence Phage display
library peptides 26 Met His His Thr Arg Phe Tyr 1 5 27 7 PRT
Artificial Sequence Phage display library peptides 27 Ala Pro Val
His Arg Pro Leu 1 5 28 7 PRT Artificial Sequence Phage display
library peptides 28 His Ala Ile Tyr Pro Arg His 1 5 29 7 PRT
Artificial Sequence Phage display library peptides 29 Gly Glu Thr
Arg Ala Pro Leu 1 5
* * * * *