U.S. patent application number 12/563201 was filed with the patent office on 2010-04-01 for polypeptide ligands for targeting cartilage and methods of use thereof.
This patent application is currently assigned to Ecole Polytechnique Federale de Lausanne. Invention is credited to Jeffrey A. Hubbell, Dominique A. Rothenfluh.
Application Number | 20100080850 12/563201 |
Document ID | / |
Family ID | 39417214 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100080850 |
Kind Code |
A1 |
Hubbell; Jeffrey A. ; et
al. |
April 1, 2010 |
POLYPEPTIDE LIGANDS FOR TARGETING CARTILAGE AND METHODS OF USE
THEREOF
Abstract
Ligands that specifically bind to articular cartilage tissues
are disclosed, including uses for targeting therapeutics towards
articular cartilage tissue and new materials for articular
cartilage. The ligands are effective in vivo to target therapeutic
materials to articular cartilage.
Inventors: |
Hubbell; Jeffrey A.;
(Morges, CH) ; Rothenfluh; Dominique A.;
(Lausanne, CH) |
Correspondence
Address: |
DARDI & HERBERT, PLLC
220 S. 6TH ST., SUITE 2000, U.S. BANK PLAZA
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Ecole Polytechnique Federale de
Lausanne
|
Family ID: |
39417214 |
Appl. No.: |
12/563201 |
Filed: |
September 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11545819 |
Oct 10, 2006 |
7592009 |
|
|
12563201 |
|
|
|
|
Current U.S.
Class: |
424/489 ;
435/320.1; 514/1.1; 530/324; 530/325; 530/326; 530/327; 530/328;
530/329; 530/350; 536/23.1 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 19/02 20180101; C07K 7/06 20130101 |
Class at
Publication: |
424/489 ;
530/329; 530/328; 530/327; 530/326; 530/325; 530/324; 530/350;
536/23.1; 435/320.1; 514/16; 514/15; 514/14; 514/13; 514/12 |
International
Class: |
A61K 9/14 20060101
A61K009/14; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12N 15/74 20060101 C12N015/74; A61K 38/08 20060101
A61K038/08; A61K 38/10 20060101 A61K038/10; A61K 38/16 20060101
A61K038/16; A61P 19/02 20060101 A61P019/02 |
Claims
1. A substantially pure polypeptide not found in nature comprising
an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or
a conservative substitution thereof, wherein the polypeptide
specifically binds a cartilage tissue.
2. The polypeptide of claim 1 wherein the sequence length is
between 6 and 1000 residues.
3. The polypeptide of claim 1 comprising a therapeutic agent
polypeptide.
4. The polypeptide of claim 3 wherein the therapeutic agent
polypeptide is tissue inhibitor of matrix metalloproteinases-3
(TIMP-3).
5. The polypeptide of claim 1 comprising a synthetic backbone
linkage.
6. A substantially pure nucleic acid not found in nature that
comprises a nucleic acid sequence that encodes SEQ ID NO:1, SEQ ID
NO:2 or SEQ ID NO:3, or a conservative substitution thereof,
wherein a polypeptide encoded by the nucleic acid specifically
binds to a cartilage tissue.
7. The nucleic acid of claim 6 wherein the polypeptide that is
encoded comprises a therapeutic agent polypeptide.
8. A vector comprising the nucleic acid of claim 6.
9. A method of treating cartilage of a mammal comprising
administering to the mammal a pharmaceutically acceptable
composition that comprises the polypeptide of claim 1, wherein the
polypeptide targets the therapeutic agent to the cartilage tissue
by specifically binding cartilage tissue of the mammal.
10. A delivery system for delivering a therapeutic agent
comprising: a substantially purified preparation that comprises a
pharmaceutically acceptable excipient, a therapeutic agent, and a
polypeptide ligand not found in nature comprising an amino acid
sequence in the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, and conservative substitutions thereof, wherein the
polypeptide ligand specifically binds a cartilage tissue for
targeted delivery of the therapeutic agent to cartilage
tissues.
11. The delivery system of claim 10 wherein the therapeutic agent
comprises a drug, a visualization agent, or a therapeutic
polypeptide.
12. The delivery system of claim 10 comprising a fusion polypeptide
that comprises the polypeptide ligand and the therapeutic
agent.
13. The delivery system of claim 10 comprising a molecule that
comprises covalent bonds to the polypeptide ligand and the
therapeutic agent.
14. The delivery system of claim 10 wherein the polypeptide ligand
is covalently bonded to a biocompatible polymer that is associated
with the therapeutic agent.
15. The delivery system of claim 14 wherein the biocompatible
polymer is free of amino acids.
16. The delivery system of claim 10 comprising a collection of
nanoparticles having an average diameter of between about 10 nm and
about 200 nm, wherein the nanoparticles comprise the therapeutic
agent and the polypeptide ligand.
17. A method of treating cartilage of a mammal comprising
administering to the mammal a pharmaceutically acceptable
composition that comprises the delivery system of claim 13, wherein
the polypeptide targets the therapeutic agent to the cartilage
tissue by specifically binding cartilage tissue of the mammal.
18. The method of claim 17 wherein the composition is administered
intra-articularly.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/545,819 filed Oct. 10, 2006, which is hereby incorporated herein
by reference.
TECHNICAL FIELD
[0002] The technical field of the invention is generally related to
delivery of therapeutic agents to cartilage tissues using
polypeptides that specifically bind cartilage.
BACKGROUND
[0003] Cartilage lesions are common and can pose difficulties both
in diagnosis and treatment. A lesion can either be a defect or a
focal cartilage degradation without visible disruption of the
cartilage matrix. Such lesions can result from an injury as in
sports, disease, or aging. The prognosis of an articular cartilage
defect varies according to age, mechanism of injury, site, size,
associated injuries and treatment received.
SUMMARY OF THE INVENTION
[0004] The invention, however, provides treatments for cartilage
injury. Some aspects of the inventions are substantially pure
polypeptides comprising an amino acid sequence of WYRGRL (SEQ ID
NO:1), DPHFHL (SEQ ID NO:2), or RVMLVR (SEQ ID NO:3), or a
conservative substitution thereof, or a nucleic acid encoding the
same. Such polypeptides specifically bind cartilage tissue. Such
polypeptides may also include a therapeutic agent.
[0005] Some inventive methods are related to treating cartilage of
a mammal comprising administering to the mammal a pharmaceutically
acceptable composition that comprises a nucleic acid encoding a
polypeptide that specifically binds a cartilage tissue. Such
polypeptide may also encode a therapeutic agent that, for example,
treats a joint or tissue.
[0006] Other aspects of the invention relate to a delivery system
for delivering a therapeutic agent comprising: a substantially
purified preparation that comprises a pharmaceutically acceptable
excipient, a therapeutic agent, and a polypeptide ligand comprising
an amino acid sequence in the group consisting of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, and conservative substitutions thereto. The
polypeptide ligands may specifically bind to cartilage tissue for
targeted delivery of the therapeutic agent to cartilage tissues.
The therapeutic agent may comprise, for example, a drug, a
visualization agent, or a therapeutic polypeptide. The delivery
system may include, for example, a collection of nanoparticles
having an average diameter of between about 10 nm and about 200 nm,
wherein the nanoparticles comprise the therapeutic agent and the
polypeptide ligand.
[0007] Other embodiments relate to a biomaterial comprising a
polymer and a substantially pure polypeptide comprising an amino
acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a
conservative substitution thereof, wherein the polypeptide
specifically binds to cartilage tissue and the polymer is free of
amino acids and has a molecular weight of at least 400. A variant
of WYRGRL (SEQ ID NO:1) is WYRGRLC (SEQ ID NO:4), with the
C-terminal residue being used as a chemical linker.
BRIEF DESCRIPTION OF FIGURES
[0008] FIG. 1a is a bar graph that shows amplification of three
phage clones identified by biopanning and demonstrates that they
all grow at equal rates comparable to the random library. Final
selection of C1-3 was therefore not affected by differences in
growth during amplification.
[0009] FIG. 1b is a bar graph that demonstrates the competitive
binding assay in which only phage clone C1-3 and C1-C1 could be
recovered.
[0010] FIG. 1c is a bar graph that shows binding specificity of
C1-3 and C1-C1 to articular cartilage. Binding of these phage
clones to synovial membrane resulted in a lower phage recovery by
two orders of magnitude, reflecting non-specific binding. Error
bars indicate mean.+-.standard deviation from three independent
experiments.
[0011] FIG. 2 is a graph showing inhibition of cartilage binding of
C1-3 by the synthetic polypeptide WYRGRLC (SEQ ID NO:4). Data
represents the percentage of maximal phage binding of clone C1-3
obtained in the absence of synthetic polypeptide. Error bars
indicate mean.+-.standard deviation from three independent
experiments.
[0012] FIG. 3 is a bar graph showing inhibition of cartilage
binding of C1-3 phage by 10.sub.1 .mu.M of synthetic polypeptide
vs. WYRGRLC (SEQ ID NO:4)--PPS nanoparticles and 10 .mu.M of
synthetic mismatch polypeptide. WYRGRLC (SEQ ID NO:4)--PPS
nanoparticles exhibit similar binding than the synthetic
polypeptide, whereas the synthetic mismatch polypeptide does not
result in a significant decrease of phage titer. Error bars
indicate mean.+-.standard deviation from three independent
experiments.
[0013] FIG. 4 is a bar graph showing relative accumulation in
articular cartilage in vivo of nanoparticles decorated with WYRGRLC
(SEQ ID NO:4) compared to control nanoparticles. Error bars
indicate mean.+-.standard deviation from three independent
experiments.
DETAILED DESCRIPTION
Introduction
[0014] Ligands that specifically bind to articular cartilage
tissues have been discovered.
[0015] These articular cartilage tissue-binding ligands have given
rise to new techniques to target therapeutics towards articular
cartilage tissue and new materials for treatment of articular
cartilage defects. The ligands are effective in vivo to target
therapeutic materials to articular cartilage.
[0016] Three of the cartilage tissue-binding ligands are
polypeptides with the amino acid sequence of WYRGRL (SEQ ID NO:1),
DPHFHL (SEQ ID NO:2), or RVMLVR (SEQ ID NO:3). Other ligands are
polypeptides with sequences that have conservative substitutions of
one of SEQ ID NOs. 1, 2, or 3. Ligand is a term that refers to a
chemical moiety that has specific binding to a target molecule. A
target refers to a predetermined molecule, tissue, or location that
the user intends to bind with the ligand. Thus targeted delivery to
a tissue refers to delivering a molecule to the intended target
tissue; a therapeutic agent delivered to a target may be intended
to act on the target itself or on some other molecule or cell,
e.g., a cell or tissue that is near the target.
[0017] One application of the cartilage tissue-binding ligands is
for targeting therapeutic agents to articular cartilage, commonly
referred to as intra-articular drug delivery when drugs are
delivered. Intra-articular drug delivery has been termed a major
challenge due to the short residence times of intra-articular
injected drugs. Drugs injected by themselves tend to diffuse away
rapidly or be otherwise rapidly taken up into the circulation, thus
causing low bioavailability of the drug at the cartilage and
unwanted systemic effects. Despite sustained-release drugs, only a
few reports exist on the intra-articular use of any
sustained-release formulations.sup.20-26. Mostly albumin and
poly(lactic-co-glycolic acid) (PLGA) have been used as
biocompatible and biodegradable polymers for this purpose either in
the form of gels.sup.22 or as microspheres.sup.23-26. Such
approaches rely, however, on degradation over time to achieve
sustained-release within the joint, a strategy that is limited by
the properties of available biomaterials, and which makes the drugs
available only when the material degrades. Targeting the delivery
system to articular cartilage with tissue-binding ligands makes the
tissue itself a reservoir for drug release to the site of the
disease process as opposed to release in the joint cavity.
[0018] The cartilage tissue-binding ligands are also useful to bind
cartilage tissue in vitro for diagnostic, assay, or imaging
purposes. For instance, sections of tissue may be exposed to
cartilage tissue-binding ligands that are also bound to a
fluorescent molecule or other imaging agent to visualize the
location of the cartilage tissue. Or, for instance, the cartilage
tissue-binding ligands may be used in affinity chromatography to
isolate the tissue.
[0019] In some aspects, therefore, articular cartilage
tissue-binding ligands described herein enable new techniques for
controlled release by enabling sustained localization through
specific binding to cartilage, and their accompanying formulations
increase intra-articular bioavailability of delivered drugs or
other therapeutic agents, which is beneficial for various disease
processes, e.g., those involving the synovium such as rheumatoid
arthritis or inflammation in clinically manifest osteoarthritis.
Because convective transport of solutes into cartilage is impaired
due to the inherent properties of this tissue.sup.27,28, the
bioavailability of drugs in the cartilage matrix, which is the
primary site of the disease process in osteoarthritis, can be
enhanced by sustained release systems that reside in the matrix
itself. Targeting of the cartilage matrix as described herein is
therefore useful for general intra-articular therapeutic agent
delivery, such as targeting of the cartilage matrix for delivery of
therapeutic agents to treat cartilage degradation in osteoarthritis
and protect cartilage in conditions like rheumatoid arthritis,
bacterial and reactive arthritis.
[0020] The cartilage tissue-binding ligands are thus useful to
direct therapeutic agents to a target. A therapeutic agent refers
to a molecule for delivery to a target to accomplish a desirable
medical or scientific function, and the term includes drugs and
imaging agents. Examples of therapeutic agents are matrix
metalloproteinase (MMP) inhibitors, aggrecanase inhibitors,
COX-inhibitors and other non-steroidal anti-inflammatory drugs
(NSAIDs), glucosamin, diacerhein, methotrexate, steroids,
immunosuppressing drugs (rapamycin, cyclosporine), protein
therapeutics (growth factors, tissue inhibitors of matrix
metalloproteinases), and oligonucleotides (e.g., siRNA, shRNA,
miRNA). Examples of imaging agents are fluorescent markers,
radio-opaque materials, magnetic resonance imaging contrast agents,
x-ray imaging agents, radiopharmaceutical imaging agents,
ultrasound imaging agents, and optical imaging agents.
Ligand Discovery and Experimental Data
[0021] The development of phage display of random peptides on the
minor coat proteins (pIII) of bacteriophages has allowed use of
affinity purification, in a process called biopanning.sup.5, to
identify specific peptides for precise targeting of multiple
tissues, both in vitro.sup.6 and in vivo in animals and even
humans.sup.7-9. As an example, Arap et al. have succeeded in
mapping the human vasculature and specifically targeting the
microvasculature of adipose tissue in mice. Another study
identified a synovium-specific homing peptide by phage display.
Human synovial grafts were transplanted into SCID mice and
biopanning was carried out in vivo which resulted in the
identification of phages with homing peptides specific for the
microvascular endothelium of synovial tissue.sup.11.
[0022] In order to identify peptide sequences which bind to the
articular cartilage extra-cellular matrix, it should first be
appreciated that an in vivo approach to biopanning must address the
challenges presented by the dense organization of extra-cellular
matrix molecules. Indeed, the collagen II fiber network of
articular cartilage has a reported mesh size of 60 nm in the
superficial zone.sup.12 and largest gaps between the side chains of
proteoglycan aggregates have been described to be as low as 20
nm.sup.13. Biopanning was therefore carried out not in vivo, but
instead was adapted for use with ex vivo materials. Specifically,
sliced bovine cartilage was used, wherein the extra-cellular matrix
was exposed for affinity purification of binding phage virions.
Materials The phage display library fUSE5/6-mer based on
filamentous phage strain fd-tet was received from the University of
Missouri, Columbia. Cartilage grafts, synovial fluid and synovial
membrane were harvested from bovine shoulders obtained from the
local slaughterhouse. Cartilage grafts were stored in 0.1% sodium
azide and protease inhibitors at 4.degree. C. and used within 72
hours. Buffers and solutions used for phage-display screening:
Blocking solution (0.1M NaHCO.sub.3, 1% BSA, pH 8.5), wash buffer
(PBS, Tween 20 0.1-1%), elution solution (50 mM glycine-HCl, pH
2.0), neutralisation buffer (0.2M NaHPO.sub.4). Solvents and
reagents for nanoparticle synthesis were purchased from
Sigma-Aldrich (Buchs, Switzerland). All peptides (synthesis
chemicals from Novabiochem, Lautelfingen, Switzerland) were
synthesized on solid resin using an automated peptide synthesizer
(CHEMSPEED PSW 1100, Augst, Switzerland) with standard F-moc
chemistry. Purification was performed on a Waters ultrapurification
system using a Waters ATLANTIS dC.sub.18 semi-preparative column
and peptides collected according to their molecular mass analyzed
by time-of-flight (TOF) mass spectrometry. Screening of
phage-displayed combinatorial peptide library and binding assays
Peptides for binding to the articular cartilage matrix were
selected by exposing a fUSE5/6-mer library to bovine cartilage
grafts, which provided 6.4.times.10.sup.7 different phage clones
with 6-amino acid linear-peptide inserts displayed on the minor
coat protein of filamentous phage.sup.5,18. Cartilage grafts were
harvested with 8 and 4 mm biopsy cutters (two-sided surface 1
cm.sup.2 and 0.25 cm.sup.2). A slice with the intact articular
surface was removed to expose the phages only to the cartilage
matrix below the surface. Affinity selection was preformed in
polystyrene 48-well plates, which were blocked for nonspecific
adhesion with a blocking solution containing 1% BSA for 2 hours
prior to screening. A total of 5 screening rounds was carried out.
In the first round 10.sup.13 and in subsequent rounds 10.sup.12
phage virions per ml were exposed to the cartilage graft, washed
with PBS/Tween 20 and eluted at low pH. While the first round was
supposed to give a high yield at low stringency, subsequent rounds
were carried out with increasing stringency to select stronger
binders. Conditions were increased from 4 hours of binding at RT
and washing with PBS/0.1% Tween to 30 minutes binding at 37.degree.
C. with 220 RPM, washing with PBS/1% Tween 20 and the cartilage
surface decreased from 1 cm.sup.2 to 0.25 cm.sup.2 in round five.
Eluted phage was amplified overnight in E. coli strain TG1 in
2.times.YT medium and purified by two times PEG/NaCl (2M, 25%)
precipitation. In the first round of screening, negative screens
against the intact articular surface as well as synovial fluid (50%
diluted in PBS to lower viscosity) were carried out in order to
eliminate phages binding to these targets. Quantitative titer
counts were obtained by spot titering of 15 .mu.l of
phage/bacterial culture onto LB-agar/tetracyclin plates and are
given in transducing units (TU)/ml. The polypeptide sequences of
affinity selected phage specific to the articular cartilage matrix
was determined by DNA sequencing (Microsynth AG, Balgach,
Switzerland) using the sequencing primer 5'-CAT GTA CCG TAA CAC TGA
G (SEQ ID NO:5). Binding specificity was determined by exposing
selected phage clones (10.sup.8 TU/ml) to articular cartilage (0.25
cm.sup.2) with and without synovial fluid, to synovial membrane
(3.times.4 mm, 0.24 cm.sup.2) and to polystyrene after blocking.
Titer counts were obtained by spot titering. Competetive binding
was probed by exposing a mix of selected phage clones and
fUSE5/6-mer library (each 10.sup.8 TU/ml) in the presence of
synovial fluid to articular cartilage for 30 min. at 37.degree. C.,
220 RPM and washed with PBS/1% Tween 20. The phage clones were
identified by DNA sequencing and the corresponding titer counts
calculated. For competitive binding against free polypeptide or
nanoparticles and to obtain a dose-response curve, 10.sup.8 TU/ml
of phage and its free polypeptide in different concentrations or
nanoparticles were mixed, exposed to articular cartilage and titer
counts determined. All experiments have been carried out in
triplicate and repeated for confirmation. Nanoparticle Synthesis
Poly(propylene sulfide) nanoparticles were prepared as described
elsewhere.sup.14. Briefly, for nanoparticles between 30 and 40 nm,
a monomer emulsion is prepared by dissolving 1.6% (w/v) of Pluronic
F-127 (MW 12600) in 10 ml of degassed, double-distilled and
filtered water. The system is continuously stirred and purged with
argon for 60-90 min. Propylene sulfide is added at a
Pluronics/monomer ratio of 0.4 (w/w). The initiator,
pentaerythritol tetrathioester (TTE) (synthesized as described
previously.sup.14), is deprotected by mixing with a molar
equivalent of 0.5M sodium methanoate and stirred under argon for 5
minutes. The deprotected initiator is then added to the emulsion.
Five minutes later, 60 .mu.l of diaza[5.4.0] bicycloundec-7-ene
(DBU) is added and the reaction stirred under inert conditions for
6 hours. Exposure to air yields disulfide crosslinking of the
particle core. Particles are subsequently purified from remaining
monomers and base by 2 days of repeated dialysis against ultrapure
water through a membrane with a MWCO of 6-8 kDa (Spectra/Por). Free
Pluronic F-127 is removed in a second dialysis step through 300 kDa
membranes. Particle size is measured after dialysis by dynamic
light scattering (ZETASIZER NANO ZS, Malvern Instruments, Malvern,
UK). For preparation of surface-functionalized nanoparticles,
polypeptides are conjugated to Pluronics-F127 prior to nanoparticle
synthesis. In order to derivatize Pluronics F-127 with vinyl
sulfone, 400 ml of toluene and 15 g of Pluronic F-127 were
introduced in a 3-neck round bottom flask connected to a Soxhlett
filled with glass wool and dry molecular sieves and a cooling tube.
Pluronic was dried azeotropically during 4 h. The dried Pluronic in
toluene was cooled in an ice-bath and sodium hydride was added in a
5 equimolar excess compared with Pluronic-OH-groups. The reaction
was stirred for 15 minutes and divinyl sulfone was added in a 15
molar excess and the reaction was carried out in the dark for 5
days at RT under argon. The reaction solution was filtrated through
a celite filter cake, concentrated by rotary evaporation and then
precipitated 5 times in ice-cold diethylether. The polymer was
dried under vacuum and stored under argon at -20.degree. C.
Derivatization was confirmed with .sup.1H-NMR (CDCl.sub.3):=1.1 (m,
PPG CH.sub.3), 3.4 (m, PPG CH), 3.5 (m, PPG CH.sub.2), 3.65 (m, PEG
CH.sub.2), 6.1 and 6.4 (d, 1H each, CH.sub.2.dbd.CH--SO.sub.2--),
6.85 (q, .sup.1H, CH.sub.2.dbd.CH--SO.sub.2--) ppm. A degree of end
group derivatization of 88% was determined by .sup.1H-NMR. All
polypeptides were acetylated at the N-terminus to prevent reaction
with the .alpha.-amine and synthesized with a cysteine at the
C-terminus to be conjugated to Pluronics-di-vinyl sulfone by
Michael-type addition via the free thiol.sup.29. For conjugation,
1.6 mM Pluronics-di-vinyl sulfone is stirred in triethanolamine
buffer at pH 8.5 until it is completely dissolved and 2 mM of
polypeptide is added and stirred for 3 hours at RT. Conjugation is
confirmed by absent vinyl sulfone peaks by .sup.1H-NMR in methanol.
Nanoparticles are prepared as described above with 10% surface
functionalisation corresponding to a fraction of 0.16% (w/v) of
conjugated Pluronic. The presence of the polypeptide on the
particle is confirmed by measuring the zeta potential by dynamic
light scattering (ZETASIZER NANO ZS, Malvern Instruments, Malvern,
UK) after particle synthesis. The nanoparticles were fluorescently
labelled with 6-iodoacetamide fluorescein (6-IAF, Molecular Probes,
Eugene, Oreg.) or Alexa FLUOR 488 maleimide by adding label at 1
mg/ml of nanoparticle solution in 10 mM Tris-HCl pH 8.5 and stirred
in the dark for 2 h at room temperature. Unreacted label was
quenched by adding 5 mg L-Cysteine. Purification was accomplished
by dialysis for 24 hours against a membrane with MWCO of 24 kDa in
5 mM PBS with two buffer shifts. In vivo injection Labeled
nanoparticles were injected into knee joints of 4-6 weeks old
C57BL/6 mice at a concentration of 1% (w/v) in a volume of 5 .mu.l
using a 25 .mu.l Hamilton syringe with Cheney reproducibility
adapter and 30 G needles (animal experimentation protocol approved
by the local review board, authorisation no. 1894). The mice were
anesthetized with isoflurane. The animals were euthanized by
CO.sub.2-asphyxiation after 24 hours. The knee joints were
harvested and freed from surrounding muscle by microdissection.
Confocal microscopy and image analysis The sections were analyzed
by confocal laser scanning microscopy using a Zeiss LSM510 meta.
Fluorescence was extracted by emission fingerprinting to reduce
autofluorescence of cartilage tissues. The pinhole was adjusted
using TETRASPECK fluorescent microspheres (Invitrogen, T14792,
Carlsbad, Calif.). Images for analysis were obtained using a Zeiss
63.times. APOCHROMAT objective in 10 different locations per joint
with a z-stack of 10 images each. Image deconvolution was
accomplished by Huygens software. Image analysis was done by
ImageJ.
Affinity Selection of Phage Display Library and Binding Assay
[0023] Cartilage grafts were incubated with the fUSES peptide on
phage display library, which expressed linear 6-mer random peptides
on minor coat proteins (pIII) with a diversity of
6.4.times.10.sup.7. The sequences corresponding to the polypeptides
displayed on the phage virions were determined by DNA sequencing
after rounds 3 and 5 of affinity selection (panning). After round
3, sequencing did not reveal a consensus motif in the selected
polypeptides. Sequencing of round 5 yielded three different phage
clones C1-3 (having SEQ ID NO:1), C1-C1 (having SEQ ID NO:2) and
C1-F1 (having SEQ ID NO:3), whereas C1-3 appeared in 94 out of 96
sequenced clones and C1-C1 and C1-F1 both only appeared once. To
ensure that the selection of the three phage clones was not the
result of differences in their amplification rates compared to
other phages, overnight amplification of 10.sup.6 particles/ml of
the three selected clones and the random fUSE5 phage library was
performed in a bacterial culture. As shown in FIG. 1a, all
amplification rates are equal to the rate of the random library.
This suggests that the three phage clones were not selected in the
panning process because of differences in their amplification
speed, which would have biased the library in their favor. A
competetive binding assay was carried out to assess the relative
binding strength of the selected phage clones against each other.
Equal amounts of the three phage clones and the random fUSES
library were mixed and exposed to cartilage grafts (0.25 cm.sup.2).
The corresponding titer counts of recovered phage virions were
determined by DNA sequencing of 96 colonies in triplicate. It is
demonstrated in FIG. 1b that only C1-3 and C1-C1 have been
recovered, thereby indicating their superior binding strength over
C1-F1 and the random library. In addition, C1-3 has a higher titer
count of almost one order of magnitude than C1-C1 which further
demonstrates its dominant binding which was already suggested by
its frequent appearance after round 5.
[0024] The binding specificity of C1-3 and C1-C1 to articular
cartilage was evaluated by exposing the phage clones to articular
cartilage (0.25 cm.sup.2) and synovial membrane (0.24 cm.sup.2) and
comparing to random binding of the fUSES phage library.
Furthermore, the effect of the presence of synovial fluid on phage
binding was probed by adding an equal volume of bovine synovial
fluid to the phages, which dilutes the synovial fluid by a factor
of 2. FIG. 1c shows that both C1-3 and C1-C1 exhibit specific
binding to articular cartilage over synovial membrane by two orders
of magnitude and that the addition of synovial fluid does not yield
a significant drop in phage binding to articular cartilage. Binding
of the specific phage clones to synovial membrane seems to reflect
background phage binding as the random phage library fUSES bound to
the synovial membrane to the same extent. In addition, phage titers
of fUSE5 to articular cartilage were at the same level as to
synovial membrane, further indicating specific binding of C1-3 and
C1-C1. Phage was exposed to polystyrene wells after blocking them
with BSA because it is contained in the plasticware in which
affinity selection was carried out. No background phage binding to
polystyrene was detectable, however.
[0025] Affinity selection of fUSE5/6-mer phage display library
resulted in the discovery the phage clone C1-3 (polypeptide WYRGRL,
SEQ ID NO:1) which exhibits both binding specificity to articular
cartilage and dominant binding compared to other phage clones.
Specific binding in the context of a polypeptide refers to the
binding of the polypeptide specifically to the target of interest
as opposed to other molecules.
Competetive Binding of C1-3 Against Free Polypeptides WYRGRLC and
YRLGRWC
[0026] Based on affinity selection and the binding assays reported
herein, the 6-mer polypeptide insert WYRGRL (SEQ ID NO:1) of clone
C1-3 as well as its scrambled mismatch YRLGRW (SEQ ID NO:6) were
synthesized on solid resin using standard Fmoc chemistry. The
N-terminal amino acids are acetylated and a cysteine was added to
the C-terminus of the polypeptides for bioconjugation to vinyl
sulfone by Michael-type addition via the free thiol. In order to
further characterize the binding properties of WYRGRLC (SEQ ID
NO:4) to articular cartilage, a competetive binding assay against
the phage clone C1-3 was performed by exposing the C1-3 and the
WYRGRLC (SEQ ID NO:4) to the cartilage. A dose-response curve was
determined by serial dilutions of the polypeptide ranging from 50
nM to 10 .mu.M and mixing them with 10.sup.8 TU/ml of phage. The
titer counts of phage recovered gradually decreased by two orders
of magnitude as the concentration of the free polypeptide in
solution was increased (FIG. 2). An IC50 of about 200 nM can be
estimated from the curve in FIG. 2.
Conjugation of Polypeptide to Pluronic F-127 and Nanoparticle
Synthesis
[0027] In order to functionalize poly(propylene sulfide) (PPS)
nanoparticles, Pluronic F-127 was derivatized with vinyl sulfone.
The polypeptides were conjugated to Pluronic-di-vinyl sulfone via
the free thiol in the C-terminal cysteine by Michael-type
addition.
[0028] Conjugation was confirmed by .sup.1H-NMR in methanol by the
absence of peaks specific to vinyl sulfone. PPS nanoparticles were
then prepared by inverse emulsion polymerization with Pluronic
F-127 (90%) and polypeptide-conjugated Pluronic F-127 (10%) serving
as the emulsifier. Because Pluronic as the emulsifier remains on
the particle surface, the conjugated polypeptide is displayed on
the surface of the nanoparticles, thereby adding the targeting
functionality of the polypeptide to the particles. Size
measurements by dynamic light scattering (ZETASIZER NANO ZS,
Malvern Instruments, Malvern, UK) revealed a size by volume of 38
nm for PPS particles displaying WYRGRLC (SEQ ID NO:4)
(polydispersity index PDI 0.221), 31 nm for particles displaying
YRLGRC (SEQ ID NO:7) PDI 0.412) and 37 nm for non-conjugated PPS
particles (PDI 0.212). The zeta-potential of non-conjugated PPS
nanoparticles is about neutral (-2.64.+-.8.97 mV).
[0029] Due to the positive charges of the polypeptide, the
zeta-potential of conjugated nanoparticles shifted to +17.8.+-.3.45
mV, which further confirms the presence of the polypeptide on the
nanoparticle surface.
[0030] WYRGRLC (SEQ ID NO:4)--PPS nanoparticles at 2% (w/v) were
subjected to a competetive binding assay against the free
polypeptides WYRGRLC (SEQ ID NO:4) and YRLGRC (SEQ ID NO:7) at
concentrations of 10 .mu.M each. Accordingly, phage clone C1-3 was
exposed to the cartilage in the presence of WYRGRLC (SEQ ID
NO:4)--PPS nanoparticles, WYRGRLC (SEQ ID NO:4) or YRLGRC (SEQ ID
NO:7), and the amount of C1-3 phage binding to the cartilage
relative to a control C1-3 phage without competitive inhibitors was
measured. The results in FIG. 3 show that WYRGRLC (SEQ ID
NO:4)--PPS nanoparticles exhibit similar binding as the
corresponding free polypeptide (WYRGRLC (SEQ ID
NO:4)--PPS10.4.+-.6% and WYRGRLC (SEQ ID NO:4)13.9.+-.2.8% of
control), whereas the YRLGRC (SEQ ID NO:7) did not bind
competetively and thus did not result in a significant drop of
phage titer (92.+-.12% of control). Conjugated PPS nanoparticles at
a degree of surface functionalisation of 10% at 2% (w/v) therefore
seem to have similar binding to articular cartilage as the free
polypeptide WYRGRLC (SEQ ID NO:4) at 10 .mu.M.
Active Targeting of Articular Cartilage In Vivo
[0031] WYRGRLC (SEQ ID NO:4)--PPS and PPS nanoparticles were
labeled with 6-IAF and dialysed for 2 days with at least 2 buffer
shifts to ensure that no free label is still in the solution. A
volume of 5 .mu.l of the nanoparticles was injected into the knee
joints of 4-6 weeks old C57BL/6 mice. Three mice were injected with
WYRGRLC (SEQ ID NO:4)--PPS in the right and PPS particles in the
left knee joint. In order to do reproducible injections a 250
Hamilton syringe (Hamilton Europe, Bonaduz, Switzerland) with a
Cheney reproducibility adapter was used for antero-lateral
parapatellar injection with a 30 G needle. Cryosections which were
obtained after 24 hrs were analysed by confocal laser scanning
microscopy. Quantification of fluorescent dots per cartilage volume
as determined by sampling of z-stacks with 10 planes in 10
different locations per joint revelead an increase in particle
accumulation from 29.0.+-.1.5% for PPS particles to 83.8.+-.4.0%
for WYRGRLC (SEQ ID NO:4)--PPS particles (FIG. 4). While there is
an obvious favorable accumulation of functionalized nanoparticles
in the articular cartilage matrix after 24 hours, nanoparticles
accumulate in the whole joint at a concentration of 2% (w/v) and
enter meniscal and ligamentous tissues in addition to the synovial
membrane.
Discussion
[0032] Several methods exist for affinity selection of binding
proteins or polypeptides such as phage display.sup.s, yeast surface
display.sup.15, mRNA display.sup.16 or peptide-on-bead
display.sup.17. Herein, phage display using the fUSE5/6-mer library
based on the filamentous phage vector fd-tet.sup.18 was used to
select short peptides which bind to the articular cartilage matrix.
Embodiments of the invention include using affinity selection of
binding proteins or peptides against cartilage ex vivo.
[0033] In this case, biopanning was carried out against slices of
bovine cartilage. Conditions in the binding step were chosen with
increasing stringency from round 1 to 5 in order to favor binding
of high affinity polypeptides. The selected sequences obtained from
DNA sequencing of 96 clones have been evaluated in a competetive
binding assay.
[0034] Two sequences C1-3 and C1-C1 exhibited stronger competetive
binding than C1-F1 and random phages from the fUSE5 library (FIG.
1b). Therefore, specificity of binding for C1-3 and C1-C1 to
cartilage was further assessed. The phage clones were subjected to
physiological conditions (37.degree. C. and shaking) and binding
specificity probed for cartilage, cartilage in the presence of
synovial fluid and the synovial membrane. Phage titers of both C1-3
and C1-C1 were higher for cartilage than for synovial membrane by
two orders of magnitude. More importantly, binding to the cartilage
target was not impaired by the addition of synovial fluid (FIG.
1c). This is likely to be the result of the negative screening
which was carried out during the first round of biopanning. In
order to eliminate phages with polypeptide sequences that
potentially bind to constituents of the synovial fluid, the first
screening was carried out in the presence of synovial fluid, which
was discarded including the phages before the binding phages were
eluted off the cartilage slice. In order assess the relative
binding affinity of the polypeptide sequence, the corresponding
polypeptide WYRGRLC (SEQ ID NO:4) was synthesized. In a competetive
binding assay against the phage C1-3 displaying WYRGRL (SEQ ID
NO:1) (10.sup.8 TU/ml) an IC.sub.50 in the high nanomolar range of
about 200 nM can be demonstrated (FIG. 2).
[0035] Binding specificity was conferred by the polypeptide
sequence rather than the net positive charge of the polypeptide
which sticks non-specifically to negatively charged proteoglycans.
This is demonstrated in FIG. 3 in that 10 .mu.M of WYRGRLC (SEQ ID
NO:4) resulted in a decrease of phage titers close to 100fold. By
contrast, 10 .mu.M of YRLGRC (SEQ ID NO:7), which comprises the
same amino acids just in scrambled order, did not result in a
significant decrease in phage titer as compared to control phage
titers without polypeptide. Thus WYRGRLC (SEQ ID NO:4) is a short
polypeptide with specific binding to articular cartilage which
shows a dose dependent decrease in competetive binding against
phage C1-3. The other polypeptides, DPHFHL (SEQ ID NO:2) and RVMLVR
(SEQ ID NO:3), which were discovered using the same experimental
methods used for WYRGRL, could also be shown to have specific
binding using these same techniques.
[0036] The polypeptides were synthesized such that they contain a
cysteine at the C-terminus. The free thiol of cysteine is used for
bioconjugation by Michael-type addition to Pluronic-di-vinyl
sulfone which serves as the emulsifier in nanoparticle synthesis
and therefore remains displayed on the particle surface. While this
is a straightforward scheme for surface functionalisation of
nanoparticles synthesized by inverse emulsion polymerization,
conjugation of Pluronic requires excess of polypeptide despite the
favorable kinetics of Michael-type addition of free thiols to vinyl
sulfone, if conjugation close to 100% is to be achieved as
evidenced by .sup.1H-NMR. In addition, some Pluronic is always lost
during nanoparticle synthesis. Alternative synthetic schemes
therefore add surface functionality to already-synthesized
nanoparticles in order to limit the amount of polypeptide
needed.
[0037] Presence of the polypeptide on the nanoparticle surface as
indicated by a shift in zeta-potential from neutral to positive was
confirmed by competitive binding. WYRGRLC(SEQ ID NO:4)--PPS
nanoparticles with a degree of surface functionalisation of 10%
(w/w of total Pluronic used) at a concentration of 2% (w/v) against
phage clone C1-3 (10.sup.8 TU/ml) to articular cartilage resulted
in a similar decrease of phage titers as 10 .mu.M of free
polypeptide WYRGRLC (SEQ ID NO:4, FIG. 3).
[0038] Targeting the extra-cellular matrix of articular cartilage
essentially depends on the ability of the drug delivery system to
enter the cartilage matrix and to stay there. In passive targeting,
the distribution of nanoparticles in the joint is mainly governed
by the capability of tissue penetration and cellular uptake. While
larger particles do not enter, smaller ones are able to penetrate
and reside in the cartilage ECM. It is demonstrated in herein that
nanoparticles (in this case, nanoparticles with a mean volume
diameter of 36 and 38 nm) are able to enter the articular cartilage
ECM and meniscal tissue in addition to the synovium. It is
consistent with the literature that small particles possess the
ability to enter the cartilage ECM. It has been demonstrated that
adeno-associated viruses (AAV) with a mean diameter of 20-25 nm
enter the articular cartilage matrix up to a depth of penetration
of 450 .mu.m in normal and 720 .mu.m in degraded cartilage.sup.19.
While both surface-functionalised and non-functionalised PPS
nanoparticles enter the articular cartilage matrix, there is a
marked increase in the accumulation of WYRGRLC (SEQ ID NO:4)--PPS
nanoparticles over non-functionalised and therefore non-targeted
PPS nanoparticles 24 hours after intra-articular injection into the
knee joint of mice (FIG. 4). WYRGRLC(SEQ ID NO:4)--PPS
nanoparticles therefore exhibit specific targeting capability for
the articular cartilage matrix. Although the basic research has
been performed with bovine cartilage, the polypeptide sequences
described herein are expected to bind to human articular cartilage
due to the general homology of these tissues. Bovine cartilage is
an accepted model in cartilage research due to the very limited
availability of healthy human cartilage.
Polypeptide Ligands Specific for Articular Cartilage
[0039] Three of the cartilage tissue-binding ligands are
polypeptides with the amino acid sequence of WYRGRL (SEQ ID NO:1),
DPHFHL (SEQ ID NO:2), or RVMLVR (SEQ ID NO:3). Other ligands are
polypeptides or functional polypeptides with sequences that have
conservative substitutions of one of SEQ ID NOs. 1, 2, or 3.
[0040] Certain embodiments are directed to the subset of
polypeptides that have a certain percentage identity to the
disclosed sequences, or a certain degree of substitution, with the
subset being primarily, or only, functional polypeptides.
[0041] The binding activity of a polypeptide to cartilage may be
determined simply by following experimental protocols as described
herein. For instance, a polypeptide variant of one of the
polypeptide ligands may be labeled with a marker (e.g., radioactive
or fluorescent) and exposed to bovine cartilage to determine its
binding affinity using well-known procedures. A binding assay may
be performed using a simple fluorescence readout using a plate
reader by labeling polypeptide variants with a fluorescent marker,
e.g., 6-fluorescein iodoacetamide which reacts with the free thiol
of the cysteine. Using such a method, the binding strengths of
polypeptide variants relative to e.g., WYRGRLC (SEQ ID NO:4) under
given physiological conditions can be determined, e.g., sequences
made using conservative substitutions, truncations of the sequences
to 5 or less amino acids, addition of flanking groups, or changes
or additions for adjusting sequences for solubility in aqueous
solution.
[0042] Polypeptides of various lengths may be used as appropriate
for the particular application. In general, polypeptides that
contain the polypeptide ligand sequences will exhibit specific
binding if the polypeptide is available for interaction with
cartilage in vivo. Protein folding can affect the bioavailability
of the polypeptide ligands. Accordingly, certain embodiments are
directed to polypeptides that have a polypeptide ligand but do not
occur in nature, and certain other embodiments are directed to
polypeptides having particular lengths, e.g., from 6 to 3000
residues, or 6-1000, or 6-100, or 6-50; artisans will immediately
appreciate that every value and range within the explicitly
articulated limits is contemplated. Moreover, the lower limits may
be 4 or 5 instead of 6.
[0043] While polypeptides of 6 residues were extensively, tested,
variants that have 3, 4, or 5, residues can also be active and
exhibit specific binding, as well as conservative substitutions
thereof. Accordingly, every contiguous 3, 4, and 5 residues in each
sequence can be rapidly screened and tested for binding using the
methods set forth herein, e.g., using the sequencing and binding
assays, or with competitive inhibition. Thus, in the case of SEQ ID
NO:1, with W being the first residue and L the sixth residue,
binding activity may be expected in the three residues 1-3, 2-4,
3-5, and 4-6; for four residues, binding activity may be expected
in 1-4, 2-5, and 3-6; for five residues, binding activity may be
expected in positions 1-5 or 2-6. The ordinary artisan, after
reading this disclosure, will be able to quickly assay this limited
number of sequences. While some decrease in binding activity might
be observed when the 6-residue sequences are truncated, a core
group is expected to exhibit substantial binding. This expectation
is based on general observations made with binding moieties in
these arts. For example, in phage display experiments, the peptide
sequences often exhibit a consensus motif which usually does not
involve all residues displayed on the phage virion, e.g., in Arap
et al. (Nature Medicine 2002; 8:121). Less stringent conditions or
sequencing of previous rounds are more likely to give a consensus
motif in different peptide sequences than one very strong binding
sequence.
[0044] Certain embodiments provide various polypeptide sequences
and/or purified polypeptides. A polypeptide refers to a chain of
amino acid residues, regardless of post-translational modification
(e.g., phosphorylation or glycosylation) and/or complexation with
additional polypeptides, synthesis into multisubunit complexes,
with nucleic acids and/or carbohydrates, or other molecules.
Proteoglycans therefore also are referred to herein as
polypeptides. As used herein, a "functional polypeptide" is a
polypeptide that is capable of promoting the indicated function.
Polypeptides can be produced by a number of methods, many of which
are well known in the art. For example, polypeptides can be
obtained by extraction from a natural source (e.g., from isolated
cells, tissues or bodily fluids), by expression of a recombinant
nucleic acid encoding the polypeptide, or by chemical synthesis.
Polypeptides can be produced by, for example, recombinant
technology, and expression vectors encoding the polypeptide
introduced into host cells (e.g., by transformation or
transfection) for expression of the encoded polypeptide.
[0045] There are a variety of conservative changes that can
generally be made to an amino acid sequence without altering
activity. These changes are termed conservative substitutions or
mutations; that is, an amino acid belonging to a grouping of amino
acids having a particular size or characteristic can be substituted
for another amino acid. Substitutes for an amino acid sequence may
be selected from other members of the class to which the amino acid
belongs. For example, the nonpolar (hydrophobic) amino acids
include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and tyrosine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Such alterations are not expected to substantially affect
apparent molecular weight as determined by polyacrylamide gel
electrophoresis or isoelectric point. Exemplary conservative
substitutions include, but are not limited to, Lys for Arg and vice
versa to maintain a positive charge; Glu for Asp and vice versa to
maintain a negative charge; Ser for Thr so that a free --OH is
maintained; and Gln for Asn to maintain a free NH.sub.2. Moreover,
point mutations, deletions, and insertions of the polypeptide
sequences or corresponding nucleic acid sequences may in some cases
be made without a loss of function of the polypeptide or nucleic
acid fragment. Substitutions may include, e.g., 1, 2, 3, or more
residues. The amino acid residues described herein employ either
the single letter amino acid designator or the three-letter
abbreviation. Abbreviations used herein are in keeping with the
standard polypeptide nomenclature, J. Biol. Chem., (1969), 243,
3552-3559. All amino acid residue sequences are represented herein
by formulae with left and right orientation in the conventional
direction of amino-terminus to carboxy-terminus.
[0046] In some cases a determination of the percent identity of a
peptide to a sequence set forth herein may be required. In such
cases, the percent identity is measured in terms of the number of
residues of the peptide, or a portion of the peptide. A polypeptide
of, e.g., 90% identity, may also be a portion of a larger peptide
Variations of the disclosed polypeptide sequences include
polypeptides or functional polypeptides having about 83% identity
(e.g., 1 of 6 substituted) or about 67% identity (e.g., 2 of 6
substituted).
[0047] The term purified as used herein with reference to a
polypeptide refers to a polypeptide that either has no naturally
occurring counterpart (e.g., a peptidomimetic), or has been
chemically synthesized and is thus substantially uncontaminated by
other polypeptides, or has been separated or purified from other
most cellular components by which it is naturally accompanied
(e.g., other cellular proteins, polynucleotides, or cellular
components). An example of a purified polypeptide is one that is at
least 70%, by dry weight, free from the proteins and naturally
occurring organic molecules with which it naturally associates. A
preparation of the a purified polypeptide therefore can be, for
example, at least 80%, at least 90%, or at least 99%, by dry
weight, the polypeptide. Polypeptides also can be engineered to
contain a tag sequence (e.g., a polyhistidine tag, a myc tag, or a
Flag.RTM. tag) that facilitates the polypeptide to be purified or
marked (e.g., captured onto an affinity matrix, visualized under a
microscope). Thus a purified composition that comprises a
polypeptide refers to a purified polypeptide unless otherwise
indicated.
[0048] Polypeptides may include a chemical modification; a term
that, in this context, refers to a change in the
naturally-occurring chemical structure of amino acids. Such
modifications may be made to a side chain or a terminus, e.g.,
changing the amino-terminus or carboxyl terminus. In some
embodiments, the modifications are useful for creating chemical
groups that may conveniently be used to link the polypeptides to
other materials, or to attach a therapeutic agent.
Nanoparticles
[0049] As demonstrated by the foregoing examples, the cartilage
tissue-binding ligands may be used to target nanoparticles to a
cartilage tissue, and such nanoparticles may include a therapeutic
agent, for example, one or more of the therapeutic agents described
herein. While certain polymer-therapeutics have been described
elsewhere, these have mainly been designed to augment drug
concentrations in tumor tissues.sup.1. Such alteration of the
biodistribution of anticancer drugs through delivery systems aims
at reducing the drug's toxicity and at improving therapeutic
effects.sup.2,3. To be effective, a drug delivery system must
escape non-specific systemic accumulation and phagocytotic
clearance by the host defense immune system. Moreover, after
accumulation at the target site, penetration of the often avascular
tissue must be achieved and the drugs released in active forms in
order to exert the therapeutic effect. For targeting articular
cartilage, non-specific systemic accumulation can be avoided by
direct intra-articular injection. While this is an attractive
treatment approach because it minimizes systemic effects, small
compounds are prone to rapid lymphatic clearance and possess a
residence time of as short as 1-5 hours.sup.4.
[0050] In order to minimize intra-articular injections and to
increase the bioavailability of drugs in articular cartilage, the
nanoparticle-based therapeutic agent delivery system described
herein exhibits active targeting functionality for the cartilage,
specifically, cartilage extra-cellular matrix. The combination of
targeting functionality and a nanoparticle-based delivery system
enables better control of bioavailability and biodistribution,
particularly for intra-articular drug delivery to the cartilage
matrix. Surface functionalisation of nanoparticles with the
selected peptides controls the biodistribution by specific
accumulation of nanoparticles in the articular cartilage,
specifically in the extra-cellular matrix. The cartilage matrix
itself therefore serves as a reservoir of nanoparticle encapsulated
therapeutic molecules, which are delivered to the site of the
disease process.
[0051] As explained herein, articular cartilage can be targeted in
vivo with nanoparticles by the use of polypeptides which have been
characterized to exhibit specific homing activity to articular
cartilage. While PPS nanoparticles are used herein for
demonstrative purposes, other techniques for making nanoparticles
may also be adapted. As exemplified with the inverse emulsion
polymerisation technique for PPS nanoparticle preparation by which
the emulsifier remains displayed on the surface.sup.14, the
targeting polypeptides were made bioavailable by exposure at a
surface of the nanoparticles. Size control was achieved in this
particular technique by adjusting the emulsifier to monomer ratio
and yielded sizes ranging from about 20 nm to about 200 nm.sup.14.
While the density of the cartilage extra-cellular matrix represents
a relevant obstacle not just for the screening of combinatorial
peptide libraries but potentially also for drug delivery to the
cartilage, the use of suitably-sized nanoparticles and/or ligands
with specific binding to the cartilage enhances delivery
efficiency.
[0052] Nanoparticles are be prepared as collections of particles
having an average diameter of between about 10 nm and about 200 nm,
including all ranges and values between the explicitly articulated
bounds, e.g., from about 20 to about 200, and from about 20 to
about 40, to about 70, or to about 100 nm, depending on the
polydispersity which is yielded by the preparative method. Detailed
methods for making and delivering nanoparticles are set forth below
and in U.S. Pat. Ser. No. 60/775,132, filed Feb. 21, 2006, which is
hereby incorporated by reference herein. Numerous nanoparticle
systems can be utilized, such as those formed from copolymers of
poly(ethylene glycol) and poly(lactic acid), those formed from
copolymers of poly(ethylene oxide) and poly(beta-amino ester), and
those formed from proteins such as serum albumin. Other
nanoparticle systems are known to those skilled in these arts. See
also Devalapally et al., Cancer Chemother Pharmacol., 07-25-06;
Langer et al., International Journal of Pharmaceutics, 257:169-180
(2003); and Tobio et al., Pharmaceutical Research, 15(2):270-275
(1998).
[0053] Larger particles of more than about 200 nm average diameter
incorporating the cartilage tissue-binding ligands may also be
prepared, with these particles being termed microparticles herein
since they begin to approach the micron scale and fall
approximately within the limit of optical resolution. For instance,
certain techniques for making microparticles are set forth in U.S.
Pat. Nos. 5,227,165, 6,022,564, 6,090,925, and 6,224,794.
[0054] Functionalization of nanoparticles to employ targeting
capability requires association of the targeting polypeptide with
the particle, e.g., by covalent binding using a bioconjugation
technique, with choice of a particular technique being guided by
the particle or nanoparticle, or other construct, that the
polypeptide is to be joined to. In general, many bioconjugation
techniques for attaching peptides to other materials are well known
and the most suitable technique may be chosen for a particular
material. For instance, additional amino acids may be attached to
the polypeptide sequences, such as a cysteine in the case of
attaching the polypeptide to thiol-reactive molecules. Herein is
described an example of conjugation of the polypeptide WYRGRL (SEQ
ID NO:1) with a cysteine at the C-terminus to Pluronic-F127 which
was derivatized with thiol-reactive vinyl sulfone. The polypeptide
was covalently bound to Pluronic by Michael-type addition and can
subsequently be used for nanoparticle synthesis.
Proteins Targeted to Articular Cartilage Tissue
[0055] Therapeutic agents such as therapeutic polypeptides can be
furnished with targeting capability by the use of the polypeptide
ligands described and advantageously exhibit longer retention times
in a joint, for instance by making a fusion protein of a
polypeptide ligand and a therapeutic protein. By designing gene
specific primers for the therapeutic polypeptide to be expressed,
the polypeptide ligands can be attached to the N- or C-terminus in
normal or reverse order. One of the primers, forward or reverse
depending whether the polypeptide ligand is supposed to be
localized at the N- or C-terminus, contains a gene sequence of the
appropriate therapeutic polypeptide. For example, to make a fusion
protein of a given therapeutic polypeptide and (GGG)WYRGRL (SEQ ID
NO:8) ligand at the C-terminus, the reverse primer corresponds to
5'-ctgatgcggccgctcTCACAGCCTGCCCCTATACCAGCCGCCGCCxxxxx-3' (SEQ ID
NO:9) which contains the codon sequence for WYRGRL in reverse
complement with a N-terminal glycine (GGG) linker (capital
letters), as well as a stop codon and a restriction site for NotI
and an overhang. The Xs correspond to the therapeutic polypeptide
specific sequence and may be, e.g., around 20 base pairs long,
although other lengths may be used as per conventional practice in
these arts. The same example with same restriction site for a
N-terminal localization of WYRGRL(GGG) (SEQ ID NO:10) corresponds
to 5'-atcaggageggccgcTGGTATAGGGGCAGGCTGGGCGGCGGCxxxxx-3' (SEQ ID
NO:11) or conservative substitutions of the codon sequence. Instead
of the three glycines as a linker, other linkers appropriate to the
properties of the therapeutic polypeptides can be chosen.
Similarly, other target ligands may be encoded, e.g., using thea
nucleic acid sequence
5'-ctgatgeggccgctcAAGATGGAAATGAGGATCGCCGCCGCCxxxxx-3' (SEQ ID NO:
12) that endocdes DPHFHLGGG (SEQ ID NO:13) or the nucleic acid
5'-ctgatgcggccgctcACGAACAAGCATAACACGGCCGCCGCCxxxxx-3'(SEQ ID NO:14)
that encodes RVMLVRGGG (SEQ ID NO:15). Thus a DNA sequence for
WYRGRL (SEQ ID NO:1) is TGGTATAGGGGCAGGCTG (SEQ ID NO: 16), and for
DPHFHL (SEQ ID NO:2) is AAGATGGAAATGAGGATC (SEQ ID NO:17), and for
RVMLVR (SEQ ID NO:3) is ACGAACAAGCATAACACG (SEQ ID NO:18).
[0056] Certain therapeutic polypeptides include proteins present in
cartilage, e.g., tissue inhibitors of matrix metalloproteinase-3
(TIMP-3), growth factors (e.g., Transforming growth factor-beta
(TGF-.beta.), growth developmental factor-5 (GDF-5), CYR61
(Cystein-rich61)/CTGF (connective tissue growth factor)/NOV
(Nephroblastoma overexpressed) (CCN2), insulin-like growth factor-1
(IGF-1), and bone morphogenic proteins (BMPs). Certain embodiments
are molecules for viscosupplementation, e.g., molecules found in
human cartilage that include chondroitin sulfate, keratane sulfate,
hyaluronic acid, proteoglycans.
[0057] In some embodiments, a fusion protein is prepared and
introduced into the body as a purified composition in a
pharmaceutically acceptable condition, or with a pharmaceutical
excipient. In certain embodiments, the fusion protein is produced
using a cell, either a procaryotic or a eucaryotic cell. In other
embodiments, nucleic acids encoding a fusion protein are introduced
into a patient, in which case the nucleic acids may be "naked" or
part of a larger construct, e.g., a vector. In other embodiments,
transfected cells are introduced into a patient. The site of
introduction may be, e.g., systemic, in a joint, or in a cartilage
tissue.
Cartilage Binding Fusion Protein: Targeted Recombinant TIMP-3
[0058] Tissue inhibitor of matrix metalloproteinase 3 (TIMP-3) is a
relatively insoluble matricellular protein.sup.30 which inhibits
several matrix metalloproteinases (MMP-1, -2, -9).sup.31 in
addition to aggrecanase 1 and 2, i.e. ADAMTS4 and ADAMTS5.sup.32.
As such, this molecule is beneficial in the treatment of
osteoarthritis in any joint, specifically in a setting where the
initiating mechanical cause is surgically corrected. Because TIMP-3
blocks these matrix degradative enzymes, the equilibrium in matrix
turn-over of articular cartilage may be restored by preventing
further degradation. TIMP-3 is not specifically expressed in
articular cartilage, however, and due to its ability of inhibiting
several enzymes may have severe potential adverse effects upon
systemic administration or systemic dissemination following
intra-articular injection. Targeted delivery of the inhibitory
domain of TIMP-3 to articular cartilage can prevent potential
systemic dissemination while increasing the therapeutic effect of
the molecule in the cartilage matrix, i.e. at the site of the
disease process in osteoarthritis. Moreover, the inhibitory domain
of TIMP-3 also serves as a protectant against cartilage degradation
in conditions such as rheumatoid arthritis, bacterial arthritis or
reactive arthritis. Disclosed herein is a targeted recombinant
TIMP-3 (trTIMP-3) (fusion protein which contains one of the
sequences (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) or conservative
substitutions thereof, e.g, at the N- or C-terminus. While TIMP-3
has been selected as an embodiment, other protease inhibitors or
matrix metalloproteinase inhibitors may also be made and used by
following the general procedures disclosed herein.
[0059] TIMP-3 contains a signal domain, residues 1 to 24, the
N-terminal inhibitory domain, residues 24 to 143, and an
extra-cellular matrix binding domain, residues 143 to 211
(SwissProt entry P35625). It has been demonstrated that N-TIMP-3,
i.e. residues 24 to 143, is sufficient for exhibiting inhibitory
activities on ADAMTS4 and ADAMTS5 as well as MMP-1 and MMP-2.sup.3.
Accordingly, it is possible to engineer a fusion protein of
N-TIMP-3 (Cys24 to Asn 143) which contains the sequence WYRGRL (SEQ
ID NO:1) at the C-terminus. The natural non-specific C-terminal
extra-cellular matrix binding domain is substituted with a
targeting polypeptide specific for articular cartilage.
[0060] Human (P35625) and mouse N-TIMP-3 (P39876) share 97%
sequence homology differing in three amino acid residues. Two of
these residues represent conservative substitutions, Thr vs. Ser at
residue 74 and Asp vs. Glu at residue 110. At residue 126 His
substituted for Gln, which lies in a domain linking two
.alpha.-chains.
[0061] For cloning, the sequence in NM.sub.--000362 was used for
primer design. The primers were designed as explained above (see
SEQ ID NO:9 for reversed primer and SEQ ID NO:11 for the forward
primer). RNA was isolated using a RNeasy MinElute spin column
(Qiagen, Hombrechikon, Switzerland) from immature murine articular
chondrocytes which have been isolated from epiphyseal cartilage of
neonatal mice using a two step digestion protocol with collagenase
D (Roche, Basel, Switzerland). First-strand cDNA was generated
using SuperScript III (Invitrogen, Carlsbad, Calif.) with an
Oligo-dT(20) primer (Microsynth, Balgach, Switzerland). RT-PCR for
amplifying the required DNA fragments was carried out with a
proof-reading DNA polymerase (Pfu Turbo polymerase, Stratagene,
LaJolla, Calif.) and the fragments with a required length of 420 bp
checked by agarose-gel electrophoresis. The DNA fragments were gel
purified with a Nucleospin II column (Macherey-Nagel, Duren,
Germany), cut with the corresponding restriction enzymes BamHI and
NotI (New England BioLabs Inc., Ipswich, Mass.) and ligated into
the bacterial protein expression vector pGEX-4T-1 (Amersham
Biosciences, GE Healthcare Europe, Otelfingen, Switzerland), which
expresses trTIMP-3 as a fusion protein with
glutathione-S-transferase (GST) for purification, total MW 40 kDa.
For bacterial expression, E. coli strain BL21 was transformed with
the trTIMP-3-pGEX construct by electroporation. An optimal
expression clone was selected by anti-GST ELISA.
[0062] Bacterial cultures were grown in 2.times.YT until they
reached an A.sub.600 of 0.6 to 0.7. Protein expression was then
induced by 0.5M IPTG for 3 hours. To collect the protein from
inclusion bodies, the bacteria were centrifuged, lysed by
sonication and centrifuged again. The pellet was resuspended in a
denaturing buffer (0.1M Tris-HCl, 50 mM glycine, 8 mM
.beta.-mercaptoethanol, 8M urea, 0.2M PMSF, pH 8.0) and stirred
overnight, centrifuged for 20 min. at 48000.times.g and the
supernatant collected for refolding. Refolding was performed by
dialysing (24 kDa MWCO) the supernatant against a large volume of
refolding buffer at 4.degree. C. with decreasing amounts of urea to
slowly remove the denaturing agent (0.1M Tris-HCl, 1 mM EDTA, 0.2
mM PMSF, pH 8.0 supplemented with 4M, 2M, 1M and 0M urea each for
24 hrs). The last step of dialysis was carried out against PBS for
24 hours. The refolded protein was freed from precipitations by
centrifugation and subsequently purified by FPLC with a GST binding
column (GSTrap FF, Amersham Biosciences, GE Healthcare Europe,
Otelfingen, Switzerland). The GST tag of purified trTIMP-3-GST
fusion protein was cleaved of by incubation with thrombin for 24
hours and purified by FPLC from GST with the GSTrap FF column and
from thrombin with a HiTrap Benzamidine FF column (Amersham
Biosciences, GE Healthcare Europe, Otelfingen, Switzerland).
Overall yield of trTIMP-3 was about 1.5 mg/l of bacterial
culture.
[0063] The activity of purified trTIMP-3 was assessed by MMP-2
zymography in which an equimolar amount of trTIMP-3 completely
inhibits MMP-2 activity indicating a nearly 100% activity of the
purified protein. Further experiments could be used to demonstrate
the aggrecanase inhibiting activity of trTIMP-3 and the targeting
specificity to articular cartilage in a similar fashion as shown
for the nanoparticles described herein. In addition, its
therapeutic potential to prevent cartilage degradation could be
demonstrated in a suitable animal model, e.g., in a mouse knee
instability model.
Vectors
[0064] Accordingly, certain embodiments are directed to vectors for
expression of a therapeutic protein of interest, e.g., a
therapeutic agent and a polypeptide ligand. Nucleic acids encoding
a polypeptide can be incorporated into vectors. As used herein, a
vector is a replicon, such as a plasmid, phage, or cosmid, into
which another nucleic acid segment may be inserted so as to bring
about replication of the inserted segment. Vectors of the invention
typically are expression vectors containing an inserted nucleic
acid segment that is operably linked to expression control
sequences. An expression vector is a vector that includes one or
more expression control sequences, and an expression control
sequence is a DNA sequence that controls and regulates the
transcription and/or translation of another DNA sequence.
Expression control sequences include, for example, promoter
sequences, transcriptional enhancer elements, and any other nucleic
acid elements required for RNA polymerase binding, initiation, or
termination of transcription. With respect to expression control
sequences, "operably linked" means that the expression control
sequence and the inserted nucleic acid sequence of interest are
positioned such that the inserted sequence is transcribed (e.g.,
when the vector is introduced into a host cell). For example, a DNA
sequence is operably linked to an expression-control sequence, such
as a promoter when the expression control sequence controls and
regulates the transcription and translation of that DNA sequence.
The term "operably linked" includes having an appropriate start
signal (e.g., ATG) in front of the DNA sequence to be expressed and
maintaining the correct reading frame to permit expression of the
DNA sequence under the control of the expression control sequence
to yield production of the desired protein product. Examples of
vectors include: plasmids, adenovirus, Adeno-Associated Virus
(AAV), Lentivirus (FIV), Retrovirus (MoMLV), and transposons. There
are a variety of promoters that could be used including, but not
limited to, constitutive promoters, tissue-specific promoters,
inducible promoters, and the like. Promoters are regulatory signals
that bind RNA polymerase in a cell to initiate transcription of a
downstream (3' direction) coding sequence.
Targeted Deliver of Viscosupplementation
[0065] Molecules for viscosupplementation may be conjugated with
polypeptide ligands described herein to form a conjugate for
enhanced delivery and effect in the cartilage. The formation of
such conjugates is within the skill of ordinary artisans and
various techniques are known for accomplishing the conjugation,
with the choice of the particular technique being guided by the
materials to be conjugated. Such conjugates may be delivered
systemically or locally, e.g., orally or by injection to a joint.
Thus hyaluronic acid conjugation with a polypeptide ligand
disclosed herein may prolong the retention time of hyaluronic acid
in the joint and therefore enhance the efficacy of intra-articular
viscosupplementation with hyaluronic acid. Conjugation of the
polypeptide to hyaluronic acid can be performed either directly as
described above, or by the use of a polymer linker. Examples of
polymer linkers are biocompatible hydrophilic polymers, including
polymers free of amino-acids. For instance, a polymer linker may be
a polyethylene glycol (PEG). Hyaluronic acid can be functionalized
with acrylated PEG-Arg-Gly-Asp conjugates created by Michael-type
addition chemistry (Park et al. Biomaterials 2003; 24:893-900). In
general, polymers described herein for use with the polypeptide
ligands may be free of amino acids, meaning that such polymers do
not contain a natural or synthetic amino acid.
[0066] In some embodiments, the conjugate is prepared and
introduced into the body as a purified composition in a
pharmaceutically acceptable condition, or with a pharmaceutical
excipient. In certain embodiments, the conjugate is produced using
a cell, either a procaryotic or a eucaryotic cell, as in the case
of a biopolymer. In other embodiments, transfected cells are
introduced into a patient. The site of introduction may be, e.g.,
systemic, in a joint, or in a cartilage tissue.
Targeting Genes Condensed with Polymers with the Polypeptides
Attached
[0067] A small gene delivery system will have advantages with
respect to penetrating the dense matrix of cartilage tissue. In
some embodiments, therefore, the delivery system uses significantly
condensed DNA, to enter the cartilage matrix and transfect
non-dividing quiescent chondrocytes or other cells. In some
embodiments, therefore, polypeptide ligands are attached to
polymers which contain a nuclear localisation sequence to form a
conjugate and are used to condense DNA to overcome challenges to
gene transfection in chondrocytes embedded in the cartilage matrix.
Some aspects of these techniques have been described by in Trentin
et al. PNAS 2006; 103:2506-11 and J Control Release 2005;
102:263-75. Thus in certain embodiments the conjugate associated
with nucleic acids encoding a therapeutic polypeptide is prepared
and introduced into the body as a purified composition in a
pharmaceutically acceptable condition, or with a pharmaceutical
excipient. In certain embodiments, such a conjugate is produced
using a cell, either a procaryotic or a eucaryotic cell, as in the
case of a biopolymer. In other embodiments, transfected cells are
introduced into a patient. The site of introduction may be, e.g.,
systemic, in a joint, or in a cartilage tissue.
Therapeutic Agents Associated with Ligands for Delivery to
Cartilage
[0068] Polypeptides as described herein can be attached to other
polymers through bioconjugation. The formation of such conjugates
is within the skill of ordinary artisans and various techniques are
known for accomplishing the conjugation, with the choice of the
particular technique being guided by the materials to be
conjugated. The addition of amino acids to the polypeptide (C- or
N-terminal) which contain ionizable side chains, i.e. aspartic
acid, glutamic acid, lysine, arginine, cysteine, histidine, or
tyrosine, and are not contained in the active portion of the
polypeptide sequence, serve in their unprotonated state as a potent
nucleophile to engage in various bioconjugation reactions with
reactive groups attached to polymers, i.e. homo- or
hetero-bi-functional PEG (e.g., Lutolf and Hubbell,
Biomacromolecules 2003; 4:713-22, Hermanson. Bioconjugate
Techniques. London. Academic Press Ltd; 1996). An application where
this may be useful is again for targeted delivery of a therapeutic
agent. In some embodiments, the agent is attached to a soluble
polymer, and may be administer to a patient in a pharmaceutically
acceptable form. Or a drug may be encapsulated in polymerosomes or
vesicles or covalently attached to polymers. In the latter case,
drugs are attached to the polymer backbone with a degradable
site-specific spacer or linker (Lu et al. J Control Release 2002;
78:165-73).
[0069] In general, soluble hydrophilic biocompatible polymers may
be used to ensure that the conjugate is soluble and will be
bioavailable after introduction into the patient. Examples of
soluble polymers are polyvinyl alcohols, polyethylene imines, and
polyethylene glycols (a term including polyethylene oxides) having
a molecular weight of at least 100, 400, or between 100 and 400,000
(with all ranges and values between these explicit values being
contemplated). Solubility refers to a solubility in water or
physiological saline of at least 1 gram per liter. Domains of
biodegradable polymers may also be used, e.g., polylactic acid,
polyglycolic acid, copolymers of polylactic and polyglycolic acid,
polycaprolactones, polyhydroxy butyric acid, polyorthoesters,
polyacetals, polydihydropyrans, and polycyanoacylates.
[0070] In some embodiments, a polypeptide-polymer association,
e.g., a conjugate, is prepared and introduced into the body as a
purified composition in a pharmaceutically acceptable condition, or
with a pharmaceutical excipient. The site of introduction may be,
e.g., systemic, in a joint, or in a cartilage tissue.
Cartilage Defect Treatment Using Polypeptide Ligands with Specific
Binding for Cartilage
[0071] The polypeptides ligands were discovered based on their
binding affinity for the cartilage matrix but are not specifically
bound to the articular surface. Therefore, besides embodiments such
as targeting the cartilage matrix by therapeutic agent delivery
systems or with engineered fusion proteins, the polypeptides are
particularly well suited to target a defect because extracellular
matrix is exposed at the defect. This feature is useful for
delivering biomaterials for gene, protein and/or cell delivery to
mediate cartilage defect repair. Accordingly, embodiments include
treating a defect in an articular cartilage using one of the
embodiments set forth herein. In fact, giving a biomaterial the
capability of adhering or otherwise specifically binding to a
cartilage defect does not only allow for injectable defect repair
strategies but also may enhance retention of the biomaterial in the
cartilage defect. A variety of chemical schemes can be used to
incorporate the cartilage-binding polypeptide into the biomaterial.
For example, using a material as described by Sawhney et al., a
chemical approach for incorporation as described by Hern et al. can
be employed (Sawhney et al. Macromolecules 1993; 26:581-587 and
Hern et al. J. Biomed. Mater. Res. 1998; 39:266-276). As another
example, using a material as described by Lutolf et al., a chemical
approach for incorporation as described therein can be employed
(Lutolf et al. Nature Biotechnol. 2003; 21:513-518). In general,
the modification of such biomaterials is within the skill of
ordinary artisans and various techniques are known for
accomplishing the modification, with the choice of a particular
technique being guided by the biomaterial and peptides to be
conjugated.
[0072] Specific binding, as that term is commonly used in the
biological arts, generally refers to a molecule that binds to a
target with a relatively high affinity compared to non-target
tissues, and generally involves a plurality of non-covalent
interactions, such as electrostatic interactions, van der Waals
interactions, hydrogen bonding, and the like. Specific binding
interactions characterize antibody-antigen binding,
enzyme-substrate binding, and specifically binding protein-receptor
interactions; while such molecules may bind tissues besides their
targets from time to time, such binding is said to lack specificity
and is not specific binding. The peptides of SEQ ID NOs 1, 2, and 3
may bind non-cartilage tissues in some circumstances but such
binding has been observed to be non-specific, as evidenced by the
much greater binding of the peptides to the targeted tissue as
opposed to surrounding joint tissues (data not shown).
[0073] Accordingly, embodiments include biomaterials comprising at
least one of the ligands disclosed herein that are used to fill or
augment a defect in a cartilage. A defect refers to a void in a
surface (e.g., a pit, tear, or hole) or a pathological
discontinuity in a surface (e.g., a tear or eroded member). Fill
refers to essentially filling or covering the defect or bridging
the discontinuity. Augment refers to at least partial filling. In
some embodiments, the biomaterial is a solid prior to placement in
a patient, while in other embodiments the material is made is situ,
meaning it is formed from precursors at the site of the defect.
Thus biomaterials for cartilage defects may be supplemented with a
ligand or other embodiment set forth herein. Examples of such
biomaterials include U.S. Pat. Nos. 5,874,500, and 5,410,016, and
which include materials formed by in-situ polymerization.
Biomaterials for targeting cartilage defects, i.e. adhering to a
cartilage defect, are suited for the delivery of therapeutic agents
to mediate cartilage repair such as growth factors and for cell
delivery to the repair site. In accordance with techniques for
autologous cartilage transplantation/implantation the use of a
biomaterial for cell delivery which adheres to the defect by the
use of polypeptide ligands (SEQ ID NO:1 through 3) and acts as a
morphogenic guide may improve cartilage defect repair.
Nucleic Acids
[0074] Certain embodiments are directed to nucleic acids. As used
herein, the term nucleic acid refers to both RNA and DNA, including
siRNA, shRNA, miRNA, cDNA, genomic DNA, synthetic (e.g., chemically
synthesized) DNA, as well as naturally occurring and chemically
modified nucleic acids, e.g., synthetic bases or alternative
backbones. A nucleic acid molecule can be double-stranded or
single-stranded (i.e., a sense or an antisense single strand). An
isolated nucleic acid refers to a nucleic acid that is separated
from other nucleic acid bases that are present in a genome,
including nucleic acids that normally flank one or both sides of a
nucleic acid sequence in a vertebrate genome (e.g., nucleic acids
that flank a gene). A conservatively substituted nucleic acid
refers to the substitution of a nucleic acid codon with another
codon that encodes the same amino acid and also refers to nucleic
acids that encode conservatively substituted amino acids, as
described herein with respect to polypeptides. Significantly, the
combination of potential codons for a polypeptide of only about six
residues is manageably small.
[0075] The nucleic acid sequences set forth herein are intended to
represent both DNA and RNA sequences, according to the conventional
practice of allowing the abbreviation "T" stand for "T" or for "U",
as the case may be, for DNA or RNA. Polynucleotides are nucleic
acid molecules of at least three nucleotide subunits.
Polynucleotide analogues or polynucleic acids are chemically
modified polynucleotides or polynucleic acids. In some embodiments,
polynucleotide analogues can be generated by replacing portions of
the sugar-phosphate backbone of a polynucleotide with alternative
functional groups. Morpholino-modified polynucleotides, referred to
herein as "morpholinos," are polynucleotide analogues in which the
bases are linked by a morpholino-phosphorodiamidate backbone (see,
e.g., U.S. Pat. Nos. 5,142,047 and 5,185,444). In addition to
morpholinos, other examples of polynucleotide analogues include
analogues in which the bases are linked by a polyvinyl backbone,
peptide nucleic acids (PNAs) in which the bases are linked by amide
bonds formed by pseudopeptide 2-aminoethyl-glycine groups,
analogues in which the nucleoside subunits are linked by
methylphosphonate groups, analogues in which the phosphate residues
linking nucleoside subunits are replaced by phosphoroamidate
groups, and phosphorothioated DNAs, analogues containing sugar
moieties that have 2' O-methyl group). Polynucleotides of the
invention can be produced through the well-known and routinely used
technique of solid phase synthesis. Alternatively, other suitable
methods for such synthesis can be used (e.g., common molecular
cloning and chemical nucleic acid synthesis techniques). Similar
techniques also can be used to prepare polynucleotide analogues
such as morpholinos or phosphorothioate derivatives. In addition,
polynucleotides and polynucleotide analogues can be obtained
commercially. For oligonucleotides, examples of pharmaceutically
acceptable compositions are salts that include, e.g., (a) salts
formed with cations such as sodium, potassium, ammonium, etc.; (b)
acid addition salts formed with inorganic acids, for example,
hydrochloric acid, hydrobromic acid (c) salts formed with organic
acids e.g., for example, acetic acid, oxalic acid, tartaric acid;
and (d) salts formed from elemental anions e.g., chlorine, bromine,
and iodine.
Pharmaceutical Carriers
[0076] Pharmaceutically acceptable carriers or excipient may be
used to deliver embodiments as described herein. Excipient refers
to an inert substance used as a diluent or vehicle for a
therapeutic agent. Pharmaceutically acceptable carriers are used,
in general, with a compound so as to make the compound useful for a
therapy or as a product. In general, for any substance, a
pharmaceutically acceptable carrier is a material that is combined
with the substance for delivery to an animal. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners
and the like may be necessary or desirable. In some cases the
carrier is essential for delivery, e.g., to solubilize an insoluble
compound for liquid delivery; a buffer for control of the pH of the
substance to preserve its activity; or a diluent to prevent loss of
the substance in the storage vessel. In other cases, however, the
carrier is for convenience, e.g., a liquid for more convenient
administration. Pharmaceutically acceptable salts of the compounds
described herein may be synthesized according to methods known to
those skilled in this arts. Thus a pharmaceutically acceptable
composition has a carrier, salt, or excipient suited to
administration to a patient. Moreover, inert components of such
compositions are biocompatible and not toxic.
[0077] The compounds described herein are typically to be
administered in admixture with suitable pharmaceutical diluents,
excipients, extenders, or carriers (termed herein as a
pharmaceutically acceptable carrier, or a carrier) suitably
selected with respect to the intended form of administration and as
consistent with conventional pharmaceutical practices. Thus the
deliverable compound may be made in a form suitable for oral,
rectal, topical, intravenous injection, intra-articular injection,
or parenteral administration. Carriers include solids or liquids,
and the type of carrier is chosen based on the type of
administration being used. Suitable binders, lubricants,
disintegrating agents, coloring agents, flavoring agents,
flow-inducing agents, and melting agents may be included as
carriers, e.g., for pills. For instance, an active component can be
combined with an oral, non-toxic, pharmaceutically acceptable,
inert carrier such as lactose, gelatin, agar, starch, sucrose,
glucose, methyl cellulose, magnesium stearate, dicalcium phosphate,
calcium sulfate, mannitol, sorbitol and the like. The compounds can
be administered orally in solid dosage forms, such as capsules,
tablets, and powders, or in liquid dosage forms, such as elixirs,
syrups, and suspensions. The active compounds can also be
administered parentally, in sterile liquid dosage forms. Buffers
for achieving a physiological pH or osmolarity may also be
used.
[0078] All patent applications, patents, and publications mentioned
herein are hereby incorporated by reference herein to the extent
they do not directly contradict the explicit disclosures set forth
herein.
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[0110] This application discloses various inventive embodiments
that each have certain features. In general, these features may be
mixed-and-matched with each other to create additional functional
embodiments.
Sequence CWU 1
1
1816PRTmammalian 1Trp Tyr Arg Gly Arg Leu1 526PRTmammalian 2Asp Pro
His Phe His Leu1 536PRTmammalian 3Arg Val Met Leu Val Arg1
547PRTmammalian 4Trp Tyr Arg Gly Arg Leu Cys1 5519DNAmammalian
5catgtaccgt aacactgag 1966PRTmammalian 6Tyr Arg Leu Gly Arg Trp1
577PRTmammalian 7Tyr Arg Leu Gly Arg Trp Cys1 589PRTmammalian 8Gly
Gly Gly Trp Tyr Arg Gly Arg Leu1 5945DNAmammalian 9ctgatgcggc
cgctctcaca gcctgcccct ataccagccg ccgcc 45109PRTmammalian 10Trp Tyr
Arg Gly Arg Leu Gly Gly Gly1 51142DNAmammalian 11atcaggagcg
gccgctggta taggggcagg ctgggcggcg gc 421242DNAmammalian 12ctgatgcggc
cgctcaagat ggaaatgagg atcgccgccg cc 42139PRTmammalian 13Asp Pro His
Phe His Leu Gly Gly Gly1 51442DNAmammalian 14ctgatgcggc cgctcacgaa
caagcataac acggccgccg cc 42159PRTmammalian 15Arg Val Met Leu Val
Arg Gly Gly Gly1 51618DNAmammalian 16tggtataggg gcaggctg
181718DNAmammalian 17aagatggaaa tgaggatc 181818DNAmammalian
18acgaacaagc ataacacg 18
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