United States Patent: 6,090,911
Inventors: Petka; Wendy A. (St. Paul, MN); Tirrell; David A. (Sunderland, MA); McGrath; Kevin P. (Alpharetta, GA)Assignee: University of Massachusetts (Boston, MA)
Appl. No.: 956307Filed: October 22, 1997
The invention is based on the discovery that a block copolymer that includes .alpha.-helical blocks, e.g., terminal blocks, which form intermolecular coiled-coil structures, and one or more random-coil blocks, which link the .alpha.-helical blocks, can form suspensions that can reversibly gel to form monodisperse hydrogels. The transition between the gel and liquid phases depends on pH, temperature, concentration, and chemical structure. The copolymers can be synthesized biologically through genetic engineering.
SUMMARY OF THE INVENTION
The invention is based on the discovery that a synthetic
block copolymer that includes .alpha.-helical blocks, e.g., terminal
blocks, which form intermolecular coiled-coil structures, and one or more
random-coil blocks, which link the .alpha.-helical blocks, can form
suspensions that can reversibly gel to form monodisperse hydrogels. The
transition between the gel and liquid phases depends on pH, temperature,
concentration, and chemical structure. The copolymers can be synthesized
chemically and biologically, e.g., through genetic engineering.
One embodiment of the invention features a synthetic block copolymer XYZ.
The block copolymer XYZ includes two .alpha.-helical protein blocks X and
Z, each having an amino acid sequence and conformation that allow each
.alpha.-helical protein block to form a coiled-coil with an
.alpha.-helical protein block on another block copolymer XYZ; and a
water-soluble, random-coil protein block Y, the random-coil protein block
covalently linking the two .alpha.-helical protein blocks. For example,
the first .alpha.-helical block, the random-coil block, and the second
.alpha.-helical block can form a continuous peptide chain. Optionally,
other amino acid sequences such as .beta.-sheet or turn sequences can be
included in the peptide chain, either at the ends of the chain or between
the other blocks (e.g., between the .alpha.-helical and random-coil
blocks).
Another embodiment of the invention features a synthetic block copolymer
having at least two .alpha.-helical protein blocks, each having an amino
acid sequence and conformation that allow each .alpha.-helical protein
block to form a coiled-coil with an .alpha.-helical protein block on
another molecule of the block copolymer; and at least one water-soluble,
random-coil protein block, the random-coil protein block linking at least
two of the .alpha.-helical protein blocks.
The random-coil block Y can have the sequence [(AlaGly).sub.p
ProGluGly].sub.n (SEQ ID NO: 23), where p is 0 to 4 (e.g., 1, 2, or 3) and
n is 5 to 100 (e.g., 8 to 54).
The sequences of amino acids that make up X and Z have an (ABCDEFG).sub.m
(SEQ ID NO: 24) pattern, where m is 4 to 100 (e.g., 6 to 18); A and D are
hydrophobic amino acids; E and G are polar amino acids; and B, C, and F
can be any amino acid. In some cases, more than 80% of the E and G amino
acids in X and Z are acidic amino acids. In other cases, more than 80% of
the E and G amino acids in X and Z are basic amino acids. Alternatively,
more than 80% of the E and G amino acids of X can be acidic amino acids
while more than 80% of the E and G amino acids of Z can be basic amino
acids. In another alternative, more than 80% of the E amino acids of X and
G amino acids of Z can be acidic amino acids while more than 80% of the E
amino acids of Z and G amino acids of X can be basic amino acids. In still
another alternative, more than 80% of the E amino acids of X and Z can be
acidic amino acids while more than 80% of the G amino acids of X and Z can
be basic amino acids.
More than 80% of the D amino acids of X and Z can be leucine, for example,
or trifluoroleucine.
The block copolymer can also include linker proteins that link the
.alpha.-helical protein blocks to the random-coil block protein.
In some cases, X and Z are at least 90% identical to each other. X and Z
can be non-identical.
The block copolymer can also include a recognition element that
specifically binds to a cell (e.g., a fibroblast) or to a macromolecule
(i.e., the element binds preferentially to the target cell or molecule in
a sample including the target, but does not bind to other cells or
molecules in the sample). Examples of such recognition elements include
the heparin-binding domain, the endothelial-binding domain, or the
sequence ArgGlyAsp. The recognition element is generally continuous with
the peptide chain that makes up the copolymer, and can be incorporated
either within the random-coil block, between separate blocks (e.g.,
linking the .alpha.-helical block and the random-coil block), or at an end
of the peptide sequence. Alternatively, the recognition element can be
bound to the copolymer via hydrophobic interactions, electrostatic
interactions, disulfide bonds, or hydrogen bonds.
The invention also features a gel that includes a liquid (e.g., an aqueous
liquid such as water) and a block copolymer suspended in the liquid. The
suspension can be monodisperse.
In another aspect, the invention features a method for making a block
copolymer. The method includes the steps of obtaining host cells including
an expression vector having a DNA sequence that encodes the amino acid
sequence of the block copolymer; culturing the host cells under conditions
and for a time sufficient to express the block copolymer; and isolating
the block copolymer from the host cells.
Yet another embodiment of the invention is a method of using a block
copolymer to stimulate regeneration of tissue around a wound. The method
includes the steps of dissolving the copolymer in a liquid to form a
solution, and treating the wound with the solution to form a gelatinous
scaffold for tissue regeneration.
Still another embodiment of the invention is a wound dressing that
includes a block copolymer and an antibiotic compound (e.g., bacitracin,
neosporin, erythromycin), where the copolymer and the antibiotic are both
dissolved in a liquid.
In another aspect, the invention features nucleic acids encoding the new
block copolymers. Examples of suitable nucleic acids can include the
following sequences:
GGT GAC CTG GAA AAC GAA GTG GCC CAG CTG GGA AGG GAA
GTT AGA TCT CTG GAA GAT GAA GCG GCT GAA CTG GAA CAA
AAA GTC TCG AGA CTG AAA AAT GAA ATC GAA GAC CTG AAA
GCC GAA (SEQ ID NO:21); and
GGT GAC CTG AAA AAC AAA GTG GCC CAG CTG AAA AGC AAA
GTT AGA TCT CTG AAA GAT AAA GCG GCT GAA CTG AAA CAA
GAA GTC TCG AGA CTG GAA AAT GAA ATC GAA GAC CTG AAA
GCC AAA (SEQ ID NO:20).
The invention also features a vector that includes this nucleic acid operatively linked to a promoter. As used herein, the term "operatively linked" means that selected DNA, e.g., encoding the copolymers, is in proximity with a promoter, e.g., a tissue-specific promoter, to allow the promoter to regulate expression of the selected DNA. In addition, the promoter is located upstream of the selected DNA in terms of the direction of transcription and translation. Suitable promoters include the P.sub.lac promoter, the T5 promoter, the adenovirus major late promoter, early and late promoters of SV40, CMV promoter, TH promoter, RSV promoter, or B19p6 promoter (Shad et al., J. Virol., 58:921, 1986). The promoter may additionally include enhancers or other regulatory elements.
The invention also features a host cell (e.g., a prokaryote such as E. coli or other bacteria, or a eukaryote such as a fungus, e.g., yeast).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patents, manufacturers' technical information, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The new polymers have numerous advantages over many existing gel-forming polymers. For example, since any given batch of the new polymers can be produced biologically from a single template, virtually all of the molecules in that batch will be of equal size; gels formed by intermolecular binding of the .alpha.-helical blocks are therefore monodisperse. Monodisperse gels have a uniform pore size that typically depends on the length of the random-coil block.
Standard molecular biological techniques (e.g., automated DNA synthesis) allow any amino acid sequence to be encoded by a gene and expressed in vivo. These techniques enable many characteristics of the new polymers, and therefore of the new suspensions, to be precisely controlled. Examples of the characteristics that can be controlled include: the lengths of the helical and random-coil blocks, the hydrophilicity or hydrophobicity of any of the blocks, the pore size of the gels formed from the polymer suspensions, and the responsiveness of the gels to pH and temperature changes.
Microorganisms can synthesize, in relatively large quantities, high molecular weight materials that are inherently stereoregular, monodisperse, and of controlled sequence. Stereoregularity and monodispersity are rarely achieved by conventional methods of polymerization such as step, chain, ring-opening, and coordination methods. Biosynthetic polymers, on the other hand, can exhibit both of these advantages, and the degree of structural control afforded by biosynthesis extends to the secondary, tertiary, and quaternary levels. The physical properties associated with these biomaterials can be developed on the bases of shape, hydrophilic/hydrophobic character, and charge placement. Moreover, designing and synthesizing polymeric materials biosynthetically allows control over the structure of the materials on both the microscopic and macroscopic levels.
Additionally, a recognition sequence or other peptidic target sequence can advantageously be inserted into, for example, the random-coil blocks of the new block copolymers by splicing a gene encoding that sequence into the template for the new copolymers. The splicing procedure can simply involve digestion of the template with a restriction enzyme followed by ligation with the gene encoding the sequence. Gels having an integral target sequence can be used, for example, in affinity chromatography.
Yet another advantage of the new copolymers is that they have relatively low molecular weight when compared to many other gel-forming molecules. Their low molecular weight can result in decreased viscosity of solutions of the copolymers while still affording high viscosity gels under suitable conditions.
Claim 1 of 30 Claims
1. A synthetic block copolymer XYZ, comprising:
two .alpha.-helical protein blocks X and Z, each having an amino acid
sequence and conformation that allow each .alpha.-helical protein block to
form a coiled-coil with an .alpha.-helical protein block on another block
copolymer XYZ;
a water-soluble, random-coil protein block Y, the random-coil protein
block linking the two .alpha.-helical protein blocks; and
linker proteins that link the .alpha.-helical protein blocks to the
random-coil protein block.
____________________________________________
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