WO2003006068A1 - Therapie genique pour le syndrome d'oeil sec - Google Patents

Therapie genique pour le syndrome d'oeil sec Download PDF

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Publication number
WO2003006068A1
WO2003006068A1 PCT/US2001/021785 US0121785W WO03006068A1 WO 2003006068 A1 WO2003006068 A1 WO 2003006068A1 US 0121785 W US0121785 W US 0121785W WO 03006068 A1 WO03006068 A1 WO 03006068A1
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Prior art keywords
nucleic acid
dihydrazide
hyaluronic acid
sequence
bioconjugate
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PCT/US2001/021785
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English (en)
Inventor
Philip Dehazya
Weiliam Chen
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Clear Solutions Biotech, Inc.
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Priority to PCT/US2001/021785 priority Critical patent/WO2003006068A1/fr
Publication of WO2003006068A1 publication Critical patent/WO2003006068A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the use of derivatized hyaluronic acid/nucleic acid
  • compositions preferably dihydrazide derivatized hyaluronic acid/nucleic acid compositions, and
  • microsphere, film, wafer, matrix, gel and sol formulations comprising these compositions.
  • compositions of the invention also relates to the use of the compositions of the invention, preferably, wherein the
  • nucleic acid is a hyaluronan synthase (HAS) gene, in gene therapy applications to treat dry eye
  • HAS hyaluronan synthase
  • Dry eye is a chronic condition characterized, simplistically, by a paucity of
  • the cause of dry eye can be infectious disease, the aging process,
  • eye include scarcity of moisture and lubrication or tear film, itching, burning, inflammation,
  • the tear film of the eye protects ocular tissues by providing moisture and lubrication. It is composed of three major components: water (whose primary source is the
  • the tear volume may be determined by the
  • ter test This is a test using a filter paper strip with graduations. The strip is placed at the
  • the distance the tear fluid travels determines whether the
  • Dry eye syndrome is a common, chronic condition. It is defined and clinically
  • lacrymal gland dysfunction can all result in dry eye. Thus, there is not one, but many causes,
  • HA hyaluronic acid
  • Hyaluronic acid has been shown to be anti-inflammatory and to stimulate the growth of
  • HA corneal epithelial cells in in vitro and in in vivo tests.
  • HA is also lubricious and hygroscopic.
  • Hyaluronan synthase (HA synthase) is the enzyme that synthesizes hyaluronic acid
  • the enzyme is found in the cell membrane and extrudes polymerized sugar
  • HA synthase (HAS1, HAS2, and HAS3) that differ in catalytic activity and in the type of
  • HA is a component of the tear film, and has anti-inflammatory and lubricating properties, enhancement of HA secretion in the eye of dry eye patients is of clinical
  • One way to enhance HA secretion is by transfection of the tissues of the eye with HA
  • tear film This provides relief of the symptoms of dry eye which is longer-lived than that provided by therapies which are currently available.
  • the HAS 1-3 genes encode a family of proteins which are highly conserved in
  • HAS 1-3 homologues which are known in the art.
  • the numbers represent the percentage of nucleotide/amino acid identity or
  • SEQ ID NO. 1 Mouse HAS1 DNA
  • SEQ ID NO. 2 Mouse HAS2 DNA
  • SEQ ID NO. 3 Mouse HAS3 TmA
  • SEQ ID NO. 4 Mouse HAS 1 Protein
  • SEQ ID NO. 5 Mouse HAS2 Protein
  • SEQ ID NO. 6 Mouse HAS3 Protein
  • the number on the left indicates the level of sequence identity between the specified sequences and the number to the right specifies the level of sequence homology between the specified sequences.
  • the present invention relates to a method for transfecting a cell (in cell culture or
  • nucleic acid e.g. , plasmid DNA
  • hyaluronan a nucleic acid (e.g. , plasmid DNA), preferably hyaluronan
  • nucleic acid has a nucleotide sequence of about 70% to about 100% identity to a reference
  • nucleotide sequence selected from the group consisting of SEQ ID NOs. 1-3 or wherein the
  • nucleic acid has a nucleotide sequence which encodes a hyaluronan synthase enzyme (e.g, HAS 1 -
  • amino acid sequence of the enzyme comprises about 70% to about 100% homology or identity to a reference amino acid sequence selected from the group consisting of
  • a derivatized hyaluronic acid/nucleic acid bioconjugate which comprises said nucleic acid or, preferably, with a dihydrazide derivatized hyaluronic acid/nucleic acid bioconjugate comprising hyaluronic acid crosslinked with adipic dihydrazide wherein the adipic dihydrazide is further crosslinked to the nucleic acid.
  • the method is used in the
  • the present invention also includes the above-described
  • the invention also includes a method of producing a dihydrazide derivatized hyaluronic acid/nucleic acid bioconjugate comprising;
  • nucleic acid comprises a nucleotide sequence of at least
  • hyaluronan synthase protein comprising an amino acid sequence which has about 70% to about
  • step (d) adjusting the pH of the suspension of step (c) to the acidic range and
  • incubating the mixture for a period of time e.g., 6 hours, 24 hours, or 48 hours;
  • step (e) isolating the suspended material from the suspension of step (d); and (f) washing the isolated material from step (e) with an alcohol.
  • cross linking maybe increased by increasing the incubation period of step (d) or decreased by
  • step (d) decreasing the incubation period of step (d).
  • the extent of crosslinking may also be increased by further decreasing the pH at step (d) or decreased by raising the pH at step (d). Increasing the extent of crosslinking
  • the concentration will decrease the extent of crosslinking.
  • lyophilization is performed after cross linking in step (d).
  • FIGURE 1 A representative HAS construct containing the HAS2 gene, a CMV
  • FIGURE 2 The expression of green fluorescent protein (GFP) in the conjunctiva
  • FIGURE 3 The expression of green fluorescent protein in the conjunctiva of
  • FIGURE 4 The expression of green fluorescent protein in the conjunctiva of
  • FIGURE 5 Schematic diagram of formulation steps for HA-DNA films
  • Microspheres were made using an emulsion polymerization method by mixing HA
  • microspheres were weighed and resuspended in physiological phosphate saline buffer. Bovine testicular hyaluronidase was added (e.g., 1 unit/ ml) as appropriate. At the indicated time
  • DNA was quantified using a thiazole orange assay by measuring the fluorescence of DNA bound
  • FIGURE 7 Chemical structures of dihydrazides which may be used in the present invention.
  • FIGURE 8 ADH cross-linked, lyophilized, HA-DNA matrices. Left: low power
  • FIGURE 9 Three formulations of cross-linked HA/DNA matrices.
  • the invention relates to the introduction of hyaluronan synthase genes to tissues
  • Hyaluronan synthase is an enzyme that causes the
  • HAST, HAS2 and HAS3 are the products of 3 distinct but related genes (HAST, HAS2 and HAS3).
  • HAS 1-3 hyaluronan synthase genes
  • HAS 1-3 synthases
  • hyaluronan synthase refers to any enzyme or gene encoding an enzyme which comprises hyaluronan synthase activity.
  • hyaluronan synthase is derived from
  • a protein may comprise hyaluronan synthase activity if said
  • Hyaluronan synthase assays are discussed below.
  • the HAS gene encodes the form of the enzyme that synthesizes HA and secretes
  • the invention uses hyaluronan synthase genes and
  • bioconjugate In these bioconjugates, the nucleic acid is crosslinked to the derivatized hyaluronic
  • the hyaluronic acid is dihydrazide derivativized, more preferably the hyaluronic
  • bioconjugate allows for the sustained delivery
  • the nucleic acid may be any nucleic acid into the surrounding mileau. Once released from the biconjugates, the nucleic acid may
  • nucleic acid wherein a nucleic acid is crosslinked to derivatized hyaluronic acid may be referred to as a " derivatized hyaluronic acid/nucleic acid bioconjugate", “cross-linked HA", “DNA/HA bioconjugate” or “bioconjugate”.
  • the biconjugates of the invention comprise a "dihydrazide derivativized hyaluronic acid/nucleic acid bioconjugate". These terms include all formulations of this composition including microspheres, films, wafers, matrices, gels, sols and
  • patient or "subj ect” refers to any organism, preferably an animal, more preferably a mammal and most preferably a human.
  • a “DNA molecule”, “nucleic acid molecule” or “nucleic acid” refers to the phosphodiester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine , or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix.
  • nucleic acid molecule and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms or to any particular
  • oligonucleotide refers to a nucleic acid molecule of 20 bases
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a
  • a "DNA sequence” or “nucleotide sequence” is a series of nucleotide bases (also
  • nucleotides in DNA and RNA, and means any chain of two or more nucleotides.
  • nucleotide sequence typically carries genetic information, including the information used by
  • DNA or cDNA DNA or cDNA, RNA, any synthetic and genetically manipulated nucleic acid, and both sense and
  • anti-sense nucleic acids This includes single- and double-stranded molecules, i.e. , DNA-DNA,
  • DNA-RNA and RNA-RNA hybrids as well as “protein nucleic acids” (PNA) formed by PNA.
  • PNA protein nucleic acids
  • conjugating bases to an amino acid backbone This also includes nucleic acids containing
  • modified bases for example thio-uracil, thio-guanine and fluoro-uracil.
  • protein refers to any peptide or polypeptide containing two or more amino acids, modified amino acids, or amino acid derivatives.
  • Protein by way of example, and without excluding other types of proteins, includes enzymes (e.g., HAS 1-3) and structural
  • heterologous refers to a combination of elements not naturally occurring.
  • heterologous DNA refers to DNA not naturally located in the cell, or
  • the heterologous DNA includes a gene foreign to
  • a heterologous expression regulatory element is such an element operatively associated
  • nucleic acids and “nucleic acid molecules” herein maybe flanked by natural
  • regulatory (expression control) sequences may be associated with heterologous sequences,
  • promoters including promoters, internal ribosome entry sites (IRES) and other ribosome binding site
  • nucleic acids may also be any nucleic acids, introns, 5'- and 3'- non-coding regions, and the like.
  • the nucleic acids may also be any nucleic acids, introns, 5'- and 3'- non-coding regions, and the like.
  • the nucleic acids may also be any nucleic acids, introns, 5'- and 3'- non-coding regions, and the like.
  • the nucleic acids may also be any nucleic acids
  • a dihydrazide e.g., adipic
  • Nucleic acids may contain one or more additional covalently
  • linked moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal
  • nucleic acids may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • nucleic acids herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
  • Exemplary labels include
  • radioisotopes fluorescent molecules, biotin, and the like.
  • host cell means any cell of any organism that is selected, modified,
  • a host ocular cell is transfected with a hyaluronan synthase gene (e.g. , HAS1-3)
  • Proteins are made in the host cell using instructions in DNA and RNA, according to
  • RNA sequence having instructions for a particular protein or enzyme is "transcribed" into a corresponding sequence of RNA.
  • the RNA sequence in turn is
  • nucleotide triplet or codon AAA can be coded by the nucleotide triplet or codon AAA or by the codon AAG. Codons
  • translation stop signals e.g., TGA, TAG or TAA. Because the nucleotides in
  • DNA and RNA sequences are read in groups of three for protein production, it is important to begin reading the sequence at the correct nucleotide, so that the correct triplets are read.
  • gene refers to a DNA sequence that encodes or corresponds to a particular sequence of amino acids that comprise all or part of one or more proteins, and may or
  • gene also includes DNA sequences which are transcribed from DNA to RNA, but are not translated into
  • a "coding sequence” or a sequence “encoding” an expression product such as a
  • RNA, polypeptide, or protein is a nucleotide sequence that, when expressed, results in the
  • RNA, polypeptide, or protein i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide or protein.
  • a coding sequence for a protein may include a
  • a nucleic acid may also "encode” a gene or DNA
  • nucleotide sequence of the gene or DNA sequence is contained within the
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell or in vitro and initiating transcription of a downstream (3 1 direction) coding
  • a promoter sequence is bounded typically at its 3' terminus by the transcription
  • initiation site and extends upstream (5' direction) to include bases or elements necessary to
  • RNA polymerase domains responsible for the binding of RNA polymerase.
  • a coding sequence may be "under the control of, "operatively associated with”
  • RNA polymerase transcribes the coding sequence into mRNA, which may then be spliced
  • a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • a substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • gene transfer refers broadly to any process by which nucleic acids are
  • gene therapy refers to the use of a gene transfer
  • HAS gene (e.g., HASl, 2 or 3) into the cells of a subject (e.g., ocular cells) constitutes a gene transfer. Transfer of a HAS gene into the ocular cells of a patient for the purpose of treating DES
  • transfection or "transformation” means the introduction of a foreign
  • nucleic acid into a host cell by any means. Transfection or transformation may cause the host
  • the introduced gene or sequence typically a protein coded by the introduced gene or sequence.
  • telomere may also be called a "cloned” or “foreign” gene or sequence and may include regulatory or
  • control sequences such as start, stop, promoter, signal, secretion, or other sequences used by a
  • the gene or sequence may include nonfunctional sequences or sequences with no known function.
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different
  • vector means the vehicle by which a DNA or RNA sequence (e.g., a
  • foreign gene can be introduced into a host cell, so as to transform or transfect the host.
  • Transformation or transfection may promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors may include plasmids (e.g., pcDNA 3.1/HisC12-13).
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign
  • DNA is inserted.
  • a common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes, which cleave DNA at specific sites
  • restriction sites specifically groups of nucleotides
  • DNA ligase which joins pieces of
  • DNA such as a restriction enzyme digested nucleic acid and a restriction enzyme digested
  • a "cassette” refers to a DNA coding sequence or segment of DNA that
  • the cassette restriction sites are designed to ensure insertion of the cassette in the proper reading
  • segment or sequence of DNA having inserted or added DNA such as an expression vector
  • Plasmid also be called a "DNA construct.”
  • a common type of vector is a "plasmid”, which generally is
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction
  • Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms.
  • a large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non- limiting examples include pcDNA3.1, pcDNA/HisC 12-13, pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WT), pRSET or pREP plasmids
  • Recombinant cloning vectors will often include one or more
  • replication systems for cloning or expression one or more markers for selection in the host, e.g.
  • sequence identity refers to exact matches between the two sequences
  • proteins may be determined by use of a BLASTP or CLUSTALW sequence comparison
  • sequence similarity As used herein, the term “sequence similarity”, “similarity”, “sequence homology”
  • proteins may be determined using a CLUSTALW or BLASTP algorithm. A conserved match
  • threonine, tyrosine, serine, glycine no charge/hydrophilic (cysteine, asparagine, glutamine, threonine, tyrosine, serine, glycine); aromatic (tryptophan, tyrosine, phenylalanine); negatively charged/hydrophilic (aspartic acid, glutamic acid); positively charged/hydrophilic (histidine, lysine, arginine).
  • BLAST ALGORITHMS BLAST ALGORITHMS
  • the present invention includes bioconjugates which comprise nucleic acids comprising a hyaluronan synthase gene (i.e., a gene comprising hyaluronan synthase activity),
  • the nucleic acid comprises a nucleotide sequence of at least about 70% identity to a
  • the encoded amino acid sequence comprises at least about 70% homology or identity to a sequence selected from the group consisting of SEQ ID NOs. 4-6, (preferably at
  • parameters of the algorithms are selected to give the largest match between the respective
  • the level of identity or homology mentioned above is greater than about
  • the present invention also includes bioconjugates which comprise nucleic acids comprising a hyaluronan synthase gene comprising a nucleotide sequence of at least about 70% identity to a reference nucleotide sequence of any hyaluronan synthase gene disclosed in U.S.
  • hyaluronan synthase proteins comprising amino acid sequences with at least about 70%) homology or identity to the hyaluronan synthase protein amino acid sequences
  • the level of identity or homology mentioned above is greater than about 70%, preferably about 80% or greater, more
  • induce'Or induction refers to an increase by a measurable amount.
  • hyaluronic acid which has been derivatized with a dihydrazide; a nucleic acid
  • a dihydrazide modified hyaluronic acid in which a pendent hydrazide moiety is still available for reaction may be referred to as a hydrazido hyaluronic acid.
  • a hydrazido hyaluronic acid Alternatively, a
  • any chemical crosslinker e.g., carbodiimides
  • any chemical crosslinker e.g., carbodiimides
  • acid/nucleic acid bioconjugate of the invention may include hyaluronic acid which is crosslinked to a dihydrazide wherein the a dihydrazide portion of the molecule is further crosslinked to a nucleic acid.
  • Adipic dihydrazide is the preferred dihydrazide with which to derivatize hyaluronic acid, however, other dihydrazide molecules may be used for this purpose (see FIGURE 7).
  • Said nucleic acids may be in the form of linear DNA, oligonucleotides or RNA, however, in a preferred embodiment, the nucleic acid is plasmid DNA. Accordingly, a preferred embodiment
  • bioconjugates comprising plasmid DNA conjugated to adipic dihydrazide derivatized hyaluronic acid.
  • the bioconjugates of the invention may be further
  • bioconjugates of the invention may be further conjugated to ligands which allow the bioconjugates to be targeted to a particular location in the subject's
  • this location may comprise a particular type of cell such as conjunctival cells
  • the additional conjugates may be used to prevent or
  • R is any compound.
  • ethyl-, propyl-, isopropyl-, butyl-, pentyl-, hexyl-, heptyl-, or octyl) are preferred, however other
  • dihydrazides are within the scope of the invention, such as sulfonodihydrazides and
  • FIGURE 7 illustrates non- limiting examples of preferred dihydrazides that may be used in the present invention.
  • the carbodiimide used in the present invention comprise ED AC (l-ethyl-3-(3-dimethyl-
  • moiety refers to a chemical entity which may be a part, portion or
  • crosslinked or “conjugated” refers to the attachment of two substances via any type of bond or force.
  • substances may include covalent bonds, ionic bonds or hydrogen bonds, van der Waals forces,
  • Nucleic acids can be released from
  • cross-linked HA microspheres films, wafers, matrices, hydrogels, gels, sols, or any other form
  • gel is intended to refer to viscous or semi-solid and jelly-like material.
  • hydrogel is intended to mean macromolecular networks, which swell in water.
  • film is intended to mean macromolecular networks, which swell in water.
  • matrix is intended to mean a substance formed by lyophilizing a gel.
  • microsphere refers to microscopic particles used to deliver substances, such as nucleic
  • microspheres of this invention may have a diameter (e.g., less than
  • microspheres of the invention are between
  • microspheres of any size wherein the essential elements of the present invention are preserved are preserved
  • a wafer is a matrix like structure which generally has a
  • the derivatized hyaluronic acid/nucleic acid bioconjugates may include any gene. Preferred embodiments include a gene which produces hyaluronic acid or produces a substance which
  • this gene causes the production of hyaluronic acid; in preferred embodiments, this gene is hyaluronan
  • hyaluronic acid/nucleic acid bioconjugates that include said genes may also include, within the nucleic acid that contains the gene, additional
  • nucleotides whose sequence causes expression of a protein or RNA, which corresponds to the
  • bioconjugates which include derivatized hyaluronic acid that is
  • nucleic acids may be produced by any means which yields nucleic acids of sufficient quality and purity so as to allow the successful practice of the invention.
  • nucleic acids may be produced by any means which yields nucleic acids of sufficient quality and purity so as to allow the successful practice of the invention.
  • nucleic acids may be produced by any means which yields nucleic acids of sufficient quality and purity so as to allow the successful practice of the invention.
  • nucleic acids may be produced
  • conjugate of hyaluronic acid and nucleic acid gels or hydrogels may be topically applied for sustained gene transfer.
  • formulations may be produced
  • physiological buffer usually contain a physiological buffer and/or salts such as sodium chloride, sugars such as
  • HA-DNA solution containing the appropriate gene
  • the dosage unit could be extracted and packaged using conventional blister packaging
  • packaged dosage forms may be sterilized by an appropriate method (e.g., ETO, EB, gamma irradiation).
  • the dosage form may be manufactured by an aseptic batch process using sterile (filter sterilized) solutions of reagents and hyaluronan.
  • sterile solution is
  • lyophilization processes are routinely practiced by the pharmaceutical and medical device industries.
  • the sterile, packaged dosage form is stored at room temperature. At the point of use, the package is opened and the dosage form is rehydrated with an appropriate ophthalmic solution. It is then placed in or on the eye in an appropriate position that will allow gene transfer
  • solid dosage forms such as films, wafers and matrices may be
  • a suitable solvent such as buffers or solutions routinely used in ophthalmic practice prior to implantation (e.g., distilled water, phosphate buffer and/or
  • Sols or gels may be applied using syringes, droppers or other devices capable of
  • Microspheres if dried or lyophilized, maybe
  • solvents e.g., distilled water phosphate buffer and/or saline
  • the biconjugate may be placed between the eye and conjunctiva for a period of time which is sufficient to allow a gene transfer process to occur (e.g., 1 hour, 5 hours or 10
  • the bioconjugate may be placed directly over the surface of the eye, after
  • the eye lid may be closed and covered with a patch.
  • the bioconjugates may be allowed to incubate for an extended period (e.g., 12 or 24 hours), after which the patch
  • the eye lid may be opened.
  • bioconjugates of the invention is given to a patient.
  • the dose given to the patient is sufficient to cause a therapeutic effect.
  • patients may require a large amount of new hyaluronan to be produced.
  • a physician may apply two doses simultaneously to increase the number of eye cells transfected, or administer the standard dose more frequently.
  • the derivatized hyaluronic acid/nucleic acid bioconjugates of the invention may
  • DES used in the treatment of DES may comprise an expression plasmid (a closed, circular piece of
  • the promoter may be a human tissue specific promoter which will function in the target
  • a promoter which may be used in this invention is an epithelial cell promoter (e.g. , the ED-LEE promoter), a human papilloma virus promoter, a B2LF 1 promoter from Epstein
  • the construct may also have enhancer
  • nucleic acid is then available for uptake by the target eye cells.
  • FIGURE 1 An example of an experimental construct which may be used to express hyaluronan synthase in cells is shown in FIGURE 1.
  • Expression of the gene in the ocular tissues will increase the hyaluronic acid content of the tear film of the eye. This will ease the symptoms of dry eye.
  • Another aspect relates to the provision of genes to other tissues of the eye that would benefit from localized therapy. Diseases of the retina, for example, could be treated by
  • angiogenesis for the treatment of macular degeneration or genes related to lipid biosynthesis that
  • the present invention may be used to treat medical conditions of the eye wherein delivery of a nucleic acid to the cells of the eye would have a desirable therapeutic effect.
  • eye refers to the visual organ as commonly known including all
  • eye includes, but is not limited to, epithelial
  • the cells of the conjunctiva are
  • bioconjugates of the present invention are useful reagents
  • any cell preferably a eukaryotic cell, more preferably a human eye cell (e.g, a conjunctival or corneal epithelial cell) with a nucleic acid which, preferably,
  • the nucleic acid has a nucleotide sequence of
  • nucleic acid consisting of SEQ ED NOs. 1-3 or wherein the nucleic acid has a nucleotide sequence which encodes a hyaluronan synthase enzyme (e.g, HAS 1 -3), preferably the amino acid sequence of the
  • enzyme comprises about 70% to about 100% homology or identity to a reference amino acid sequence selected from the group consisting of SEQ ED NOs.4-6 wherein homology is
  • the bioconjugates provide a means by which a nucleic acid may be gradually
  • bioconjugate is degraded, the nucleic acid portion of the bioconjugate is liberated and made available for uptake by the host cells.
  • the host cells are used for in vitro cell transformation.
  • liposome mediated conjugates instead of, or in addition to, free, unconjugated, nucleic acids.
  • transformation protocols using the bioconjugates may include seeding a 35 mm plate with about
  • a mixture comprising the bioconjugates (e.g, comprising l ⁇ g or more
  • hyaluronidase e.g, 10, 15, 20, or 25 units/ml bovine testicular hyaluronidase
  • cationic liposomes e.g., lipofectin
  • hyaluronidase is believed to degrade the hylauronic acid of the bioconjugate and release the
  • the rate at which the nucleic acid is released may be modulated by controlling the
  • Hyaluronic acid determination One way to determine the level of hyaluronan synthase activity in a cell is to determine the level of hyaluronic acid in the cell. Cells with a
  • high level of hyaluronan synthase activity may comprise a high level of hyaluronic acid.
  • level of hyaluronan synthase activity which is associated with a given gene or protein e.g., human, Xlaevis, B. taurus, M.musculus, R. norvegicus or G.gallus, HASl, HAS2
  • HAS3 can be determined by introducing the gene (e.g., by use of the bioconjugates of the
  • the level of hyaluronic acid in the cell may be compared to that of a cell into which a
  • Hyaluronic acid production, in cells which have been transformed with the bioconjugates of the invention, may be determined by measuring the
  • hyaluronic acid in a transformed cell maybe measured by a Particle
  • the Assay includes contacting transformed cells with fixed and suspended
  • the erythrocytes may be obtained commercially. After the cells are allowed to settle
  • Hyaluronic acid production by transformed cells may also be measured by a Biotinylation Reporter Assay.
  • transformed cells are contacted with biotinylated
  • biotin-HABP Hyaluronic Acid Binding Protein
  • Biotin-HABP will bind to
  • streptavidin conjugated alkaline phosphatase in added to the biotin-HABP bound cells and an alkaline phosphatase substrate is added.
  • streptavidin moiety of streptavidin conjugated alkaline phosphatase will be described in detail below.
  • biotin-HABP-streptavidin-alkaline phosphatase complex binds to the biotin moiety of biotin-HABP on the surface of the cells.
  • the presence of the biotin- HABP-streptavidin-alkaline phosphatase complex on the cells will be apparent when alkaline phosphatase catalyzes the substrate to produce a colored product.
  • the substrate forms an insoluble product, when catalyzed by alkaline phosphatase, which deposits on the surface of
  • the color change may be observed microscopically or it can be measured colorimetrically in a clear plastic microtiter plate using a microplate reader.
  • Hyaluronan synthase activity may also be determined using the assays disclosed
  • Another aspect relates to the commercial feasibility of manufacture of the
  • aqueous solution of HA is mixed with the DNA, preferably a
  • the pH of the solution is adjusted to an acidic range below 7, and preferably between about 1 to about 5, and the HA-DNA mixture
  • FIGURE 5 illustrates a flow chart depicting the method described above. Gels and sols having approximately 97-99.5% (w/v) water content are made by
  • Matrices and wafers are made by either mixing all of the
  • One aspect of the invention relates to the control of the release of DNA by the
  • linking can be controlled by the concentration of the reactants, the pH of the reaction mix, the
  • the extent of crosslinking may be increased by decreasing the pH of the
  • crosslinking reaction extending the period of time for which the hyaluronic acid is allowed to crosslink in the presence of crosslinker or by increasing the concentration of the crosslinking reagent (e.g., the dihydrazide) in the reaction.
  • the extent of crosslinking may be decreased by increasing the pH of the crosslinking reaction, shortening the period of time for which the hyaluronic acid is allowed to crosslink in the presence of crosslinker or by decreasing the concentration of the crosslinking reagent in the crosslinking reaction.
  • FIGURE 9 demonstrates that DNA/HA matrices including HA incubated in the presence of crosslinker for 6 hours (#2) are less refractory to degradation and release of DNA than that of
  • matrices including HA crosslinked for 12 hours (#1) or 24 hours (#3).
  • the release of DNA can also be controlled by using a variety of cross-linkers
  • dihydrazide produces gels that are more easily degraded.
  • the kinetics of DNA release can be
  • DNA can be evaluated.
  • the assay described in this invention can also be performed in the
  • the method of drying is lyophilization. Lyophilization refers to the process of freezing a liquid and drying it
  • EXAMPLE 1 EXPRESSION OF MURINE HYALURONAN SYNTHASE GENES IN
  • HAS2 and HAS3 in mammalian cells were prepared as follows. Plasmids encoding
  • mouse hyaluronan synthase cDNAs HAS2 SEQ TD NOs: 2 and 5; Spicer et al, J. Biol.
  • the insert DNA was purified from the gel slices by solubilizing in a chaotropic buffer, binding to a commercially available (Qiagen;
  • silica matrix spin filter followed by elution with a Tris buffer at pH8.5.
  • primers were designed to conserve the previously inserted optimized Kozak initiation sequence
  • thermostable polymerase pfu Turbo, Stratagene; La Jolla, CA
  • the resulting PCR products were analyzed by gel electrophoresis and purified (Qiagen; Valencia, CA). All four sets of PCR products were digested sequentially with Xhol and BamH ⁇ ,
  • the vector was double cut with
  • V5 is a viral epitope engineered
  • the volume of the solution was about 90 % of the final volume to allow for the addition
  • the DNA expression plasmid of EXAMPLE 1 was added and mixed well at slow speed (about 100 rpm). To effect a 1% weight per volume loading, 10 mg of DNA was added
  • the liquid mixture may be dispensed into a mold that will contain one dose or multiple doses of the therapeutic. Alternatively, the mixture may be placed into a mold that will
  • cross-linker e.g., adipic dihydrazide or ADH
  • each mixing step was performed for 10 minutes. If buffers are not present, the
  • pH will be slightly basic after addition of reagents and prior to acidification. Because the release
  • genes can be modulated by the degree of cross-linking, this step can be varied.
  • the lyophilized matrices are cross-linked for 6 hours. This will enable a relatively long term
  • the cross-linking step can be performed for sustained gene delivery.
  • the cross-linking step can be performed for sustained gene delivery.
  • the matrices maybe extracted with alcohol while still within the
  • the molds by introducing the alcohol to the mold, and allowing the matrix to "soak" in the alcohol solution for the required amount of time.
  • the alcohol is removed by aspiration and the process is repeated 4 times. After extraction of reagents as depicted in the flow chart, the matrices are
  • the extracted matrix maybe packaged as other ophthalmic products; for example,
  • FIGURE 1 An electron micrograph of a matrix produced by this method is shown in FIGURE
  • hyaluronic acid bioconjugate maybe transferred to living rat ocular cells and that the transformed ocular cells may express functional GFP.
  • the GFP gene was functionally associated with a promoter which can cause
  • FIGURES 2-4 Micrographs showing GFP fluorescence in the rat eyes are shown in FIGURES 2-4.
  • the GFP allele used in the experiment had a short half life of only 2-4 hours and yet GFP fluorescence was observed for an extended period after initial transfer of the gene.
  • human corneal epithelial cells are transformed with matrices of
  • the invention comprising mouse HASl , HAS2 or HAS3. Furthermore, expression of hyaluronic
  • mice HASl SEQ ED NO.1
  • mouse HAS2 SEQ ED NO.2
  • mouse HAS 3 SEQ ED NO.3
  • pcDNA3.1/V5-His A invitrogen; Carlsbad, CA
  • Hyaluronic acid production is then determined for each transformed cell line.
  • plasmid pcDNA3.1/V5-His A (Invitrogen; Carlsbad, CA) which drives expression of the HAS 1, HAS2 or HAS 3 genes from a CMV promoter.
  • Matrices comprising each plasmid are
  • Human corneal epithelial cell lineHCE-2 (CRL-11135;ATCC,
  • Solution A Dilute DNA/HA matrix bioconjugates in 1 OO ⁇ l serum-free medium ( OPTI-MEM,
  • Solution B For each transfection, dilute 2-25 ⁇ l of LEPOFECTAMENE Reagent into 1 OO ⁇ l of the
  • Solutions A and B are combined and mixed gently, and incubate at room
  • Hylauronic acid determination The quantity of hyaluronic acid associated with
  • each cell line is determined using the biotin/streptavidin/horse radish peroxidase conjugated assay discussed below.
  • the transformed cells are fixed and incubated for 1 hour with
  • HABP biotinylated hyaluronic acid binding protein

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Abstract

L'invention concerne des bioconjugués comprenant des substances mettant en oeuvre une sûreté et une biodisponibilité améliorées d'acides nucléiques lorsqu'elles sont utilisées dans des applications de thérapie génique. Les compositions d'acide nucléique et d'acide hyaluronique à dérivatisation dihydrazide de l'invention contiennent de l'acide hyaluronique dérivatisé avec un dihydrazide, de préférence avec un dihydrazide de l'acide adipique, réticulé à un acide nucléique. Ces compositions peuvent être enfermées dans des formulations de microsphère, de film, de cachet, de matrice, d'hydrogel, de gel et de sol. Ces bioconjugués sont utiles dans des applications de thérapie génique concernant le traitement de plusieurs troubles médicaux y compris le syndrome d'oeil sec ou d'autres troubles médicaux dans lesquels une augmentation de la production d'acide hyaluronique dans l'oeil conduit à un effet thérapeutique. Il existe, en outre, d'autres troubles médicaux qui pourraient retirer un bénéfice d'une augmentation de la production de hyaluronane, notamment l'ostéoarthrite des articulations. Dans le traitement du syndrome d'oeil sec, les compositions de l'invention contiennent un gène humain de hyaluronane synthétase. Lorsque des cellules de l'oeil sont transfectées avec ce gène et expriment la hyaluronane synthétase, il en résulte une production d'acide hyaluronique. La production d'acide hyaluronique dans l'oeil produit un effet thérapeutique, lors du traitement du syndrome d'oeil sec, par augmentation de la production ou changement de composition du film lacrymal. Il est aussi possible de produire les compositions de l'invention grâce à un procédé facilement modifié pour la production de grandes quantités d'acide hyaluronique dérivatisé avec un dihydrazide.
PCT/US2001/021785 2001-07-10 2001-07-10 Therapie genique pour le syndrome d'oeil sec WO2003006068A1 (fr)

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US6982298B2 (en) 2003-01-10 2006-01-03 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
WO2006010066A3 (fr) * 2004-07-09 2006-07-20 Cleveland Clinic Foundation Reseau macromoleculaire reticule de hydroxyphenyle et applications associees
US7465766B2 (en) 2004-01-08 2008-12-16 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8080260B2 (en) 2008-02-13 2011-12-20 The Cleveland Clinic Foundation Molecular enhancement of extracellular matrix and methods of use
US8137688B2 (en) 2003-01-10 2012-03-20 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8138265B2 (en) 2003-01-10 2012-03-20 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8410180B2 (en) 2008-04-30 2013-04-02 The Cleveland Clinic Foundation Methods to treat urinary incontinence

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Cited By (10)

* Cited by examiner, † Cited by third party
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US6982298B2 (en) 2003-01-10 2006-01-03 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US7368502B2 (en) 2003-01-10 2008-05-06 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8021350B2 (en) 2003-01-10 2011-09-20 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8137688B2 (en) 2003-01-10 2012-03-20 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8138265B2 (en) 2003-01-10 2012-03-20 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US8207262B2 (en) 2003-01-10 2012-06-26 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
US7465766B2 (en) 2004-01-08 2008-12-16 The Cleveland Clinic Foundation Hydroxyphenyl cross-linked macromolecular network and applications thereof
WO2006010066A3 (fr) * 2004-07-09 2006-07-20 Cleveland Clinic Foundation Reseau macromoleculaire reticule de hydroxyphenyle et applications associees
US8080260B2 (en) 2008-02-13 2011-12-20 The Cleveland Clinic Foundation Molecular enhancement of extracellular matrix and methods of use
US8410180B2 (en) 2008-04-30 2013-04-02 The Cleveland Clinic Foundation Methods to treat urinary incontinence

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